Histochemistry Of Single Molecules Methods And

Protocols Methods In Molecular Biology 2566 2nd

Ed 2023 Carlo Pellicciari Editor download

https://ebookbell.com/product/histochemistry-of-single-molecules-
methods-and-protocols-methods-in-molecular-biology-2566-2nd-
ed-2023-carlo-pellicciari-editor-46507584

Explore and download more ebooks at ebookbell.com

Here are some recommended products that we believe you will be
interested in. You can click the link to download.

Histochemistry Of Single Molecules Carlo Pellicciari Marco Biggiogera

https://ebookbell.com/product/histochemistry-of-single-molecules-
carlo-pellicciari-marco-biggiogera-37739592

Histochemistry Jinsong Zhou Xian Jiaotong University Press Co

https://ebookbell.com/product/histochemistry-jinsong-zhou-xian-
jiaotong-university-press-co-50339066
Methods in
Molecular Biology 2566

Carlo Pellicciari · Marco Biggiogera

Manuela Malatesta Editors

Histochemistry
of Single
Molecules
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
 Histochemistry of Single
Molecules

Methods and Protocols

Second Edition

Edited by

Carlo Pellicciari
Department of Biology and Biotechnology, University of Pavia, PAVIA, Italy

Marco Biggiogera
Department of Biology and Biotechnology, University of Pavia, PAVIA, Italy

Manuela Malatesta
Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, VERONA, Italy
Editors
Carlo Pellicciari Marco Biggiogera
Department of Biology and Department of Biology and Biotechnology
Biotechnology University of Pavia
University of Pavia PAVIA, Italy
PAVIA, Italy

Manuela Malatesta
Department of Neurosciences,
Biomedicine and Movement Sciences
University of Verona
VERONA, Italy

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-2674-0 ISBN 978-1-0716-2675-7 (eBook)
https://doi.org/10.1007/978-1-0716-2675-7
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
of Springer Nature 2023
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and
retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter
developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Cover Illustration: Combined lectin- and immune-histochemistry on a semithin cryosection of normal human
urothelium. For labelling the proteins, the primary polyclonal rabbit antibody against transmembrane proteins
uroplakins and Alexa Flour 588 conjugated secondary goat anti-rabbit antibody were used (red fluorescence), while
for labelling the sugar residues, the FITC-conjugated Amaranthus caudatus agglutinin (ACA) was used (green
fluorescence). Colocalization of uroplakins and ACA binding is seen as orange to yellow fluorescence. Nuclear DNA
was counterstained with DAPI (blue fluorescence). (Courtesy of Daša Zupančič, Mateja Erdani Kreft and Rok Romih.)

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface

As a distinctive feature, histochemical techniques allow localizing different chemical species

in the very place (in a tissue, or a cell or an organelle) they exist, are synthesized, or function
in vivo; this makes histochemistry a unique tool for basic and applied bio-medical research,
where it provides topological evidence of the biochemical and molecular data.
The story of histochemistry goes a long way back and, in the last 150 years, has evolved
in parallel with the detection instruments, from light and electron microscopy to cytometry
and super-resolution microscopy. Since the year 2000, about 325,000 articles, in which
histochemistry was used, have been published in qualified international journals (according
to the Web of Science database). This demonstrates that histochemistry is central for inves-
tigating very different research subjects (from cell and tissue biology to anatomy and
pathology, from zoology and botany to ecology, from developmental biology to nanotech-
nology), where it is principally applied to locate and quantify single molecules or molecular
complexes in situ, in the attempt to relate structural organization and function.
This second edition of Histochemistry of Single Molecules aims at updating and improving
the first edition’s overview of histochemical techniques, through a series of discursive
chapters and lab-tested protocols for the detection of specific molecules or metabolic
processes, at light and electron microscopy.
The book opens with a review chapter on the evolution of histochemical markers in
cytometry, and then it is divided into seven parts dealing with an assortment of chemical
targets.
Part I is on vital histochemistry, and it includes four chapters on autofluorescence
imaging, lysosome imaging by carbon dots, the detection of oxidative and nitrosative stress,
and the identification of adipogenic and osteogenic differentiation of living stem cells. Part
II, Carbohydrate Histochemistry, is comprises an updated overview on lectin histochemis-
try, followed by two chapters on the histochemical and immunohistochemical labelling of
proteoglycans, and on combined lectin- and immuno-histochemistry for fluorescence
microscopy. Five chapters form Part III, Protein Histochemistry: in the first four chapters,
immunohistochemistry is used to detect proteins marking myogenic differentiation or
autophagy at light microscopy, or milk proteins at electron microscopy, while in the last
one potassium permanganate is rediscovered as a stain for basic proteins on ultrathin
sections at transmission electron microscopy. Part IV, Lipid Histochemistry, offers an update
of basics in fixation and tissue processing, and gives protocols for the staining of myelin in
the nervous system or of lipid droplets in mouse oocytes and embryos. Part V, Nuclear
Histochemistry, contains a protocol for assessing DNA damage in cervical epithelial cells and
two contrasting methods for transmission electron microscopy: a uranyl-free technique for
nuclear structures and a staining procedure for the specific visualization of RNA by terbium
citrate vapors. Part VI is on plant histochemistry: three chapters describe protocols for the
detection of different molecules in plant cell walls, one is on starch staining with iodine
solution, and the last one on plant secretory structures. The book ends with Part VII,
Histochemistry for Nanoscience: this part, which was not present in the first edition,
includes protocols for the visualization of chemically different nanoconstructs in animal
and plant cells at bright-field, fluorescence, and transmission electron microscopy.

v
vi Preface

These 28 chapters demonstrate that histochemical techniques may be effectively used to

visualize, with high specificity, a plethora of molecules in differently processed tissues and
cells, thus confirming that histochemistry still ranks high among the methodological
approaches in life science research.

Pavia, Italy Carlo Pellicciari

Pavia, Italy Marco Biggiogera
Verona, Italy Manuela Malatesta
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 Histochemistry in Advanced Cytometry: From Fluorochromes
to Mass Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Giuliano Mazzini and Marco Danova

PART I VITAL HISTOCHEMISTRY

2 Autofluorescence Label-Free Imaging of the Liver Reticular Structure. . . . . . . . . 29
Anna C. Croce, Giuseppina Palladini, Andrea Ferrigno,
and Mariapia Vairetti
3 Lysosome Imaging Based on Fluorescent Carbon Dots . . . . . . . . . . . . . . . . . . . . . . 37
Shuo Guo, Yuanqiang Sun, and Zhaohui Li
4 Oxidative and Nitrosative Stress Detection in Human Sperm
Using Fluorescent Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Sara Escada-Rebelo and João Ramalho-Santos
5 Simultaneous Labeling of Adipogenic and Osteogenic Differentiating
Stem Cells for Live Confocal Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Patrizia Vaghi, Amanda Oldani, Paola Fulghieri, Lidia Pollara,
Enza Maria Valente, and Virginie Sottile

PART II CARBOHYDRATE HISTOCHEMISTRY

6 Lectin Histochemistry: Historical Perspectives, State of the Art,

and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Susan Ann Brooks
7 Histochemical and Immunohistochemical Methods for the
Identification of Proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
David Sánchez-Porras, Juan Varas, Carlos Godoy-Guzmán,
Fabiola Bermejo-Casares, Sebastián San Martı́n, and Vı́ctor Carriel
8 Combined Lectin- and Immuno-histochemistry (CLIH) for
Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Daša Zupančič, Mateja Erdani Kreft, and Rok Romih

PART III PROTEIN HISTOCHEMISTRY

9 Immunofluorescence Labeling of Skeletal Muscle in Development,
Regeneration, and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Marie E. Esper, Kasun Kodippili, and Michael A. Rudnicki

vii
viii Contents

10 Immunohistochemical Detection of the Autophagy Markers LC3

and p62/SQSTM1 in Formalin-Fixed and Paraffin-Embedded Tissue . . . . . . . . . 133
Sabina Berezowska and José A. Galván
11 Immunohistochemical Detection of the Chaperone-Mediated
Autophagy Markers LAMP2A and HSPA8 in Formalin-Fixed and
Paraffin-Embedded Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Tereza Losmanová, Mario P. Tschan, José A. Galván,
and Sabina Berezowska
12 Immunogold Labeling of Milk Proteins at Transmission
Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Paolo D’Incecco
13 Rediscover Potassium Permanganate as a Stain for Basic Proteins
on Ultrathin Sections at Transmission Electron Microscopy . . . . . . . . . . . . . . . . . . 159
Lorena Zannino, Claudio Casali, and Marco Biggiogera

PART IV LIPID HISTOCHEMISTRY

14 Tissue Fixation and Processing for the Histological Identification

of Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
David Sánchez-Porras, Fabiola Bermejo-Casares, Ramon Carmona,
Tamara Weiss, Fernando Campos, and Vı́ctor Carriel
15 Staining Methods for Normal and Regenerative Myelin in the
Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Óscar D. Garcı́a-Garcı́a, Tamara Weiss, Jesús Chato-Astrain,
Stefania Raimondo, and Vı́ctor Carriel
16 Nile Red and BODIPY Staining of Lipid Droplets in Mouse
Oocytes and Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Simona Bisogno, Łukasz Ga˛sior, and Grażyna E. Ptak

PART V NUCLEAR HISTOCHEMISTRY

17 Chromatin Dispersion Test to Asses DNA Damage in Cervical

Epithelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Elva I. Cortés-Gutiérrez, José L. Fernández, Martha I. Dávila-Rodrı́guez,
Carlos Garcı́a de la Vega, and Jaime Gosálvez
18 Uranyl-Free Staining as a Suitable Contrasting Technique for
Nuclear Structures at Transmission Electron Microscopy . . . . . . . . . . . . . . . . . . . . 225
Maria Assunta Lacavalla and Barbara Cisterna
19 Specific RNA Visualization at Electron Microscopy via Terbium
Citrate Vapors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Claudio Casali, Lorena Zannino, and Marco Biggiogera

PART VI PLANT HISTOCHEMISTRY

20 Localizing Molecules in Plant Cell Walls Using Fluorescence Microscopy . . . . . . 243

Lloyd A. Donaldson
 Contents ix

21 Ratiometric Fluorescent Safranin-O Staining Allows the

Quantification of Lignin Contents In Muro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Oriane Morel, Corentin Spriet, Cédric Lion, Fabien Baldacci-Cresp,
Garance Pontier, Marie Baucher, Christophe Biot, Simon Hawkins,
and Godfrey Neutelings
22 Live Fluorescence Visualization of Cellulose and Pectin in
Plant Cell Walls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Youssef Chebli and Anja Geitmann
23 Staining Starch with Iodine Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Shengnan Zhao, Yinhui Ren, and Cunxu Wei
24 Histochemical Analysis of Plant Secretory Structures . . . . . . . . . . . . . . . . . . . . . . . . 291
Diego Demarco

PART VII HISTOCHEMISTRY FOR NANOSCIENCE

25 Alcian Blue Staining to Visualize Intracellular Hyaluronic

Acid-Based Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Mathieu Repellin, Flavia Carton, Giovanna Lollo,
and Manuela Malatesta
26 Prussian Blue Staining to Visualize Iron Oxide Nanoparticles . . . . . . . . . . . . . . . . 321
Valeria Bitonto, Francesca Garello, Arnaud Scherberich,
and Miriam Filippi
27 Diaminobenzidine Photooxidation to Visualize Fluorescent
Nanoparticles in Adhering Cultured Cells at Transmission
Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Manuela Costanzo and Manuela Malatesta
28 Fluorescent Labeling of Lignin Nanocapsules with Fluorol
Yellow 088 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Franco Cheli, Sara Falsini, Maria Cristina Salvatici,
Sandra Ristori, Silvia Schiff, Emilio Corti, Irene Costantini,
Cristina Gonnelli, Francesco Saverio Pavone, and Alessio Papini

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Contributors

FABIEN BALDACCI-CRESP • Université de Lille, CNRS, UMR 8576, UGSF – Unité de

Glycobiologie Structurale et Fonctionnelle, Lille, France
MARIE BAUCHER • Laboratoire de Biotechnologie Végétale (LBV), Université Libre de
Bruxelles, Gosselies, Belgium
SABINA BEREZOWSKA • Department of Laboratory Medicine and Pathology, Institute of
Pathology, Lausanne University Hospital and University of Lausanne, Lausanne,
Switzerland; Institute of Pathology, University of Bern, Bern, Switzerland
FABIOLA BERMEJO-CASARES • Department of Histology (Tissue Engineering Group), Faculty
of Medicine, University of Granada, Granada, Spain; Instituto de Investigacion
Biosanitaria, Ibs.GRANADA, Granada, Spain
MARCO BIGGIOGERA • Department of Biology and Biotechnology “Lazzaro Spallanzani”,
Laboratory of Cell Biology and Neurobiology, University of Pavia, Pavia, Italy
CHRISTOPHE BIOT • Université de Lille, CNRS, UMR 8576, UGSF – Unité de Glycobiologie
Structurale et Fonctionnelle, Lille, France
SIMONA BISOGNO • Malopolska Centre of Biotechnology, Jagiellonian University, Krakow,
Poland
VALERIA BITONTO • Department of Molecular Biotechnology and Health Sciences, University
of Turin, Torino, Italy
SUSAN ANN BROOKS • Department of Biological & Medical Sciences, Oxford Brookes
University, Oxford, UK
FERNANDO CAMPOS • Department of Histology (Tissue Engineering Group), Faculty of
Medicine, University of Granada, Granada, Spain; Instituto de Investigacion
Biosanitaria, Ibs.GRANADA, Granada, Spain
RAMÓN CARMONA • Department of Cell Biology, Faculty of Science, University of Granada,
Granada, Spain
VÍCTOR CARRIEL • Department of Histology (Tissue Engineering Group), Faculty of
Medicine, University of Granada, Granada, Spain; Instituto de Investigacion
Biosanitaria, Ibs.GRANADA, Granada, Spain
FLAVIA CARTON • Department of Neurosciences, Biomedicine and Movement Sciences,
Anatomy and Histology Section, University of Verona, Verona, Italy; University of Eastern
Piedmont, Department of Health Sciences, Novara, Italy
CLAUDIO CASALI • Department of Biology and Biotechnology “Lazzaro Spallanzani”,
Laboratory of Cell Biology and Neurobiology, University of Pavia, Pavia, Italy
JESÚS CHATO-ASTRAIN • Department of Histology (Tissue Engineering Group), Faculty of
Medicine, University of Granada, Granada, Spain; Instituto de Investigacion
Biosanitaria, Ibs.GRANADA, Granada, Spain
YOUSSEF CHEBLI • Department of Plant Science and ECP3-Multi-Scale Imaging Facility,
McGill University, Sainte-Anne-de-Bellevue, QC, Canada
FRANCO CHELI • LENS – European Laboratory for Non-linear Spectroscopy, University of
Florence, Florence, Italy
BARBARA CISTERNA • Department of Neurosciences, Biomedicine and Movement Sciences,
University of Verona, Verona, Italy

xi
xii Contributors

ELVA I. CORTÉS-GUTIÉRREZ • Universidad Autonoma de Nuevo Leon, México Faculty of

Biological Sciences, Monterrey, Mexico
EMILIO CORTI • Department of Biology, University of Florence, Florence, Italy
IRENE COSTANTINI • LENS – European Laboratory for Non-linear Spectroscopy, University of
Florence, Florence, Italy
MANUELA COSTANZO • Department of Neurosciences, Biomedicine and Movement Sciences,
Anatomy and Histology Section, University of Verona, Verona, Italy
ANNA C. CROCE • Institute of Molecular Genetics “Luigi Luca Cavalli Sforza” (IGM) –
CNR, Pavia, Italy; Department of Biology and Biotechnology “Lazzaro Spallanzani”,
University of Pavia, Pavia, Italy
PAOLO D’INCECCO • Department of Food, Environmental and Nutritional Sciences,
University of Milan, Milan, Italy
MARCO DANOVA • Department of Internal Medicine and Oncology, ASST Pavia and
LIUCC University, Castellanza, Varese, Italy
MARTHA I. DÁVILA-RODRÍGUEZ • Universidad Autonoma de Nuevo Leon, Faculty of Public
Health and Nutrition, Monterrey, Mexico
DIEGO DEMARCO • Departamento de Botânica, Instituto de Biociências, Universidade de São
Paulo, São Paulo, Brazil
LLOYD A. DONALDSON • Scion Crown Research Institute, Rotorua, New Zealand
SARA ESCADA-REBELO • PhD Programme in Experimental Biology and Biomedicine (BEB),
IIIUC- Institute for Interdisciplinary Research, University of Coimbra, Coimbra,
Portugal; Biology of Reproduction and Stem Cell Group, Center for Neuroscience and Cell
Biology, University of Coimbra, Coimbra, Portugal
MARIE E. ESPER • The Sprott Centre for Stem Cell Research, Regenerative Medicine Program,
Ottawa Hospital Research Institute, Ottawa, ON, Canada; Department of Cellular and
Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada
SARA FALSINI • Department of Biology, University of Florence, Florence, Italy
JOSÉ L. FERNÁNDEZ • Genetics Unit, INIBIC, Complejo Hospitalario Universitario A
Coruña, As Xubias, La Coruña, Spain; Laboratorio de Genética Molecular y
Radiobiologı́a Centro Oncologico de Galicia, La Coruña, Spain
ANDREA FERRIGNO • Department of Internal Medicine and Therapeutics, University of
Pavia, Pavia, Italy
MIRIAM FILIPPI • Soft Robotics Laboratory, ETH Zurich, Zurich, Switzerland
PAOLA FULGHIERI • Department of Molecular Medicine, University of Pavia, Pavia, Italy
JOSÉ A. GALVÁN • Institute of Pathology, University of Bern, Bern, Switzerland
CARLOS GARCÍA DE LA VEGA • Unit of Genetics, Department of Biology, Universidad
Autonoma de Madrid, Madrid, Spain
ÓSCAR D. GARCÍA-GARCÍA • Department of Histology (Tissue Engineering Group), Faculty of
Medicine, University of Granada, Granada, Spain; Instituto de Investigacion
Biosanitaria, Ibs.GRANADA, Granada, Spain
FRANCESCA GARELLO • Department of Molecular Biotechnology and Health Sciences,
University of Turin, Torino, Italy
ŁUKASZ GA˛SIOR • Malopolska Centre of Biotechnology, Jagiellonian University, Krakow,
Poland
ANJA GEITMANN • Department of Plant Science and ECP3-Multi-Scale Imaging Facility,
McGill University, Sainte-Anne-de-Bellevue, QC, Canada
 Contributors xiii

CARLOS GODOY-GUZMÁN • Centro de Investigacion Biomédica y Aplicada (CIBAP), Escuela

de Medicina, Universidad de Santiago de Chile, (USACH), Santiago, Chile
CRISTINA GONNELLI • Department of Biology, University of Florence, Florence, Italy
JAIME GOSÁLVEZ • Unit of Genetics, Department of Biology, Universidad Autonoma de
Madrid, Madrid, Spain
SHUO GUO • College of Chemistry, Institute of Analytical Chemistry for Life Science,
Zhengzhou University, Zhengzhou, China
SIMON HAWKINS • Université de Lille, CNRS, UMR 8576, UGSF – Unité de Glycobiologie
Structurale et Fonctionnelle, Lille, France
KASUN KODIPPILI • The Sprott Centre for Stem Cell Research, Regenerative Medicine
Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada; Department of
Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa,
ON, Canada
MATEJA ERDANI KREFT • Institute of Cell Biology, Faculty of Medicine, University of
Ljubljana, Ljubljana, Slovenia
MARIA ASSUNTA LACAVALLA • Department of Neurosciences, Biomedicine and Movement
Sciences, University of Verona, Verona, Italy
ZHAOHUI LI • College of Chemistry, Institute of Analytical Chemistry for Life Science,
Zhengzhou University, Zhengzhou, China
CÉDRIC LION • Université de Lille, CNRS, UMR 8576, UGSF – Unité de Glycobiologie
Structurale et Fonctionnelle, Lille, France
GIOVANNA LOLLO • Laboratoire d’Automatique, de Génie des Procédés et de Génie
Pharmaceutique, Université Claude Bernard Lyon 1, Villeurbanne, France
TEREZA LOSMANOVÁ • Institute of Pathology, University of Bern, Bern, Switzerland
MANUELA MALATESTA • Department of Neurosciences, Biomedicine and Movement Sciences,
Anatomy and Histology Section, University of Verona, Verona, Italy
GIULIANO MAZZINI • Institute of Molecular Genetics – CNR (National Research Council),
Pavia, Italy; Department of Biology and Biotechnology “Lazzaro Spallanzani”, University
of Pavia, Pavia, Italy
ORIANE MOREL • Université de Lille, CNRS, UMR 8576, UGSF – Unité de Glycobiologie
Structurale et Fonctionnelle, Lille, France; Institute of Biophysics, University of Natural
Resources and Life Sciences Vienna, Vienna, Austria
GODFREY NEUTELINGS • Université de Lille, CNRS, UMR 8576, UGSF – Unité de
Glycobiologie Structurale et Fonctionnelle, Lille, France
AMANDA OLDANI • PASS-Bio Med, Centro Grandi Strumenti, University of Pavia, Pavia,
Italy
GIUSEPPINA PALLADINI • Fondazione IRCCS Policlinico San Matteo, Pavia, Italy;
Department of Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy
ALESSIO PAPINI • Department of Biology, University of Florence, Florence, Italy
FRANCESCO SAVERIO PAVONE • LENS – European Laboratory for Non-linear Spectroscopy,
University of Florence, Florence, Italy
LIDIA POLLARA • Department of Molecular Medicine, University of Pavia, Pavia, Italy
GARANCE PONTIER • Université de Lille, CNRS, UMR 8576, UGSF – Unité de Glycobiologie
Structurale et Fonctionnelle, Lille, France
GRAŻYNA E. PTAK • Malopolska Centre of Biotechnology, Jagiellonian University, Krakow,
Poland
xiv Contributors

STEFANIA RAIMONDO • Dipartimento di Scienze Cliniche e Biologiche, Università di Torino,

Torino, Italy; Neuroscience Institute Cavalieri Ottolenghi (NICO), Torino, Italy
JOÃO RAMALHO-SANTOS • Biology of Reproduction and Stem Cell Group, Center for
Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Department of
Life Sciences, University of Coimbra, Coimbra, Portugal
YINHUI REN • Key Laboratory of Crop Genetics and Physiology of Jiangsu Province/Key
Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou
University, Yangzhou, China; Co-Innovation Center for Modern Production Technology of
Grain Crops of Jiangsu Province/Joint International Research Laboratory of Agriculture
and Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou,
China
MATHIEU REPELLIN • Department of Neurosciences, Biomedicine and Movement Sciences,
Anatomy and Histology Section, University of Verona, Verona, Italy; Laboratoire
d’Automatique, de Génie des Procédés et de Génie Pharmaceutique, Université Claude
Bernard Lyon 1, Villeurbanne, France
SANDRA RISTORI • Department of Chemistry, University of Florence, Florence, Italy
ROK ROMIH • Institute of Cell Biology, Faculty of Medicine, University of Ljubljana,
Ljubljana, Slovenia
MICHAEL A. RUDNICKI • The Sprott Centre for Stem Cell Research, Regenerative Medicine
Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada; Department of
Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa,
ON, Canada
MARIA CRISTINA SALVATICI • Institute of Chemistry of Organometallic Compounds
(ICCOM)-Electron Microscopy Centre (Ce.M. E.), National Research Council (CNR),
Florence, Italy
SEBASTIÁN SAN MARTÍN • Centro de Investigaciones Biomédicas, Escuela de Medicina,
Facultad de Medicina, Universidad de Valparaı́so, Valparaı́so, Chile
DAVID SÁNCHEZ-PORRAS • Department of Histology (Tissue Engineering Group), Faculty of
Medicine, University of Granada, Granada, Spain; Instituto de Investigacion
Biosanitaria, Ibs.GRANADA, Granada, Spain
ARNAUD SCHERBERICH • Department of Biomedicine, University and University Hospital of
Basel, Basel, Switzerland; Department of Biomedical Engineering, University of Basel,
Allschwil, Switzerland
SILVIA SCHIFF • Department of Biology, University of Florence, Florence, Italy
VIRGINIE SOTTILE • Department of Molecular Medicine, University of Pavia, Pavia, Italy
CORENTIN SPRIET • Université de Lille, CNRS, UMR 8576, UGSF – Unité de Glycobiologie
Structurale et Fonctionnelle, Lille, France; Université de Lille, CNRS, Inserm, CHU Lille,
Institut Pasteur de Lille, US 41-UMS 2014-PLBS, Lille, France
YUANQIANG SUN • College of Chemistry, Institute of Analytical Chemistry for Life Science,
Zhengzhou University, Zhengzhou, China
MARIO P. TSCHAN • Institute of Pathology, University of Bern, Bern, Switzerland
PATRIZIA VAGHI • PASS-Bio Med, Centro Grandi Strumenti, University of Pavia, Pavia,
Italy
MARIAPIA VAIRETTI • Department of Internal Medicine and Therapeutics, University of
Pavia, Pavia, Italy
 Contributors xv

ENZA MARIA VALENTE • Department of Molecular Medicine, University of Pavia, Pavia,

Italy; Neurogenetics Research Centre, IRCCS Mondino Foundation, Pavia, Italy
JUAN VARAS • Centro de Investigaciones Biomédicas, Escuela de Medicina, Facultad de
Medicina, Universidad de Valparaı́so, Valparaı́so, Chile
CUNXU WEI • Key Laboratory of Crop Genetics and Physiology of Jiangsu Province/Key
Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou
University, Yangzhou, China; Co-Innovation Center for Modern Production Technology of
Grain Crops of Jiangsu Province/Joint International Research Laboratory of Agriculture
and Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou,
China
TAMARA WEISS • Department of Plastic, Reconstructive and Aesthetic Surgery, Medical
University of Vienna, Vienna, Austria
LORENA ZANNINO • Department of Biology and Biotechnology “Lazzaro Spallanzani”,
Laboratory of Cell Biology and Neurobiology, University of Pavia, Pavia, Italy
SHENGNAN ZHAO • Key Laboratory of Crop Genetics and Physiology of Jiangsu Province/Key
Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou
University, Yangzhou, China; Co-Innovation Center for Modern Production Technology of
Grain Crops of Jiangsu Province/Joint International Research Laboratory of Agriculture
and Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou,
China
DAŠA ZUPANČIČ • Institute of Cell Biology, Faculty of Medicine, University of Ljubljana,
Ljubljana, Slovenia
 Chapter 1

Histochemistry in Advanced Cytometry: From

Fluorochromes to Mass Probes
Giuliano Mazzini and Marco Danova

Abstract
For over half a century, fluorescence has been the milestone of most of the quantitative approaches in
various fields from chemistry and biochemistry to microscopy. This latter also evolved into cytometry,
thanks to the development of fluorescence techniques. The dyes of classical cytochemistry were replaced by
fluorochromes, and the pioneer microphotometry was replaced by microfluorometry. The latter has great
advantages in terms of simplicity, sensitivity, and accuracy. The extensive research and availability of new
fluorochromes as well as the technological evolution contributed to the success of microfluorometry. The
development of flow cytometry in the 1960s gave a giant boost to cell analysis and in particular to the
clinical diagnostics. The synergy between flow cytometry and the subsequent development of monoclonal
antibodies allowed the setup of multiparametric analytical panels that are today popular and irreplaceable in
many clinical and research laboratories. Multiparametric analysis has required the application of an increas-
ing number of fluorochromes, but their simultaneous use creates problems of mutual contamination, hence
the need to develop new fluorescent probes. Semiconductor and nanotechnology research enabled the
development of new probes called nanocrystals or quantum dots, which offered great advantages to the
multiparametric analysis: in fact, thanks to their spectrofluorometric peculiarities, dozens of quantum dots
may be simultaneously used without appreciable crosstalk between them. New analytical horizons in
cytometry seem to be associated with a new concept of analysis that replaces fluorescence toward new
markers with (non-radiative) isotopes of heavy metals. Thus, the mass flow cytometry was born, which
seems to guarantee the simultaneous compensation-free analysis of up to 100 markers on a single sample
aliquot.

Key words Quantitative microscopy, Cytometry, Fluorescence, Flow cytometry, Mass flow cytometry

1 Introduction

The great advances of microscopic techniques, in the second half of

the last century, led to the birth of quantitative microscopy. The
microscope became not only an instrument for the observation of
microstructures and morphologies but also an instrument capable
of quantitate some of the main cellular components. Thus, cyto-
metry was born, an analytical approach that, initially based on
microscopy, was aimed to the quantitative determination of cellular

Carlo Pellicciari et al. (eds.), Histochemistry of Single Molecules: Methods and Protocols,
Methods in Molecular Biology, vol. 2566, https://doi.org/10.1007/978-1-0716-2675-7_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

1
2 Giuliano Mazzini and Marco Danova

constituents. In addition to cytometry, cytochemistry also evolved:

from the empirical science that used salts and natural pigments
(from both the animal and plant worlds), cytochemistry became a
refined technique based on defined and controlled chemical reac-
tions. The central core of this first methodological and later instru-
mental evolution is DNA on which an immense research work was
carried out in the years from 1950 to 1970 [1–7]. DNA, as a
fundamental cell component, immediately became the subject of
quantitative study. From the first observations of cell proliferation
and neoplastic transformation, the need derived for a procedure to
exactly measure the amount of DNA per cell. The first approaches
of DNA cytometry were based on the evaluation of the Feulgen
reaction products by absorption measurement [8–12]. The com-
plexity and limitations of this procedure were soon overcome by the
development of fluorescence microscopy [13] and related fluoro-
chromes. Quantitative cytochemistry thus underwent a great devel-
opment with the availability of new fluorescent molecules that have
made cytofluorometry the technique of choice for increasingly
sensitive, precise, and reliable quantitative determinations [14–16].
After decades of research on DNA, other cellular components
have also been the subject of quantitative interest, e.g., RNA and
proteins.
In addition to the quantitative fluorescence probes, several
markers have been developed to study cellular functions. In this
case, measurements are not strictly quantitative but rather aimed at
acquiring information on the cell functional states (viability, death/
apoptosis occurrence, oxidative state, membrane potential, pH,
etc.) under both physiological and pathological conditions.
A milestone in the development of cytometry, however, is
linked to the technological evolution that led to the birth of flow
cytometry (FC). The ability to directly analyze cells in suspension
led to enormous advantages. In fact, classical cytometry on a slide
(even after various attempts for automation) allowed the analysis of
a few hundred cells in very long times, whereas FC made it possible
to analyze thousands of cells in a few seconds [17–22].
A further boost (this is very great too) was linked to the
development of immunofluorescence and, immediately after, to
the advent of monoclonal antibodies. Thanks to the conjugation
with a variety of fluorochromes, monoclonal antibodies became a
very powerful investigative tool in various fields of immunology,
both in biological research and in the clinical diagnostics. The
combination of fluorescent monoclonal antibodies and FC is
today an unbeatable tool in many clinical laboratories [23–25].
Modern diagnostic approaches in the immunohematological
areas often require the use of a large panel of antibodies. These
multiparametric investigations, therefore, require the simultaneous
use of multiple fluorescent markers. To avoid errors due to the
mutual interference between probes, it is necessary that the labeling
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 3

fluorochromes are spectrally separated or, in other words, with

fluorescent emissions in fairly narrow and sufficiently distinct
bands. Today, using traditional fluorochromes (in addition to the
new ones called “tandem”), the FC techniques allow to discrimi-
nate up to 12 different colors; to do this, however, the sample must
be divided into aliquots analyzed with instruments equipped with
several sequential excitation sources (from the UV to the far-red
wavelengths).
To further expand the potential of multiparametric analysis, a
family of innovative fluorescent probes called nanocrystals
(or QDs) have been developed in recent years. Their chemical/
physical characteristics make them unique in the broad scenario of
the commercially available fluorescent markers, today.
Finally, after almost a century of fluorescence, cytometry is
evolving toward new analytical horizons offered by the use of
markers based on heavy metal isotope. FC is going to leave the
concept of optical (spectrofluorometric) analysis and uses the time-
of-flight mass spectrometry (so-called CyTOF) to recognize new
markers. This technology utilizes rare metal isotopes instead of
fluorophores to label antibodies, thus breaking the limit of multi-
plexing capability of conventional cytometry, and allows the multi-
ple simultaneous labelling of dozens of “targets” (actually near a
hundred) without mutual interference. Obviously, in parallel with
the instruments’ technological evolution, a commercial fight has
also begun for the development of new diagnostic kits based on
these markers. Terminology is also changing and the historical
“fluorescence flow cytometry” becomes today “mass flow
cytometry.”

2 Fluorochromes and Cytometry

Although the first prototypes of flow cytometers were based on the

measurement of scattered light parameters, the instrumentation
that later became commercially available was instead based on the
use of fluorochromes labeling the cellular components to be
detected and measured. It is worth noting that although the vast
majority of (clinical) applications of FC are presently directed to
measure immunofluorescence markers, DNA was instead the start-
ing cellular target.
The first experimental contributions to the automated analysis
of cells in suspension were aimed at measuring the volume (through
the evaluation of scattered light) and immediately afterward the
DNA content (through the measurement of fluorescence). The
goal was in fact to automate the cytometric analyses of DNA
(traditionally carried out on a slide), and, more or less in the same
years, FC was replacing the more laborious and imprecise cytopho-
tometric determination [8–12]. In other words, fluorochromes
4 Giuliano Mazzini and Marco Danova

were replacing the classic dyes of cytochemistry, at least as far as

quantitative aspects were concerned.
In the years around the 1970s, FC was an advanced analytical
technique for cell proliferation studies, mainly based on the analysis
of fluorescence emitted by a stoichiometrically bound fluoro-
chrome to DNA. It is important to remember the first attempts
to adapt to FC some important classical reactions of cytochemistry
in fluorescence [26–29]. Subsequently, FC was significantly
improved by extensive methodological researches that made it
available, in the timespan of a few years, a wide range of methodol-
ogies with often specially developed procedures and fluoro-
chromes. The impact of the phenanthridine fluorochromes,
ethidium bromide (EB) and propidium iodide (PI), on DNA FC
was huge [30–32], and these fluorochromes are still nowadays
among the most widely used probes for the quantitative analysis
of DNA [33–37]. From the methodological point of view, also the
so-called fluorescent antibiotics (mithramycin, olivomycin, cromo-
mycin) have provided important contributions to DNA
quantitation.
After DNA, the interest of FC was addressed to cellular pro-
teins, and the methodological evolution exactly follows what has
just been described for DNA. At first, traditional techniques were
adapted to samples of cells in suspension, and then FC-tailored
methods and fluorochromes were, on purpose, developed. Starting
from eosin, fluorescamine, and ortho-phthalaldehyde, then fluores-
cein isothiocyanate (FITC) became the leader probe in this field
(still today, it is the most widely used in immunofluorescence). The
transfer to FC of the first immunofluorescence methods (initially
used in fluorescence microscopy) then gave a major boost toward
spreading the FC techniques in most of the biomedical labora-
tories. The methodological research subsequently made a series of
fluorochromes available, either synthetic (e.g., the Hoechst series)
or derived from algae (e.g., phycobiliprotein).
Other fluorochromes were later proposed with the aim not to
strictly quantitate the amount of molecules but to monitor cellular
functions or markers of cell viability or death, oxidation state, pH,
etc. A new family of probes were recently developed, called nano-
crystals or QDs. They are characterized by very peculiar spectroflu-
orometric properties allowing a dedicated application in
multiparametric FC. Lastly, cytometry evolved toward an innova-
tive analytical strategy that would even replace fluorescence with
the labeling by heavy metal mass isotopes; hence, mass cytometry
was born.
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 5

3 Fluorescence and Autofluorescence

The evolution of cytometry in fluorescence also conditioned a series

of researches on the importance of bioluminescence with mainly
negative contributes to cytofluorimetric detections. It became
immediately evident that biological tissues can be a spontaneous
source of autofluorescence and that this can interfere with the
analytical results. Fluorescence analysis allows cells to be irradiated
by a beam of light of a certain frequency and high intensity
(depending on the type of excitation source) which, interacting
with the various biological components, can generate (in addition
to diffused and refracted light) also a spontaneous fluorescence
emission called autofluorescence.
In general, fluorescence can be distinguished into primary
(spontaneous, or autofluorescence, or natural fluorescence) and
secondary (or induced) fluorescence. The latter in turn can be
further classified into numerous “subtypes” depending on the
physicochemical treatments used to generate the emission.
Autofluorescence can be more or less high, depending on the
cell type under examination, and is generally produced by the
photoluminescence of some amino acids and therefore by the pro-
teins where they are contained in. In particular, tryptophan and
tyrosine are fairly luminescent especially when excited in ultraviolet
light. Since these amino acids are commonly present in proteins,
they can be considered the main components responsible for the
typical autofluorescence of many biological tissues observed in
ultraviolet light under a fluorescence microscope. Many other com-
ponents (or substances) physiologically or even pathologically pres-
ent in cells can increase the basic autofluorescence of certain cell
types. Many vitamins, lipids, enzymes, and coenzymes are often
characterized by specific fluorescence emissions. As far as lipids are
concerned, there are, for example, porphyrins which represent an
important family of substances naturally present in some tissues
(especially in the hematopoietic compartment); the increase in
porphyrin content under some pathological situations and the
consequent increase in the typical fluorescence intensity can be
used for diagnostic purposes. Lipids also have the peculiar charac-
teristic of being similar to many vitamins and lipofuscins (often very
fluorescent) that they are able to accumulate within cells or tissues
in the form of micro-drops or liposomes.
Other biological components of some tissues, such as elastin
and collagen, show a high level of spontaneous emission when
observed under a fluorescence microscope. In these cases, however,
since the localization of this substance is mostly in the extracellular
matrix, the incidence of this phenomenon is quite modest as far as
single cell (or nuclei) analyses are concerned. In addition, the
autofluorescence of cells or tissues can increase as a result of the
6 Giuliano Mazzini and Marco Danova

presence of non-physiological substances, among which are some

types of drugs such as some antibiotics. Virtually all anthracyclines
are so fluorescent that they can be considered as “fluorochromes,”
and some antibiotics (described below) represent a particular group
of luminescent molecules. These drugs specifically bind DNA so
that their clinical use, even at low doses, allows their accumulation
in the cell nuclei: as a consequence, when analyzed by FC or
observed under a microscope, the nuclei have a much higher spon-
taneous fluorescence. Autofluorescence in cytometry can be a diag-
nostic tool as it has been shown to increase in some tumor tissues
compared to the corresponding normal tissue. So, in particular
situations, autofluorescence can be considered as a positive occur-
rence; however, in general, spontaneous fluorescence represents a
negative aspect in many cytometric approaches, especially in static
cytometry, but also, though to a lesser extent, in FC. In static
cytometry, the sample is often observed under excitation light
with the contribution of the biological matrix surrounding the
cells; on the contrary, in FC, the cells are isolated from the tissue
milieu and even single nuclei are often processed. However, one
should always be aware of the sample autofluorescence and have an
approximate estimate of its level, to predict the possible error
incidence.
The fluorescence that mainly interest the field of cytometry are
the so-called extrinsic or induced fluorescence. All the lumines-
cence artificially induced (by means of physicochemical/chemical
treatments) in a sample belong to this class. Historically, some of
the first fluorochromization processes in histochemistry involved
the transformation of weakly luminescent substances (e.g., the
catecholamines) into compounds with stronger emission. Other
treatments transformed a substrate into a fluorescent product
(e.g., enzyme histochemistry) and still others, on the basis of
intermediate reactions, finally bound a fluorochrome (e.g., the
fluorescent Feulgen reaction). The current labeling techniques for
cytometry are essentially attributable to two types of procedures:
(a) those that lead to the binding of a fluorochrome to the
biological molecule, based on specific chemical-physical interac-
tions, and (b) those that bind a fluorochrome to the target antigen
through an immunochemical reaction.
In recent years, the progress of FC was dependent on the
technological improvement but also on the evolution of the stain-
ing procedures, among which were the manufacture and commer-
cial availability of monoclonal antibodies: thanks to their use, FC
became a routine analytical technique in many research and clinical
laboratories, in particular for leukocyte immunophenotyping.
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 7

4 DNA Probes

As already recalled, the most important chapter on fluorescent

markers undoubtedly deals with DNA cytochemistry.
The Feulgen reaction [7] was the very first attempt to quantify
a cellular component of fundamental importance for a wide range
of biomedical applications. Few years later many contributes [4, 16]
were focused on the automation of this determination (by means of
both static and flow cytometry). The first step was the modification
of the original method, replacing the conventional Shiff’ reagent
with alternative fluorochromes (which paints the nuclei
red-magenta and was used for the first absorption measurements)
[26–29]. The results were not particularly “brilliant” due to the
complex and time-consuming method that required acid hydrolysis
with many washes and centrifugations, which resulted in a high cell
loss. Nonetheless, the first applications of FC were based on the
Feulgen reaction modified with acriflavine, 2,5-bis(benzoxazol-2-
yl)thiophene (BBT), and auramine. In the meantime, a new fluo-
rescent probe acridine orange (AO) was proposed, whose meta-
chromatic property was already exploited in other histochemical
applications [38]. Together with its peculiar chemical-physical
characteristics, AO appeared to be (at least initially) the probe of
choice for the analysis of nucleic acids by FC [39]. In fact, it has an
absorption spectrum favorable for its excitation with the argon ion
laser (488 or 514 nm) which was (and still is) the typical excitation
source of the most popular instruments. AO has also a good quan-
tum yield and, above all, it provides two different information at the
same time: its peculiar characteristic is in fact to intercalate into the
DNA double helix, and in this situation (molecules spaced between
them) it emits green fluorescence, while it can also bind externally
the single RNA helix and in this other condition (molecules close
one to each other) emits red fluorescence. This peculiarity was
initially exploited in numerous applications in the hemato-
oncology field, where the DNA/RNA ratio is a powerful marker
in the study and classification of some leukemias. From the point of
view of general applications, however, this method has numerous
drawbacks, mainly related to its crucial binding mechanism that
requires extremely controlled and not always exactly reproducible
reaction conditions.

4.1 G-C Base- Other fluorochromes able to bind to DNA with different mecha-
Specific nism were later proposed under the name of “fluorescent antibio-
Fluorochromes tics.” Among these mithramycin (MM), cromomycin, and
olivomycin all react with a similar mechanism that involves the
formation of a complex with guanine (in the presence of magne-
sium ions) and are therefore base-specific probes, with a preference
for G-C pairs. Their excitation maximum is in the blue
8 Giuliano Mazzini and Marco Danova

(410–430 nm) and emission in the green-yellow (500–600 nm).

They can therefore be best excited with lamp sources of both
mercury and xenon vapor while maintaining a good emission
even when excited with argon lasers (both in blue and green).
Thanks to their base specificity, they should not bind to RNA;
therefore, they guarantee an accurate DNA determination, without
the need to enzymatic RNA digestion. In fact, MM in particular has
been used for this purpose in combination with EB [40]. Exploiting
the energy transfer between MM and EB, it is possible to excite
MM in the blue region (at 420 nm where EB does not absorb) to
obtain red fluorescence from EB, which does not receive light
directly from the lamp but receives energy from the excited MM
molecules. This phenomenon occurs only if the molecules of the
donor-acceptor pair are in close proximity, as it occurs for MM and
EB when they are bound to DNA. The collected fluorescence signal
is therefore dependent on the amount of DNA only, without the
interference of RNA.
These G-C fluorochromes in combination with other A-T-
specific dyes can provide information on DNA base composition.
Based on this strategy, a method has been applied to the differenti-
ation of single chromosomes using chromomycin in combination
with Hoechst, as well as on the discrimination of classes of bacteria
with DAPI. In more recent years, another fluorochrome belonging
to this family and derived from actinomycin-D, called 7-amino
actinomycin D (7-AD), was proposed for the analysis of DNA in
combination with other probes [24]. The physicochemical charac-
teristics of 7-AD, and in particular its deep red emission, allow a
good separation of its fluorescence even in the presence of the
popular yellow-orange probes. Since 7-AD can penetrate through
the membrane of unfixed cells, it has been proposed for the DNA
multiparametric determination in unfixed cells previously immuno-
labeled for different antigenic markers using FITC- and phycoery-
thrin (PE)-conjugated antibodies [25].

4.2 A-T Base- Some very important fluorochromes for the role they have played in
Specific FC applications belong to this class. DAPI and its analogue DIPI
Fluorochromes have contributed to the development of a family of “European
cytometers,” substantially derived from the fluorescence micro-
scope and thus using lamps as excitation sources [19, 40]. Instead,
another family of instruments born in the USA exploited the power
of the new argon ion lasers in place of lamps. Both types of instru-
ments had ideal fluorochromes fitting their different excitation
performances: FITC fits the argon line at 488 nm while DAPI is
optimally excited by the UV band of the mercury lamp. Its maxi-
mum absorption is in the ultraviolet where mercury has a very
powerful emission line (at 365 nm). DAPI has a high quantum
efficiency that, combined with the high excitation energy, leads to
an extraordinary intensity of fluorescence emission. These findings
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 9

ensued an excellent result, in terms of coefficient of variation of the

DNA data, as reported in the literature about the DAPI-DNA
analysis carried out with lamp instruments.
Other A-T-specific fluorochromes are those developed by
Hoechst and named as HO followed by a number [41]. Among
others the HO33258 e HO33342 became very popular in FC; in
particular, HO33342 is now the probe of choice for cell viability
studies [42, 43], as it can cross the cell membrane and stain the
nucleus in supravital conditions (see below Subheading 7.1). Two
other fluorochromes from the A-T family are quinacrine (QC,
already well known for its use in chromosome banding) and the
more recently proposed LC585 [44]. This latter has interaction
characteristics similar to HO3342 (therefore with the possibility to
be used as viability probe) but with excitation spectrum in the
visible light range.

4.3 Intercalating Although acridine derivatives, and AO in particular, are the most
Dyes famous intercalating fluorochromes, in the history of DNA cyto-
chemistry [39], the family of phenanthridine derivatives EB and PI
has made the most relevant contribution to FC, from the practical
point of view [33]. EB has been extensively studied from a physi-
cochemical point of view and its interactions with DNA described
in detail even at the ultrastructural level. Its analog, PI, had a wider
methodological application, thanks to its spectrofluorometric char-
acteristics that make it more suitable, especially for multiparametric
FC. Phenanthridines have the peculiar characteristic of being
almost non-fluorescent as free molecules in the solvent while
increasing considerably their quantum efficiency when intercalated
in double-stranded DNA. The explanation for this phenomenon
lies in the change of microenvironment that intercalated molecules
acquire, compared to those free in the surrounding dye solution.
The latter, after light excitation, from the higher electronic level can
disperse much of the absorbed energy, exchanging it with the
surrounding water molecules (polar and very mobile); thus, their
energy is not emitted as fluorescence. On the contrary, the inter-
calated molecules become electronically shielded from each other
and especially from the external water dipoles. In this state, much of
the energy absorbed by excitation is returned into the form of
fluorescence. It means that only the intercalated molecules contrib-
ute to the generation of the fluorescence signal emitted by the
nucleus. This peculiarity, together with its spectrofluorometric
characteristics, made PI the probe of choice for DNA quantitation
by FC. Regarding the spectral characteristics, it should be noted
that PI has a wide absorption band in the visible, with best absorp-
tion in the blue/green region and another band in the UV. It is
therefore widely used both with laser-based instruments, where it is
typically excited by the 488 nm argon line, and with lamp instru-
ments where the 546 nm mercury line is exploited. The emission
10 Giuliano Mazzini and Marco Danova

spectrum is fairly located in the red, with a maximum around

610 nm: this allows it to be widely used in multiparametric FC in
combination, usually, with FITC and PE, although with a discrete
spectral overlap with the latter. Although other fluorochromes with
narrow red emission have been recently proposed (to minimize
cross-talking between probes), PI maintain its wide popularity as a
marker of choice for DNA. Phenanthridines have a single draw-
back, as their binding mechanism does not allow an absolute speci-
ficity toward DNA: they can, in fact, bind also double-stranded
RNA (typically t-RNA). The high-resolution DNA measurement
with EB or PI therefore requires a pretreatment for the enzymatic
digestion of RNA. To overcome this event and thus avoid the use of
enzyme, a specific methodology was proposed [19] that involves
the use of a mixture of MM and EB in equimolar combination and
exploit the principle of energy transfer from one to the other. After
dual staining, the double-stranded DNA will bind EB
(by intercalation) and MM (by interaction with the G-C base
pairs), while RNA will have only EB intercalated (because MM
does not bind to RNA). Using an excitation light beam around
430 nm (e.g., by a mercury lamp), only MM will be excited,
because EB does not absorb light at this wavelength. However,
given the proximity (less than 100 A
) of the molecules of MM and
EB in DNA, the former can transfer directly the absorbed energy to
the molecules of EB that finally emit its typical fluorescence: the
intensity of this fluorescence emission is thus proportional to the
DNA amount.

5 RNA Probes

As far as RNA is concerned, its typical probe is AO, already

described in the previous section. Thanks to the very intense meta-
chromatic effect, AO can act as a versatile bifunctional probe able to
bind both DNA and RNA with two different emission signals.
Intercalated inside the double-stranded DNA may fluoresce
green, while externally bounded to RNA turn its fluorescence to
red [39]. Before the advent of the so-calledproliferation markers,
now widely used for studies of cell proliferation, the interest was in
fact focused on RNA as a cellular component (of second level after
DNA) to “monitor” cell proliferation. Many contributions refer
the importance of assessing the ratio DNA/RNA performed by FC
via AO labeling, especially in the field of oncohematology [22].
Another more tedious method was based on the use of phe-
nanthridines (as mentioned above) with a reverse procedure to
what is done for DNA. Following digestion with DNase, the resid-
ual fluorescence refers to RNA.
Another RNA-specific probe, already widely used as a conven-
tional histochemical dye, is pyronin Y (PY). Together with methyl
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 11

green, it has given rise to one of the first biparametric methods in

absorption microphotometry. In cytometry, this method was then
modified by combining PY with Hoechst, which replaced methyl
green as a DNA fluorochrome [25]. The results obtained with PY
have never been without criticism and its routine use has found the
same obstacles already mentioned for AO.
A specific clinical application of RNA probes was dealing with
the counting of reticulocytes in peripheral blood. The RNA resi-
dues in the cytoplasm of immature erythrocytes were in fact labeled
with different fluorochromes including DiOC1, thioflavin T, and
thiazole orange in addition to PY. Among these, the method most
frequently applied in the literature is the one using thiazole orange
that, despite not being exclusively specific for RNA (as it binds also
DNA), has shown good sensitivity in the automatic counting of the
anucleated reticulocytes in peripheral blood [25].

6 Protein Probes

The most popular protein-specific fluorescent probe is surely fluo-

rescein isothiocyanate (FITC), which was also one of the first
fluorochromes used in fluorescence microscopy. This probe has
practically promoted the amazing development of immunofluores-
cence techniques [22]. Like most of the protein-specific dyes, FITC
establishes a covalent bond between the thiocyanate radical and the
primary amine groups spread everywhere in the biological tissues.
Thanks to the chemical stability of the complex, all the necessary
washing procedures can be performed, in both conventional cyto-
chemistry and immunocytochemistry, without loss of the reaction
products: careful washing is necessary because free and bound
FITC are equally fluorescent, thus being crucial to eliminate
unbound FITC to make a proper quantitative analysis. The spec-
trofluorometric characteristics of FDITC are ideal for an excitation
with the 488 nm line of the argon laser, being the absorption
maximum around 490 nm, while the emission spectrum is in the
green region, with a maximum at 520 nm. FITC has metachro-
matic properties similar to those described for AO, i.e., it changes
its spectroscopic structure depending on its concentration. For
many years, FITC has been used as the classical probe for total
cellular proteins, thanks also to the biparametric method in combi-
nation with PI, for simultaneous DNA determination [33].
Other historical protein markers are ortho-phthalaldehyde and
fluorescamine, which are excited by UV light and emit blue-white
fluorescence [44]. They were not widely applied since the required
UV excitation generates a significant level of background fluores-
cence and also because more efficient fluorochromes became soon
available.
12 Giuliano Mazzini and Marco Danova

An important group of fluorochromes with a FITC-like reac-

tion mechanism is the rhodamine family. Among these, sulforoda-
mine 101 (SR101) and tetramethylrhodamine-isothiocyanate
(TRITC) have been the most widely used in FC [25]. Important
was then the setting of a method that, using SR101 together with
DAPI, with a single UV excitation, allows measuring the blue
fluorescence of DAPI and the red emission of SR101. This method
has been extensively used with mercury lamp cytometers and is still
in use in some laboratories. The results are generally very good, and
often the CV of the DNA is even smaller than in single-stained
DAPI samples (it is not easy to explain this phenomenon, and one
can only speculate that the presence of SR101 prevents the nonspe-
cific interaction of DAPI outside DNA). Even more surprising are
the SR101 data (which follow those of FITC on the same samples)
taking into account the crosstalk between the two emission spectra.
Some coumarin derivatives such as 7-amino-coumarin
(AMCA) and N-(7-dimethylamino-4-methylcoumarinyl) malei-
mide (DACM) are very interesting for their spectrofluorometric
properties: high absorption in UV, emission in the first part of the
visible light that allows obtaining a brilliant white-blue fluores-
cence. The amine-reactive AMCA was rather widely used in the
field of immunofluorescence as AMCA conjugates became com-
mercially available and proved to be useful for multiparametric
analyses in combination with other green- and red-emitting probes
[45]. Another coumarin derivative, DACM, has similar fluores-
cence properties as AMCA, but a different reaction mechanism
being specific for the protein sulfhydryl (-SH) groups
[46]. DACM was initially applied to detect SH-rich proteins in
the skin [47, 48] and to investigate the thiol-to-disulfide transition
in nuclear protamines during mouse sperm maturation
[49]. Thanks to their spectral characteristics, DACM and PI were
used as a dye pair for studying DNA and proteins in nuclear
chromatin by fluorescence resonance energy transfer [50]. This
couple of probes were also applied in lamp-based FC, thanks to
the high performance UV excitation of this type of
instruments [51].
To complete the list of protein probes, there are the so-called
“phyco” derivatives. These organic complexes, called phycobilipro-
teins, are molecules of natural origin (from marine algae or cyano-
bacteria) and exhibit very interesting photophysical properties that
make them suitable for FC application and, in particular, for immu-
nofluorescence labeling [51–55]. In nature, phycobiliproteins are
components of the photosynthetic light-harvesting antenna com-
plexes of red algae and cyanobacteria; they absorb the sun’s energy
and efficiently transfer it to chlorophyll pigments by fluorescence
resonance energy transfer, for the photosynthetic reactions. Phyco-
biliproteins are characterized by a high extinction coefficient (which
is fundamental for a suitable fluorescence emission) and are
Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 13

commercially available as three fluorochromes: B-phycoerythrin

(B-PE), R-phycoerythrin (R-PE), and allophycocyanin (ApC).
Phycobiliproteins are characterized by a high molecular weight,
resulting from the very complex organic structure, which can
include up to 34 chromophore groups per molecule. PEs reach a
molecular weight around 240 kDa, while ApC is about 104 kDa;
they have common quite broad absorption spectra in the visible
band beyond 450 nm. In particular, R-PE has a specific absorption
peak around 490 nm, which makes this fluorochrome particularly
suitable to be excited with the 488 nm line of the argon laser. B-PE
has its excitation maximum more shifted toward the green and is
therefore better excited in this spectral region. In contrast, the
excitation peak of ApC is shifted to the red region with a maximum
around 650 nm. As mentioned above the peculiarity of these fluor-
ochromes is to be very efficient in terms of fluorescence emission, as
a result of the high specific absorption of photons and an equally
high fluorescence quantum yield. The absorption is characterized
by the molar extinction coefficient, which represents the statistical
probability that the molecule has the ability to absorb a photon of a
given wavelength. This value, relative to the wavelength of 488 nm,
is, for example, for R-PE, equal to 2  106, while it is only 86  103
in the case of FITC. For ApC this value is 7  105 at 650 nm. It is
therefore evident that the ability of these fluorochromes to absorb
light is very high as compared to FITC. As far as quantum yield is
concerned, from the theoretical value of 1, PE has a value of 0.98
and ApC of 0.68, while FITC, which is known to be a brilliant
fluorochrome, is only 0.5. Thus, it is quite clear that phycobilipro-
teins are definitely the fluorochromes of choice, in terms of sensi-
tivity (i.e., for the measurement of small amount of antigens/
proteins) [25].
From the application point of view, these fluorochromes have
been very successful, having become, in a few years, the first coun-
terparts to FITC in FC. Actually, in FC the single-parameter immu-
nofluorescence analysis may be performed using just FITC as probe
of choice, while FITC may be combined with PE in dual-parameter
measurements, with the further addition of ApC in case of triple
labeling. In this latter case, a dual-laser excitation system must be
available to properly excite ApC in the red region.
In the case of single-laser blue excitation, the third probe must
be a “tandem” fluorochrome. This belongs to the family of syn-
thetic probes developed by joining together two molecules with
complementary photophysical properties, to exploit the principle
of energy transfer from one (the donor) to the other (the acceptor).
They have various commercial names and generally use PE, as a
donor coupled to another chromophore, which receives the energy
from this latter and finally emits a more red-shifted fluorescence.
They were specifically developed to solve the problem of the
so-called multilabeling immunofluorescence based on the use of a
14 Giuliano Mazzini and Marco Danova

single blue excitation, for the measurement of three signals: green

(FITC), yellow (PE), and red (tandem) [53].
Cyanines (known with the trade names Cy2–7) belong to a new
family of protein probes: they are also synthetic fluorochromes
designed to mimic the characteristics (viz., the advantages) of the
natural phycobiliproteins. They have a specific high light-
absorption coefficient (~ 2  103) and a good fluorescence quan-
tum efficiency (0.1  0.3). Compared to the natural parental
molecules, cyanines have narrower absorption-emission spectra,
which offer better emission selectivity and less mutual reabsorption,
when used in multiparametric analysis. The most interesting and
promising are those that are excited in the red (exploiting the lines
of the He-Ne laser or the solid-state lasers) and emit in the far red,
up to over 800 nm [44].

7 Cell Function Probes

7.1 Viability Probes The fluorochromes described so far are the most historical and did
contribute to the progress of the microfluorometric techniques,
having been developed to specifically and stoichiometrically bind
different cellular components to obtain their quantitative determi-
nation. Thanks to histochemists’ efforts, perfectly controlled and
reproducible conditions of dye concentration, pH, reaction time,
and temperature have been defined to ensure a perfect correlation
between fluorescence intensity and probe content, as the funda-
mental concept of any cytometric techniques is the stoichiometry of
the probe toward the cellular component to be quantified.
More recently, however, it has become interesting to use some
fluorescent probes not for “quantitative” investigations but to
obtain information on specific cellular features or functions.
Evaluating cell viability is crucial in biology and is a basic
finding in many experimental studies. Cell viability is mainly
assessed through the integrity (impermeability) of the plasma mem-
brane. Most of the fluorochromes described above are unable to
penetrate the intact cell membrane due to their chemical structure
and high molecular mass. However, there are some of them able to
do that. Among these, a specific probe has become very popular for
this property. It belongs to the “Hoechst” series of DNA-binding
fluorochromes characterized by the initials HO, which has become
the progenitor of a family of markers used for this purpose. Specifi-
cally, the HO33342 under appropriate (physiological) incubation
conditions it is easily internalized in all (both living and dead) cells.
In living cells, however, a metabolic pump is active, capable of
expelling most of the internalized fluorochrome [43], whereas in
dead cells this pump is off and all the fluorochrome molecules that
had crossed the membrane are kept inside: due to the DNA speci-
ficity of HO33342, the nuclei of dead cells will thus have a higher
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 15

fluorescence than those of living cells. In addition, the metachro-

masia of HO33342 makes a further decisive contribution to the
differentiation between life and death: at low concentration its
fluorescence emission is typically white/blue, whereas in conditions
of higher concentration the emission turns toward the yellow.
Thanks to these two coexisting phenomena (i.e., concentration
and metachromatic effect), it is simple to discriminate living from
dead cells, both by fluorescence microscope and FC.
HO33342 can also be used in combination with another
“functionally” different fluorescent probe, namely, PI that cannot
penetrate the intact membrane of living cells and therefore cannot
intercalate into nuclear DNA. Using a mixture HO33342/PI on a
cell sample, an impressive rainbow-color result is obtained that
characterizes the different cell viability status: viable intact cells
are only labeled by HO33342 and show a faint blue fluorescence;
mildly damaged cells may be only strongly labeled by HO and
fluoresce brilliant white/blue; more damaged cells are stained by
both dyes and exhibit a brilliant pink (white plus red) fluorescence;
and dead cells are strongly stained by both probes and fluoresce
deeply red.
Other DNA-specific fluorochromes can be used in microscopy
and FC as viability probes: 7-AAD is capable of crossing the cell
membrane of all cells and accumulate more in the nuclei of the dead
ones. On the contrary, LDS 751 like PI is not able to enter intact
viable cells but can instead stain only the nuclei of dead cells. The
emission spectra are in the deep red and therefore better than PI;
these fluorochromes are suitable for multiparametric analyses with
other green-emitting cell markers [56].

7.2 Proliferation Cell proliferation, among the cellular functions, is of particular

Markers interest in the biomedical field. The nuclear DNA content is cer-
tainly the most significant marker to estimate cell proliferation in
normal cell populations as well as in tumors [57, 58], and the
cytometric evaluation of DNA histograms is one of the most widely
studied biological indicators.
A limitation of this methodological approach, however, is the
fact that DNA content is a “static” probe as it just shows the cell
distribution through the cell cycle, at the time of sample collection.
The alternative may be the “active” study of cell proliferation
through the labeling of S-phase cells, which involves the use of
DNA precursors such as tritiated thymidine (3H-TdR) or bromo-
deoxyuridine (BrdU) [59–62].
The cyto-autoradiographic method based on the incorporation
of 3H-TdR is a classic approach to the study of proliferative kinet-
ics, and in recent years there has been a progressive validation of the
so-called labeling index, in the clinical field [36]. Obviously, this
method has some disadvantages including the need to use radioac-
tive material and the time-consuming procedure that makes it
16 Giuliano Mazzini and Marco Danova

unsuitable for the clinical studies that require rapid answers. The
availability of the new DNA precursor, BrdU, opened new horizons
in cell proliferation studies. BrdU tagging is comparable to that of
3
H-TdR without the disadvantages of radioactivity and with a
number of advantages related to its labeling with specific monoclo-
nal antibody, which may be detected by microscopy and
FC. Following the administration of BrdU in vivo and the analysis
by dual-parameter flow cytometry of the BrdU labeling and DNA
content, it is possible to measure the S-phase cell fraction and to
assess dynamic proliferation parameters, such as the duration of the
S phase (or DNA synthesis time, TS) and the potential doubling
time of the normal or tumor cell population (Tpot). This approach
has been the first example of DNA multiparametric analysis where
the classical “DNA content” can be associated with other kinetic
parameters that can increase the intrinsic value of the data [63–66].
Later on, other markers of potential diagnostic or prognostic
importance (surface antigens, intracytoplasmic antigens, nuclear or
nucleolar antigens, hormonal receptors, molecules related to par-
ticular cell functions, etc.) have been proposed to be simultaneously
correlated to the DNA content. This approach allows a quantitative
and statistically significant analysis of the cellular components in
direct correlation with the cell cycle, resulting in a more accurate
assessment of the potential clinical significance of these parameters.
The multiparametric FC evaluation of the expression of anti-
gens (such as the one recognized by the Ki-67 antibody, PCNA/
cyclin, and other cell cycle-related antigens) allows a precise assess-
ment of the proliferative status in various clinical diseases [67–
75]. Again, the quantification of other nuclear or nucleolar anti-
gens (e.g., p105) may be useful for a more objective assessment of
the degree of anaplasia. Some products of cellular oncogenes have
proved promising as markers of tumor aggressiveness, while few
glycoproteins localized on the cytoplasmic membrane have been
shown to be related to the process of anticancer drug
resistance [64].

7.3 Other Probes (for A large family of fluorescent tracers has become commercially
Mitochondrial available, in recent years, to study specific cellular conditions related
Intermembrane to both the plasma membrane function and the cytoplasmic orga-
Potential, pH, Ca++ nelles. These tracers are often complex molecules characterized by
Content, and Others) distinctive commercial acronyms [44].
Among the cationic lipophilic markers, JC1 is widely used
especially in confocal microscopy but also in FC for studies of
mitochondrial function. In viable mitochondria with high inter-
membrane potential (ΔΨm), the probe is concentrated inside the
organelles in the form of aggregates and metachromatically emits
red fluorescence; in mitochondria with low ΔΨm, the dye remains
in a less concentrated status and its fluorescence emission is typically
green. Rhodamine 123 (Rh123) is also a viable fluorochrome quite
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 17

specific to mitochondria. Its non-fluorescent derivative, dihydror-

hodamine 123 (DHR), is able to penetrate viable cells where in
presence of oxidative radicals (especially H2O2) it may be converted
to Rh123: the resulting green fluorescence allows easy monitoring
of the fraction of cells in which reactive oxygen species are present
[25]. The cationic rhodamine derivative, tetramethylrhodamine
(TMRE), is able to accumulate inside mitochondria according to
their membrane potential, and the level of emitted fluorescence
allows monitoring the mitochondrial functional status. Informa-
tion on the density (or mitochondrial mass) can be obtained with
two probes whose fluorescence is independent from the potential
and/or the energy metabolism: nonyl acridine orange (NAO) and
MitoTracker Green passively accumulate inside the mitochondria,
and the emitted fluorescence by the cells is a function of the
mitochondrial mass. However, it should be kept in mind that all
these mitochondrial markers do not bind stoichiometrically the
target and may therefore provide pseudo/quantitative information
only. Other markers such as chloromethyl-X-rosamine (CMXRos)
and MitoTracker Red give information on metabolic mitochondrial
activity; the latter, in particular, turns from a non-fluorescent to a
fluorescent form induced by intracellular oxidative processes.
For the evaluation of intracellular pH, there are several probes
such as 20 ,70 -Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein
(BCECF) and seminaphtorhodafluor-1 (SNARF-1) that are called
ratiometric probes because they show different emission spectra as a
function of pH [44].
In many cell biology researches, there is the need to know the
concentration levels of the Ca++ ion that is involved in many pro-
cesses such as muscle contraction, movement, secretion, etc. For
this purpose several markers have been proposed: Indo 1 that is
excitable in UV and Fluo-3, Fluo-4, Oregon Green, and Fura that
are excitable at 488 nm, thus being of choice for argon laser-based
FC [24, 25].

8 Quantum Dots

Most of the fluorochromes described so far belong to the family of

organic compounds. Many of them are synthetic products designed
and produced by the chemical industry, but some are instead of
natural origin. The latter, playing the role of “light detectors” in
nature, are characterized by high absorption/emission efficiency of
light. All fluorochromes of organic nature are characterized by
molecules with a complex chemical structure rich in aromatic
rings and double bonds [76–79].
This structural complexity is related to a great variety of energy
levels that the molecule can assume which in turn allows the com-
plexity of their spectrofluorometric characteristics. In other words,
18 Giuliano Mazzini and Marco Danova

their absorption/emission spectra are very broad, practically

extended over most of the frequencies of the visible spectrum.
There is also frequently a significant overlap between absorption
and emission spectra (very short Stokes shift). All these are unde-
sirable characteristics in FC especially in the case of multiparametric
tests: broadband fluorescence emission obviously entails the risk of
overlapping between the emission spectra of the different probes
and may cause possible errors in their quantitative measurements.
To try to eliminate (or reduce) these drawbacks, a new family of
fluorochromes called nanocrystals or QDs has recently been devel-
oped. Unlike all the above described fluorochromes, they are inor-
ganic compounds derived from research on semiconductor
materials: their development derives in fact from the enormous
advances in the world of microelectronics and nanotechnology.
QDs are currently composed mainly of cadmium/selenide and
cadmium/telluride compounds. Compared to the large organic
molecules of traditional fluorochromes, their crystal size is in the
order of a few nanometers. Their optical characteristics are a func-
tion of their size and not of their chemical nature. In particular,
with the same composition but increasing the crystal size, the
emission of fluorescence shifts to the right in the visible spectrum.
So, a QD of 2 nm in diameter can have an emission spectrum in the
violet, whereas a crystal of 12 nm has an emission spectrum in the
red. Very peculiarly instead, their absorption spectrum is more or
less constant and mainly located in the first part of the visible (from
ultraviolet to blue). For these spectrofluorometric characteristics,
they proved to be of choice for multiparametric applications in FC
[80]. Due to their exceptionally large Stokes shifts (up to 400 nm),
these probes can contribute to the setting of very large analytical
panels with small spectral overlaps. This allows multiparametric FC
analyses even with a single excitation without the need of complex
compensation procedures [45].
As mentioned before, these probes are inorganic crystals of very
small size that do not have chemical functional groups or radicals
that could make them chemically reactive. Being chemically inert,
they cannot practically be conjugated to antibodies or specific cell
targets, hence the need to coat them with organic polymers to make
them suitable for conjugation chemistry. This organic coating
increases their solubility in water and enriches them with functional
groups (such as NH2, NH3) for subsequent conjugation to anti-
bodies, streptavidin, or nucleic acids. Their final size thus becomes
close to that of fluorochromes commonly used to label antibodies,
such as PE. The QDs’ quantum efficiency is similar to that of PE or
APC and sometimes even higher. Unlike the generally used
fluorochromes that have a rather limited emission range (in the
green-to-red spectrum), QDs are extremely versatile in terms of
spectrofluorometric properties.
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 19

There is a wide range of products characterized by commercial

labels (QD 450, QD 500, QD 510, . . .QD 630) that indicate their
emission peak, practically over the entire spectrum from blue
to red.
Theoretically, QDs are the ideal fluorescent markers and appar-
ently without any bugs or limitations; obviously, all these merits
have a price and QDs are currently more expensive than the classic
fluorochromes. They are still subject to development especially in
the coating: those of the latest generation have a shell modified
with long polyethylene glycol (PEG) chains rich in amino groups
that allow QDs to more easily conjugate to antibodies [81, 82].

9 Mass Cytometry

We have seen in previous sections how the increasing demand for

new markers has contributed to the design of more sophisticated
panels for multiparametric analyses, especially in the field of clinical
diagnostics. Nevertheless, the use of conventional fluorescent
probes is limited by the mutual crosstalk, which limits the number
of those that can be simultaneously used on a single sample. The
instrumental strategy of using multiple excitation sources (lasers)
only partially allows the number of markers to be increased. It is
also possible to divide the sample into several aliquots, subjected to
different analytical panels of probes, and finally sequentially ana-
lyzed. This obviously increases the costs and, primarily, the time
required for the analyses.
A new FC approach has recently been proposed [83–86] using
a fully innovative labeling strategy. It uses heavy metal isotopes
instead of fluorochromes. The detection of these probes obviously
cannot be performed by an optical mode (through spectrofluorom-
etry) but is made by emulating what happens in mass spectrometry
by “time-of-flight” detection (FC is thus replaced by cytometry by
time-of-flight, CyTOF).
The mass isotopes used as probes are not naturally present in
biological samples and are therefore used to tag specific reagents
(e.g., antibodies) similar to what is done with fluorochromes. Bio-
chemical research has made available several binding processes for
specific custom-labeling recipes.
The sample preparation is very similar to that required for the
more traditional FC mode, but the analytical strategy is completely
different. The labeled sample (the cell suspension) once introduced
into the system is nebulized and further completely ionized. Clouds
of molecules and particles are then further disrupted at the atomic
level. The sequence of atomic particles generated by each single cell
is analyzed by CyTOF, which allows associating a specific set of
markers to each single cell. The data collection, handling, and
display software are completely similar to the ones of the
20 Giuliano Mazzini and Marco Danova

conventional cytometers. The three-dimensional representation of

complex multiparametric panels characterizing the immunopheno-
type analyses is now routine in many clinical laboratories [87–89].
The great commercial boost driving the field of clinical diag-
nostics has already made a very wide range of specific markers
available [90, 91]. The first field of interest is certainly that of
monoclonal antibodies. It is therefore foreseeable in the short
period a sort of war between the two fronts: “fluorescence versus
mass.” The panels with conventional fluorescent probes have
become increasingly sophisticated and efficient but still require
operator adjustments (compensation) on the acquired data. On
the contrary, the panels with mass markers should not have this
problem.
Obviously, both analytical approaches (fluorescence vs mass)
have specific advantages and limitations:
1. Fluorescence FC allows the analysis of thousands of cells in very
short times and reliable results in many fields of clinical diag-
nostics. Classical FC is nowadays the routine analytical tech-
nique in research centers and hospitals. The instrumentation
and the related methodology, after more than 40 years of
development and progress, have reached absolute levels of
performances and reliability. The analytical power covers a
very wide range of particle sizes, from whole cells to bacteria,
to subcellular constituents such as the microvesicles. Even the
various cellular functions have been considered for specific
analytical approaches. The cells analyzed can also be separated
(with flow sorters) into subpopulations identified with specific
markers to be subjected to further molecular investigations.
Some instruments (image in flow) even allow storing image
files for subsequent correlation between quantitative fluores-
cence data and detailed morphology of the relevant cells. This
technology has a great diagnostic power as it combines the
information of the microscope with those from the FC.
2. The CyTOF boasts as a special feature the possibility to per-
form multi-parameter analysis (in a compensation-free mode)
with dozens of markers in simultaneous analysis. Of great
strength is the detection without mutual probe crosstalk and
the consequent ability to measure a very high number of mar-
kers (actually 40–100) on a single sample aliquot. This
approach seems therefore to be of choice for the complex
panels of immunophenotype analyses that are nowadays very
popular (and powerful) in various fields of immunohematolo-
gical diseases. However, one of the main limitations is the need
to destroy the cells to be measured and the consequent loss of
morphometric information. Of course in the wide panel of
results, the classical scatter parameters are missed (worldwide
used in classical CF analysis) which must be surrogated by other
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 21

markers. The analysis time is longer than in FC because the

sample undergoes a series of destructive treatments to get from
cells to clouds of atoms.
In summary, CyTOF could be defined as a “high-content
analysis,” compared to conventional CF, as it allows acquiring a
very high number of information on a more limited number of
cells. In the decade to come, we will see if CyTOF may replace FC
or if, more likely, these two approaches will integrate.

10 Concluding Remarks

For over half a century, fluorescence has been the milestone for all
quantitative analyses both at chemical/biochemical level (in tubes,
vials, cuvettes) and in cytometry (in both sections, cell smears, and
suspensions). In particular, FC (especially thanks to immunofluo-
rescence) promoted the development of multiparametric analytical
panels that are now routinely used in many areas of clinical diag-
nostics. Due to the continuous development of new fluorescent
molecules, a wide variety of markers have become available for the
determination of both cell components and cellular functions. The
fluorescent markers of the latest generation have chemical/physical
characteristics that can ensure quantitative multiparametric analysis
with high sensitivity, efficiency, and reliability.
New horizons in cytometry are now emerging, following the
development of a new analytical strategy based on the use of mass
isotope markers. These probes seem to represent the ideal way for
the setting of high-performance multiparametric analyses. How-
ever, we will have to wait a few years of application to understand if
the costs/benefits of this new analytical strategy will really be able
to replace the classic “old fluorescence.”

References
1. Franklin RE, Gosling RG (1953) Evidence for 5. Gil JE, Jotz MM (1976) Further observations
2-chain helix in crystalline structure of sodium on the chemistry of pararosaniline-Feulgen
Deoxyribonucleate. Nature 172:156–157 staining. Histochemistry 46:147–160
2. Watson JD, Crick FH (1953) Molecular struc- 6. Kjellstrand PTT (1977) Temperature and acid
ture of nucleic acids: a structure for deoxyri- concentration in the search for optimum Feul-
bose nucleic acid. Nature 171:737–738 gen hydrolysis conditions. J Histochem Cyto-
3. Kasten FH (2003) Robert Feulgen and his chem 25:129–134
histochemical reaction for DNA. Biotech His- 7. Chieco P, Derenzini M (1999) The Feulgen
tochem 78:45–49 reaction 75 years on. Histochem Cell Biol
4. Böhm N, Sprenger E (1968) Fluorescence 111:345–358
cytophotometry: a valuable method for the 8. Vialli M, Reggiani M (1948) Dispositivo per lo
quantitative determination of nuclear studio colorimetrico e fotometrico di preparati
Feulgen-DNA. Histochemie 16:100–118 microscopici. Boll Soc Med Chirur Pavia 62:
299–301
22 Giuliano Mazzini and Marco Danova

9. Vialli M, Romanini G (1950) Dispositivi sem- 26. Prenna G, De Paoli AM (I964) Derivati tiazo-
plificati di istofotometria nel visibile. Boll Soc lici come reagenti tipo Schiff fluorescenti. Rend
Ital Biol Sperim 26:1633 Ist Lomb Sc Lett B 98:267–273
10. Vialli M, Perugini S (1954) Due nuovi modelli 27. Prenna G, Bianchi UA (1964) Reazioni di
di apparecchiature istofotometriche. Riv Istoch Feulgen fluorescenti e loro possibilità citofluor-
Norm Pat 1(2):149–170 ometriche quantitative. 5) Citofotometria
11. Deeley EM (1955) An integrating microdensi- quantitativa in fluorescenza ed in assorbimento
tometer for biological cells. J Sci Instrum 31: della reazione di Feulgen eseguita con
263–267 acriflavina-SO2. Riv Istoch Norm Pat 10:
12. Benedetti PA, Viola-Magni MP (1966) A scan- 667–676
ning integrating histophotometer. J Sci 28. Prenna G, De Paoli AM (1968) Impiego del
Instrum 43:141–143 Rivanol come reagente tipo Schiff fluorescente
13. Ploem JS (1967) The use of vertical illuminator nella reazione di Feulgen. Riv Istoch Norm Pat
with interchangeable dichroic mirrors for fluo- 14:169–170
rescence microscopy with incident light. Z Wiss 29. Trujillo TT, Van Dilla MA (1972) Adaptation
Mikrosk 68:129–142 of the fluorescent Feulgen reaction to cells in
14. Prenna G, Mazzini G, Cova S (1974) Method- suspension for flow microfluorometry. Acta
ological and instrumentational aspects of cyto- Cytol 16:26–30
fluorometry. Histochem J 6:259–278 30. Mazzini G, Giordano P (1980) Effects of some
15. Cova S, Prenna G, Mazzini G (1974) Digital solvents on the fluorescence intensity of phe-
microscpectrofluorometry by multichannel nantridinic derivatives-DNA complexes: flow
scaling and single photon detection. Histo- cytofluorometric application. In: Laerum OD,
chem J 6:279–299 Lindmo T, Thorud E (eds) Flow cytometry
IV. Universitetsforlaget, Bergen/Oslo/
16. Prenna G, Leiva S, Mazzini G (1974) Quanti- Trondheim
tation of DNA by cytofluorometry of the con-
ventional Feulgen reaction. Histochem J 6: 31. Mazzini G, Giordano P, Riccardi A, Monte-
467–489 cucco CM (1980) Biological significance of
flow cytometric application of phenantridinic
17. Kamentsky LA, Melamed MR, Derman H dyes at low concentration. Basic Appl Histo-
(1969) Spectrophotometer: new instrument chem 24:264
for ultrarapid cell analysis. Science 150:630–
631 32. Mazzini G, Giordano PA (1981) Flow cytome-
try: a methodologic approach for fast quantita-
18. Van Dilla MA, Trujillo TT, Mullaney PF, Coul- tive cytochemical measurements and its use for
ter JR (1969) Cell microfluorometry: a method the study of the chromatin structure. Basic
for rapid fluorescence measurement. Science Appl Histochem 25:303
163:1213
33. Crissman HA, Steinkamp JA (1973) Rapid
19. Dittrich W, Göhde W (1969) Impulsfluorome- simultaneous measurement of DNA, protein,
trie bei einzelzellen in suspension. Z Natur- and cell volume in single cells from large mam-
forsch 24b:360–361 malian cell populations. J Cell Biol 59:766–
20. Göhde W, Dittrich W (1970) Simultane 771
Impulsfluorimetrie des DNS und Proteinge- 34. Krishan A (1975) Rapid flow cytofluorometric
haltes von Tumorzellen. Z Anal Chem 352: analysis of mammalian cell cycle by propidium
328–330 iodide staining. J Cell Biol 66:188–193
21. Ormerod MG (1990) Flow cytometry: a prac- 35. Fried J, Perez AG, Clarkson BD (1976) Flow
tical approach. IRL Press, Oxford/New York/ cytofluorometric analysis of cell cycle distribu-
Tokyo tions using propidium iodide. Properties of the
22. Melamed MR, Lindmo T, Mendelsohn ML method and mathematical analysis of the data. J
(1991) Flow cytometry and sorting. Wiley- Cell Biol 71:172–181
Liss, New York 36. Giordano P, Mazzini G, Riccardi A, Monte-
23. Laerum OD, Farsund T (1981) Clinical appli- cucco CM, Ucci G, Danova M (1985) Propi-
cations of flow cytometry: a review. Cytometry dium iodide staining of cytoautoradiographic
2:1–13 preparations for the simultaneous determina-
24. Robinson JP (1993) Handbook of flow cyto- tion of DNA content and grain count. Histo-
metry methods. Wiley-Liss, New York chem J 17(11):1259–1270
25. Shapiro HM (2005) Practical flow cytometry, 37. Mazzini G, Giordano P, Montecucco CM, Ric-
4th edn. Alan R Liss Inc, New York cardi A (1980) A rapid cytofluorometric
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 23

method for quantitative DNA determination study of nuclear sulphydryl and disulphide
on fixed smears. Histochem J 12:153–168 groups during sperm maturation in the
38. Rigler R Jr (1966) Microfluorometric charac- mouse. J Reprod Fertil 68(2):371–376.
terization of intracellular nucleic acids and https://doi.org/10.1530/jrf.0.0680371
nucleo-proteins by Acridine Orange. Acta 50. Bottiroli G, Croce AC, Pellicciari C, Ramponi
Physiol Scand 67(267):1–122 R (1994) Propidium iodide and the thiol-
39. Darzynkiewicz Z (1979) Acridine orange as a specific reagent DACM as a dye pair for fluo-
molecular probe in studies of nucleic acids. In: rescence resonance energy transfer analysis: an
Melamed MR, Mullaney PF, Mendelsohn (eds) application to mouse sperm chromatin. Cyto-
Flow cytometry and sorting. Wiley, New York metry 15(2):106–116. https://doi.org/10.
40. Göhde W (1972) Automation of cytofluoro- 1002/cyto.990150204
metry by use of the 51. Mazzini G, Giordano PA, Pellicciari C,
impulsmicrophotometer. In: Thaer A, Sernetz Costa A, Marchese G (1987) A double staining
M (eds) Fluorescence techniques in cell biol- method for the cytometric quantitation of
ogy. Springer, New York DNA and thiol groups. In: Burger G, Ploem
41. Latt SA, Stetten G (1976) Spectral studies on JS, Goerttler K (eds) Clinical cytometry and
33258 Hoechst and related bisbenzimidazole histometry. Academic Press, pp 149–152
dyes useful for fluorescent detection of deoxyr- 52. Bryant DA, Glazer AN, Eiserling FA (1976)
ibonucleic acid synthesis. J Histochem Cyto- Characterization and structural properties of
chem 24:24–33. https://doi.org/10.1177/ the major biliproteins of Anabaena sp. Arch
24.1.943439 Microbiol 110:61–75
42. Ellwart JW, Dormer P (1990) Viability mea- 53. Oi VT, Glazer AN, Stryer L (1982) Fluores-
surement using spectrum shift in Hoechst cent phycobiliprotein conjugates for analyses of
33342 stained cells. Cytometry 11:239–243 cells and molecules. J Cell Biol 93:981–986
43. Mazzini G, Ferrari C, Erba E (2003) Dual 54. Kronick MN, Grossman PD (1983) Immuno-
excitation multi-fluorescence flow cytometry assay techniques with fluorescent phycobilipro-
for detailed analyses of viability and apoptotic tein conjugates. Clin Chem 29(9):1582–1586
cell transition. Eur J Histochem 47:289–298 55. Telford WG, Moss MW, Morseman JP, Allnutt
44. Haugland RP, Larison KD (2002) Handbook FC (2001) Cyanobacterial stabilized phycobili-
of fluorescent probes and research chemicals, somes as fluorochromes for extracellular anti-
8th edn. Molecular Probes, Inc., Eugene, gen detection by flow cytometry. J Immunol
OR, USA Methods 254:13–30
45. Adan A, Alizada G, Kiraz Y, Baran Y, Nalbant A 56. Puzorjov A, Mccormick AJ (2020) Phycobili-
(2016) Flow cytometry: basic principles and proteins from extreme environments and their
applications. Crit Rev Biotechnol 14:1–14 potential applications. J Exp Bot 71(13):
46. Yamamoto K, Sekine T (1978) Fluorescent 3827–3842
tracer method for protein SH groups. III. Use 57. Zembruski NCL, Nadine CL, Stache V, Weiss J
of N-(7-dimethylamino-4-methylcoumarinyl) (2012) 7-Aminoactinomycin D for apoptosis
maleimide as a tracer of cysteine-containing staining in flow cytometry. Anal Biochem
peptides. Anal Biochem 90(1):300–308. 429(1):179–181. https://doi.org/10.1016/j.
https://doi.org/10.1016/0003-2697(78) ab.2012.07.005
90034-9 58. Costa A, Mazzini G, Del Bino G, Silvestrini R
47. Ogawa H, Taneda A, Kanaoka Y, Sekine T (1981) DNA content and kinetic characteris-
(1979) The histochemical distribution of pro- tics of non-Hodgkin’s lymphoma determined
tein bound sulfhydryl groups in human epider- by flow cytometry and autoradiography. Cyto-
mis by the new staining method. J Histochem metry 2(3):185–188. https://doi.org/10.
Cytochem 27(5):942–946. https://doi.org/ 1002/cyto.990020310
10.1177/27.5.90070 59. Zippel R, Martegani E, Vanoni M, Mazzini G,
48. Taneda A, Ogawa H, Hashimoto K (1980) The Alberghina L (1982) Cell cycle analysis in a
histochemical demonstration of protein-bound human cell line (EUE cells). Cytometry 2(6):
sulfhydryl groups and disulfide bonds in 426–430. https://doi.org/10.1002/cyto.
human hair by a new staining method 990020612
(DACM staining). J Invest Dermatol 75(4): 60. Danova M, Riccardi A, Gaetani P, Wilson GD,
3 6 5 – 3 6 9 . h t t p s : // d o i . o r g / 1 0 . 1 1 1 1 / Mazzini G, Brugnatelli S et al (1988) Cell
1523-1747.ep12531243 kinetics of human brain tumors: in vivo study
49. Pellicciari C, Hosokawa Y, Fukuda M, Man- with bromodeoxyuridine and flow cytometry.
fredi Romanini MG (1983) Cytofluorometric Eur J Cancer Clin Oncol 24(5):873–880
24 Giuliano Mazzini and Marco Danova

61. Riccardi A, Danova M, Wilson G, Ucci G, dyscrasias: a flow cytofluorimetric study. Br J

Dörmer P, Mazzini G et al (1988) Cell kinetics Cancer 62(5):781–785
in human malignancies studied with in vivo 72. Rosti V, Bergamaschi G, Lucotti C, Danova M,
administration of bromodeoxyuridine and Carlo-Stella C, Locatelli F et al (1995) Oligo-
flow cytometry. Cancer Res 48(21): deoxynucleotides antisense to c-abl specifically
6238–6245 inhibit entry into S-phase of CD34+ hemato-
62. Riccardi A, Danova M, Dionigi P, Gaetani P, poietic cells and their differentiation to
Cebrelli T, Butti G et al (1989) Cell kinetics in granulocyte-macrophage progenitors. Blood
leukaemia and solid tumours studied with 86(9):3387–3393
in vivo bromodeoxyuridine and flow cytome- 73. Bozzetti C, Nizzoli R, Camisa R, Guazzi A,
try. Br J Cancer 59(6):898–903 Ceci G, Cocconi G et al (1997) Comparison
63. Giordano M, Riccardi A, Danova M, Gobbi P, between Ki-67 index and S-phase fraction on
Riccardi A (1991) Cell proliferation of human fine-needle aspiration samples from breast car-
leukemia and solid tumors studied with in vivo cinoma. Cancer 81(5):287–292
bromodeoxyuridine and flow cytometry. Can- 74. Cova E, Cereda C, Galli A, Curti D, Finotti C,
cer Detect Prev 15(5):391–396 Di Poto C et al (2006) Modified expression of
64. Giordano M, Danova M, Mazzini G, Gobbi P, Bcl-2 and SOD1 proteins in lymphocytes from
Riccardi A (1993) Cell kinetics with in vivo: sporadic ALS patients. Neurosci Lett 399(3):
bromodeoxyuridine assay, proliferating cell 186–190. https://doi.org/10.1016/j.neulet.
nuclear antigen expression, and flow cyto- 2006.01.057
metric analysis. Prognostic significance in 75. Montanaro L, Mazzini G, Barbieri S, Vici M,
acute nonlymphoblastic leukemia. Cancer Nardi-Pantoli A, Govoni M et al (2007) Dif-
71(9):2739–2745 ferent effects of ribosome biogenesis inhibition
65. Erba E, Giordano M, Danova M, Mazzini G, on cell proliferation in retinoblastoma protein-
Ubezio P, Torri V et al (1994) Cell kinetics of and p53-deficient and proficient human osteo-
human ovarian cancer with in vivo administra- sarcoma cell lines. Cell Prolif 40(4):532–549.
tion of bromodeoxyuridine. Ann Oncol 5(7): https://doi.org/10.1111/j.1365-2184.2007.
627–634 00448.x
66. Mazzini G, Danova M, Ferrari C, Dionigi P, 76. Cova E, Ghiroldi A, Guareschi S, Mazzini G,
Riccardi A (1996) Cell proliferation and ploidy Gagliardi S, Davin A et al (2010) G93A SOD1
of human solid tumours: methodological expe- alters cell cycle in a cellular model of Amyo-
rience with in vivo bromodeoxyuridine and trophic Lateral Sclerosis. Cell Signal 22(10):
DNA flow cytometry. Anal Cell Pathol 10(2): 1477–1484. https://doi.org/10.1016/j.
101–113 cellsig.2010.05.016
67. Robinson JP, Roederer M (2015) History of 77. Vesey G, Deere D, Gauci MR, Griffiths KR,
science. Flow cytometry strikes gold. Science. Williams KL, Veal DA (1997) Evaluation of
https://doi.org/10.1126/science.aad6770 fluorochromes for immunofluorescent labeling
68. Pellicciari C, Danova M, Giordano M, Conti A, of microorganisms in environmental water
Mazzini G, Wang E et al (1991) Expression of samples. Cytometry 29:147–154
cell cycle related proteins proliferating cell 78. Chan WCW, Nie S (1998) Quantum dot bio-
nuclear antigen (PCNA) and statin during conjugates for ultrasensitive nonisotopic detec-
adaptation and de-adaptation of EUE cells to tion. Science 281:2016–2018
a hypertonic medium. Cell Prolif 24(5): 79. Bruchez M, Moronne M, Gin P et al (1998)
469–479. https://doi.org/10.1111/j. Semiconductor nanocrystals as fluorescent
1365-2184.1991.tb01175.x biological labels. Science 281:2013–2016
69. Giordano M, Danova M, Riccardi A, Mazzini 80. Han M, Gao X, Su JZ, Nie S (2001) Quantum-
G (1989) Simultaneous detection of cellular ras dot-tagged microbeads for multiplexed optical
p21 oncogene product and DNA content by coding of biomolecules. Nat Biotechnol 19:
two-parameter flow cytometry. Anticancer Res 631–635
9(3):799–803 81. Lee LY, Ong SL, Hu JY, Ng WJ, Feng Y, Tan X
70. Danova M, Riccardi A, Mazzini G (1990) Cell (2004) Use of semiconductor quantum dots
cycle-related proteins and flow cytometry. Hae- for photostable immunofluorescence labeling
matologica 75(3):252–264 of Cryptosporidium parvum. Appl Environ
71. Danova M, Riccardi A, Ucci G, Luoni R, Microbiol 70:5732–5736
Giordano M, Mazzini G (1990) Ras oncogene 82. Mattoussi H, Mauro JM, Goldman ER, Ander-
expression and DNA content in plasma cell son GP, Sundar VC, Mikulec FV et al (2000)
Self-assembly of CdSe-ZnS quantum dot
 Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes 25

bioconjugates using in engineered recombi- 87. Gautreau G, Pejoski D, Le Grand R, Cosma A,

nant protein. J Am Chem Soc 122:12142– Beignon A-S, Tchitchek N (2017) SPADE-
12150 VizR: an R package for visualization, analysis
83. Bendall SC, Simonds EF, Qiu P, Amir ED, and integration of SPADE results. Bioinfor-
Krutzik PO, Finck R et al (2011) Single-cell matics 33:779–781
mass cytometry of differential immune and 88. Weber LM, Robinson MD (2016) Comparison
drug responses across a human hematopoietic of clustering methods for high-dimensional
continuum. Science 332:687–696 single-cell flow and mass cytometry data. Cyto-
84. Irish J, Doxie D (2014) High-dimensional sin- metry A 89:1084–1096
gle-cell cancer biology. In: Fienberg H, Nolan 89. Qiu P, Simonds EF, Bendall SC, Gibbs KD Jr,
G (eds) High-dimensional single cell analysis. Bruggner RV, Linderman MD et al (2011)
Current topics in microbiology and immunol- Extracting a cellular hierarchy from high-
ogy. Springer, Berlin, Heidelberg dimensional cytometry data with SPADE. Nat
85. Levine J, Simonds E, Bendall S (2015) Data- Biotechnol 29:886–891
driven phenotypic dissection of AML reveals 90. Spitzer MH, Nolan GP (2016) Mass cytome-
progenitor-like cells that correlate with prog- try: single cells, many features. Cell 165:780–
nosis. Cell 162(1):184–197 791
86. Atkuri KR, Stevens JC, Neubert H (2015) 91. Huang A, Postow M, Wherry EJ (2017) T-cell
Mass cytometry: a highly multiplexed single- invigoration to tumour burden ratio associated
cell technology for advancing drug develop- with anti-PD-1 response. Nature 545:60–65
ment. Drug Metab Dispos 43:227–233
 Part I

Vital Histochemistry
 Chapter 2

Autofluorescence Label-Free Imaging of the Liver Reticular

Structure
Anna C. Croce, Giuseppina Palladini, Andrea Ferrigno,
and Mariapia Vairetti

Abstract
Autofluorescence rising from biological substrates under proper excitation light depends on the presence of
specific endogenous fluorophores and can provide information on the morpho-functional properties in
which they are strictly involved. Besides the numerous endogenous fluorophores involved in metabolic
functions, fibrous proteins may act as direct, label-free biomarkers of the tissue structural organization. The
optical properties of collagen, in particular, are currently applied as an alternative to established histochem-
ical procedures to investigate the connective tissue as well as its changes in diseased conditions. This is
particularly true in hepatology where the histochemical procedures to label the reticular structure are not
routinely applied, as they are complex and time-consuming. The morphology of the liver reticular structure
and its changes are up to now poorly considered despite the increasing awareness of the regulatory role
played by the remodeling of the reticular structure in pathological conditions. In this context, the auto-
fluorescence label-free imaging has proven to be a suitable approach.

Key words Endogenous fluorophore, Collagen, Fatty liver, Sinusoids

1 Introduction

Autofluorescence (AF) can be observed in biological substrates

because of the common presence of intrinsic biomolecules (endog-
enous fluorophores, EFs) that are able to fluoresce under light
excitation at suitable wavelengths. In cytochemistry and histo-
chemistry, AF emission is often an undesirable light signal disturb-
ing the detection of exogenous fluorophores used for the specific
labeling of target molecules. On the other hand, a proper detection
and analysis of AF emission can be a valuable analytical and diag-
nostic tool for real-time, label-free characterization and monitoring
of the morpho-functional conditions of biological substrates. This
analytical potential is allowed by the presence of specific EFs whose
chemical nature, quantity, and microenvironment are related to the

Carlo Pellicciari et al. (eds.), Histochemistry of Single Molecules: Methods and Protocols,
Methods in Molecular Biology, vol. 2566, https://doi.org/10.1007/978-1-0716-2675-7_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

29
30 Anna C. Croce et al.

metabolic and structural properties of cells and tissues. Thanks to

the continuous improvement in the detection systems, measure-
ment procedures, and analytical data processing, this approach has
wide-ranging applications in the plant and animal field, including
ecology and environmental surveillance as well as the improvement
of industrial or in-field production and control of biomass or food.
In particular, the AF features allow in situ investigation of the
biometabolic mechanisms of organs and tissues under normal and
diseased conditions, which makes AF an intrinsic diagnostic
biomarker [1].
In hepatology, the multiple and complex anabolic and catabolic
functions carried out by the liver involve various kinds of EFs,
making their AF a powerful means to characterize and monitor
the normal, physiologically altered, or diseased condition of this
organ. Among the main metabolism-related EFs in liver, we may
recall porphyrins, lipofuscins and vitamin A, as well as NAD(P)H
and flavins. These latter coenzymes have for a long time been
regarded as direct biomarkers of cell energy production, reductive
biosynthesis, and antioxidant defense, in a strict relationship with
aging, cell differentiation, and neoplastic transformation or in the
response to changes in oxygen availability [2–4]. Most recently, also
fluorescing fatty acids have been proposed as promising biomarkers
of lipid accumulation, as well as of their systemic metabolic disorder
in the progression of liver disease [5]. A valuable diagnostic mean-
ing is also commonly given to the architectural organization of liver
parenchyma, where the connective structures and their typical
fibrous proteins (namely, elastin, keratin, and collagen) are consid-
ered as potentially diagnostic EFs.
Collagens belong to a family of fibrillar polymeric proteins with
variable molecular structure, organization, and complexity, localiz-
ing in different histological sites [6]. Qualitative studies performed
on the normal liver by immunostaining procedures have allowed to
localize collagen types I and III in the blood vessel walls and around
the biliary ducts, in the portal tracts, and in perisinusoidal areas,
while collagen type IV that is a typical component of the basement
membrane was found in the portal veins and perisinusoidal areas
[7]. Further immunostaining-based investigation has confirmed
the presence of collagen types I, III, and IV in central veins and in
the perisinusoidal space of Disse in elder human livers with various
stages of fibrosis [8].
In the histopathological practice, the established picrosirius red
staining is commonly used to visualize collagen (Fig. 1), while the
overall network of thin and delicate reticular fibers in the liver can
be demonstrated by silver impregnation [9, 10]: both methods,
however, require fixed tissue sections and are rather complex and
time-consuming.
A valuable option to detect collagen directly, in a real-time and
label-free manner, is offered by its own intrinsic optical properties
 Autofluorescence of Liver Reticular Structure 31

Fig. 1 Sections of liver tissue, from unfixed frozen specimens cut at cryostat and stained with hematoxylin and
eosin (a) or picrosirius red (b). Picrosirius red staining allows to appreciate the normal parenchyma, with a
central vein (cv) with converging yellowish hepatocyte organized in plates, partly delineated by reddish thin
structures relatable to the presence of collagen associated with sinusoids. Bars: 50 μm

of birefringence and fluorescence. Birefringence is characteristic of

the collagen fibrillar forms and may be visualized by polarized light
microscopy that, however, is seldom present in a histochemical lab.
On the contrary, a conventional fluorescence microscope is often
available and allows observing the AF emission of collagen from
connective structures [11, 12]. The proper choice of fluorescence
observation conditions is able to provide a direct in situ detection of
the delicate structural fibrous system in normal, physiologically
altered, or diseased liver (Fig. 2) (see Note 1).

2 Materials

2.1 Samples and 1. Freshly collected liver specimens.

Chemicals 2. Liquid nitrogen.
3. Cryostat embedding medium (e.g., OCT, Tissue-Tek; Sakura
Finetek USA).

2.2 Equipment 1. Cryogenic liquid nitrogen containers.

2. Cryogenic vials (capacity 2 mL).
3. Cryostat.
4. High-quality, clean glass slides with minimalized self-
fluorescence.
5. Microscope equipped for bright-field and epi-fluorescence illu-
mination (see Note 2).
32 Anna C. Croce et al.

Fig. 2 Unstained cryostatic sections of unfixed liver tissue. (a–d) In the control liver, the typical organization of
normal parenchyma, with the anastomosing plates of hepatocytes converging toward central veins (cv), can
be easily appreciated under both bright-field and fluorescence conditions, where the lines of sinusoids are
evidenced by their emission brighter than that of hepatocytes, independently of the AF detection conditions
 Autofluorescence of Liver Reticular Structure 33

3 Methods

3.1 Section 1. Take tissue pieces (about 0.5 cm in diameter) from freshly
Preparation dissected liver and immediately deep frozen in liquid N2 for
10 min (see Note 3).
2. Cut 15 μm-thick cryostatic sections and collect them on glass
slides.
3. Allow the sections to air dry at room temperature for at least
48 h.
4. Observe the unstained sections without coverslip.

3.2 Imaging 1. Micrographs of the unstained liver sections are collected under
Microscopy bright-field or epi-illumination conditions.
2. For AF imaging, the light excitation/detection conditions may
be selected by optical cubes with the following filter combina-
tions: 340–390 nm excitation filter, 410 nm dichroic mirror,
420-nm-long pass filter (UV/blue-observation condition);
460–495 nm excitation filter, 505 nm dichroic mirror,
510-nm-long pass filter (green-observation condition);
530–550 nm excitation filter, 570 nm dichroic mirror,
575-nm-long pass filter (red-observation condition).
Imaging of unstained unfixed sections in the bright-field con-
dition still allows to appreciate the structural organization of nor-
mal liver (Fig. 2a). Comparable structural organization is easily
appreciable also in AF images (Fig. 2b–d). As to fatty liver, the
pattern observed under the bright-field conditions allows to appre-
ciate almost only some lipidic droplets as dark and light roundish
structures, which appear empty or filled with some fluorescing
material when observed under UV excitation in AF imaging
(Fig. 2b). As compared with the bright field, UV condition allows
to better appreciate the presence of more numerous, smaller, and
full vesicles, along with other large structures with apparently
thicker dark-blue borders, identifiable with small vessels [13]. The
ä

Fig. 2 (continued) applied. (e–h) Fatty liver, on the contrary, when observed under the bright-field conditions
(e), allows to only appreciate lipid droplets, because of their dark margins and, in some cases, their dark
content. Autofluorescence observed under the UV/blue condition (f) still reveals some lipid droplets that appear
as vesicular structures full of fluorescing material, whereas few of them result as large dark areas due to the
loss of their lipid content during cutting and collection of the sections. Empty spaces with a nearly triangular
shape and a darker border than the surrounding tissue can be ascribed to dilated central veins. The AF
patterns under green- (g) or red-observation condition (h) show almost exclusively the network of a reticular
organization that delineates the boundaries of the lipid droplets and allows appreciating some droplets neatly
distributed along part of the margin of the small vessels (arrows); the reticular network shows a tighter texture
in the areas around the vessels. Under green-observation condition, yellow emitting granules of lipofuscin-like
lipopigment are well evident (arrowheads). Bars: 150 μm
34 Anna C. Croce et al.

use of AF excitation and emission conditions at longer wavelength

shows a more detailed structural pattern of liver parenchyma. In
particular, the AF green- and red-observation conditions appear to
be greatly selective for a fluorescing network, which demarcates the
fatty droplets and appears to be organized with a tighter texture in
the areas close to the vessels. It is worth underlining that, as
compared with the conventional histochemical techniques, the AF
images of unfixed and unstained sections are free from the struc-
tural modifications that may possibly occur following fixation,
embedding, and staining (see Note 4).

4 Notes

1. The potential of the AF imaging as a valuable tool to investigate

the remodeling of the extracellular matrix in the liver paren-
chyma is here demonstrated comparing normal liver with an
experimental model of lipid accumulation, obtained from male
Wistar rats fed with a methionine- and choline-deficient diet for
8 weeks [14]. Observation under different excitation and emis-
sion wavelengths makes it possible to evidence the fine reticular
system associated with the liver sinusoids.
2. Here we used a BX53 Olympus microscope with UPlanFL 10x
(NA 0.30) or UPlanFL 20x (NA 0.50) objectives (Olympus
Optical Co. GmBH, Hamburg, Germany); the images were
recorded by an EOS 1300D Olympus camera and optimized
for presentation with the IrfanView 4.54 program (Irfan Skil-
jan copyright 1996–2020).
3. Frozen samples may be preserved at 80  C until cutting at the
cryostat.
4. The direct detection of the fluorescing reticular network in liver
parenchyma can improve studies on the mechanism affecting
the balance between metalloproteinases and their specific tissue
inhibitors in the remodeling of extracellular matrix and base-
ment membrane in the rising of liver disease [15].

Acknowledgments

We wish to particularly thank Giovanni Bottiroli, for starting the

autofluorescence-based studies applied to biomedicine. Thanks are
also due to all our colleagues who cooperated with us in studying
various subjects by the wide-ranging autofluorescence approach.
 Autofluorescence of Liver Reticular Structure 35

References
1. Croce AC (2021) Light and autofluorescence, 9. Wheater PR, Burkitt HG, Daniels VG (1979)
multitasking features in living organisms. Functional histology: a text and colour atlas.
Photochem 1:67–125. https://doi.org/10. Churchill Livingstone, Edinburgh London
3390/photochem1020007 Melbourne and New York
2. Mayevsky A, Chance B (2007) Oxidation- 10. Gömöri G (1937) Silver impregnation of retic-
reduction states of NADH in vivo: from ani- ulum in paraffin sections. Am J Pathol 13:
mals to clinical use. Mitochondrion 7: 993-1002.5
330–339. https://doi.org/10.1016/j.mito. 11. Marcu L (2000) Characterization of type I, II,
2007.05.001 III, IV, and V collagens by time-resolved laser-
3. Croce AC, Ferrigno A, Bottiroli G, Vairetti M induced fluorescence spectroscopy. Proc SPIE
(2018) Autofluorescence-based optical biopsy: 3917:93–101. https://doi.org/10.1117/12.
an effective diagnostic tool in hepatology. Liver 382720
Int 38:1160–1174 12. Wang Y, Vincent R, Yang J, Asgharpour A,
4. Kunz WS, Gellerich FN (1993) Quantification Liang X, Idowu MO et al (2017) Dual-photon
of the content of fluorescent flavoproteins in microscopy-based quantitation of fibrosis-
mitochondria from liver, kidney cortex, skeletal related parameters (q-FP) to model disease
muscle, and brain. Biochem Med Metab Biol progression in steatohepatitis. Hepatology 65:
50:103–110 1891–1903. https://doi.org/10.1002/hep.
5. Croce AC, di Pasqua LG, Berardo C, 29090
Ferrigno A, Siciliano V, Bottiroli G et al 13. Kleiner DE, Brunt EM (2012) Nonalcoholic
(2017) Real time optical biopsy: serum fatty fatty liver disease: pathologic patterns and
acids profiling in a rat model of nonalcoholic biopsy evaluation in clinical research. Semin
steatohepatitis. Lasers Surg Med 49:E5. Liver Dis 32:3–13
https://doi.org/10.1002/lsm.22672 14. Di Pasqua LG, Berardo C, Rizzo V, Richelmi P,
6. Borel JP, Monboisse JC (1993) Collagens: why Croce AC, Vairetti M et al (2016) MCD diet-
such a structural complexity? C R Seances Soc induced steatohepatitis is associated with
Biol Fil 187:124–142 alterations in asymmetric dimethylarginine
7. Voss B, Rauterberg J, Allam S, Pott G (1980) (ADMA) and its transporters. Mol Cell Bio-
Distribution of collagen type I and type III and chem 419:147–155. https://doi.org/10.
of two collagenous components of basement 1007/s11010-016-2758-2
membranes in the human liver. Pathol Res 15. Palladini G, di Pasqua LG, Berardo C,
Pract 170:50–60. https://doi.org/10.1016/ Siciliano V, Richelmi P, Perlini S et al (2019)
S0344-0338(80)80155-5 Animal models of steatosis (NAFLD) and stea-
8. Mak KM, Chu E, Lau KHV, Kwong AJ (2012) tohepatitis (NASH) exhibit hepatic lobe-
Liver fibrosis in elderly cadavers: localization of specific gelatinases activity and oxidative stress.
collagen types I, III, and IV, α-smooth muscle Can J Gastroenterol Hepatol 19:5413461.
actin, and elastic fibers. Anat Rec (Hoboken) https://doi.org/10.1155/2019/5413461
295:1159–1167. https://doi.org/10.1002/
AR.22504
 Chapter 3

Lysosome Imaging Based on Fluorescent Carbon Dots

Shuo Guo, Yuanqiang Sun, and Zhaohui Li

Abstract
Lysosomes play key roles in different cellular processes such as autophagy, phagocytosis, and apoptosis.
Lysosomal dysfunction is related to many diseases. Fluorescence lysosome staining strategy is valuable for
the researches on the lysosome involvement in different pathological diagnosis. Here we describe fluores-
cence lysosome staining methods with carbon dots for the identification of lysosomes in living and fixed
cells.

Key words Lysosome, Cell imaging, Carbon dots, Confocal fluorescence microscopy

1 Introduction

Lysosomes are membrane-bound organelles and the specific pH

(4.5–6.0) makes them “stomachs” of the cells. They possess more
than 60 acid hydrolases and generate small-molecule nutrients
through decomposing the macromolecular species [1]. Lysosomes
are fundamental for the normal function of eukaryotic cells due to
their participation in homoeostasis, plasma membrane repair, and
intracellular signal transmutation. Lysosomal dysfunction is accom-
panied by changes in their number and morphology [2]. Lysosomal
dysfunction is involved in various diseases such as Gaucher disease,
neurodegenerative diseases, and cancer. In this context, visualizing
of lysosomes in real time is essential for the diagnosis of related
diseases and exploration of the relational mechanisms [3, 4]. The
aim of this chapter is to describe the staining procedures of lyso-
somes with carbon dots in different conditions of cells such as living
cells and fixed cells.
Carbon dots (CDs) were discovered in 2004. Xu et al. found
these nanomaterials in the procedure of the purification of single-
walled carbon nanotubes, which were isolated from arc-discharge
soot [5]. In general, the size of CDs is less than 10 nm with the
spherical-like structure [6, 7]. CDs have been confirmed to possess

Carlo Pellicciari et al. (eds.), Histochemistry of Single Molecules: Methods and Protocols,
Methods in Molecular Biology, vol. 2566, https://doi.org/10.1007/978-1-0716-2675-7_3,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

37
38 Shuo Guo et al.

Fig. 1 The strategy of synthesizing CDs

Fig. 2 Fluorescent images of HeLa cells stained with (a) CDs (60 μg/mL) for 20 min, (b) LysoTracker™ Deep
Red (50 nM) for 25 min, (c) Panels a and b merged, and (d) intensity profiles within the linear region of interest
of CDs and LysoTracker™ Deep Red. Scale bar: 25 μm

many unique properties, such as high biocompatibility, robust

photostability, easy producibility, low cost, etc. [8, 9]. Thus, CDs
have been applied in many fields including photocatalysis, bio-
chemical sensing, cancer therapy, and cell imaging [10, 11]. In
addition, fluorescence analysis has attracted considerable interest
of researches, which includes the features of in situ, noninvasive,
and dynamic observation of samples. Moreover, some CDs that can
stain lysosomes have also been reported [12–15].
In this chapter we describe the method of CDs for staining
lysosomes in living cells and fixed cells. Herein, citric acid and N,N-
dimethylaniline are selected to prepare CDs through microwave-
assisted method (Fig. 1). The ultra-small size endows them to
localize in lysosomes rapidly and lysosomal movements are tracked
successfully. Moreover, the pH-independent property of CDs
makes them visible in lysosomes (Fig. 2), realizing long-term imag-
ing. In addition, the CDs could not only stain lysosomes in living
cells of different cell lines but also in fixed cells (Fig. 3) [15].

2 Materials

Deionized water is used in all the experiments. All the chemicals are
analytical grade. All reagents and buffers are stored at 4  C. We
recommend wearing lab coats, gloves, and masks throughout the
experiment.
Random documents with unrelated
content Scribd suggests to you:
The Project Gutenberg eBook of For Yardley: A
Story of Track and Field
This ebook is for the use of anyone anywhere in the United States
and most other parts of the world at no cost and with almost no
restrictions whatsoever. You may copy it, give it away or re-use it
under the terms of the Project Gutenberg License included with this
ebook or online at www.gutenberg.org. If you are not located in the
United States, you will have to check the laws of the country where
you are located before using this eBook.

Title: For Yardley: A Story of Track and Field

Author: Ralph Henry Barbour

Illustrator: C. M. Relyea

Release date: March 10, 2019 [eBook #59043]

Language: English

Credits: Produced by Donald Cummings and the Online Distributed

Proofreading Team at http://www.pgdp.net (This file was
produced from images generously made available by The
Internet Archive/American Libraries.)

*** START OF THE PROJECT GUTENBERG EBOOK FOR YARDLEY: A

STORY OF TRACK AND FIELD ***
 FOR YARDLEY

BY RALPH HENRY BARBOUR.

Each Illustrated, 12mo, Cloth, $1.50.

For Yardley.
Finkler’s Field. ($1.25.)
Winning His “Y.”
The New Boy at Hilltop.
Double Play.
Forward Pass!
The Spirit of the School.
Four in Camp.
Four Afoot.
Four Afloat.
The Arrival of Jimpson.
Behind the Line.
Captain of the Crew.
For the Honor of the School.
The Half-Back.
On Your Mark.
Weatherby’s Inning.

D. APPLETON & COMPANY, NEW YORK.

 “Wheelock cleaned the bases with a long drive
over left fielder’s head.”

FOR YARDLEY
A STORY OF TRACK AND FIELD
 By
RALPH HENRY BARBOUR
AUTHOR OF “FORWARD PASS,” “DOUBLE PLAY,”
“WINNING HIS ‘Y’,” ETC.

ILLUSTRATED

NEW YORK AND LONDON

D. APPLETON AND COMPANY
1911

Copyright, 1911, by

D. APPLETON AND COMPANY

Published September, 1911

Printed in the United States of America

 CONTENTS
CHAPTER PAGE

I.— A Rainy Saturday 1

II.— The S. P. M. 16
III.— A Call for Candidates 28
IV.— The Initiation 44
V.— The Challenge 59
VI.— Alf Becomes an Editor 66
VII.— The Scholiant 76
VIII.— Gerald Lies Low 89
IX.— A Midnight Escapade 105
X.— Pursuit and Escape 119
XI.— Gerald Visits the Office 130
XII.— Gerald Pays the Penalty 140
XIII.— The April Fools 148
XIV.— Mr. Collins Smiles 156
XV.— Back in Training 170
XVI.— Yardley Is Puzzled 184
XVII.— What Head Work Did 194
XVIII.— The Great Temptation 202
XIX.— A Falling Out 216
XX.— Harry Gets Revenge 222
 XXI.— The Stamp Albums Are Put Away 230
XXII.— Gerald Makes the Team 241
XXIII.— Sport on the River 256
XXIV.— A Tenth Inning Victory 267
XXV.— The Dual Meet 277
XXVI.— For Yardley! 288
 LIST OF ILLUSTRATIONS
FACING
PAGE
“Wheelock cleaned the bases with a long drive
over left fielder’s head” Frontispiece
“The boy on the stone never moved” 112
“‘You’ll wish you hadn’t been so smart,’ he
sneered” 220
“Alf squirmed onto one of the barrels and held
the craft” 264

FOR YARDLEY
 CHAPTER I
A RAINY SATURDAY

“Wonder why it always rains on Saturdays?” muttered Alf Loring,

laying his book face-down in his lap and staring discontentedly out
of the window beside him.
It was a cheerless outlook. Through the blurred panes his gaze
traversed the Yard, empty and bedraggled, to the back of Merle Hall
and the gymnasium. Everywhere was rotting snow or pools of water,
while from a low, leaden sky the rain fell straight and persistently. It
had been raining just this way all day and half of last night, and to
all appearances it intended to continue raining in the same manner
for another twenty-four hours. Yesterday the Yard had been a foot
deep in nice clean snow, the result of the blizzard that had swept
over Wissining and New England in general two days before, and
there had been more than one jolly battle royal out there. But now—
Alf sighed; and, turning, looked aggrievedly at his roommate.
Tom Dyer was seated at the study table, face in hands, the
droplight shedding its yellow glow on his tousled hair, paying little
heed to aught but the lesson he was striving to master. Alf scowled.
“Who invented rain, anyhow?” he demanded. There was no reply.
“Tom!”
“Eh? What?” Tom looked up from his book, blinking.
“I asked who invented rain, you deaf old haddock.”
“Oh! I don’t know,” answered Tom, vaguely. His eyes went back to
the book. Then he added, evidently as an afterthought and with a
desire to escape responsibility, “I didn’t.”
 “Well, I’d like to know what it’s good for,” grumbled Alf.
“Makes crops grow,” Tom murmured.
“There aren’t any crops the first of March, you idiot. For the love
of Mike, Tom, shut that book up and talk to a fellow!”
“What do you want to talk about?” asked Tom, without, however,
obeying his chum’s command.
“Anything. I’m sick of studying. I’m sick of everything. I’m sick of
this rotten rain.”
“Pull the curtains and you won’t know it’s raining,” advised Tom.
“Of course I’ll know it,” replied the other, crossly. “I’ve seen it. This
is a mean old time of year, anyhow. There’s nothing to do but study
and read and loaf around; no hockey, no baseball, no golf——”
“There’s chess.”
“Chess!” exclaimed Alf, derisively. “That’s not a game, that’s—
that’s hard labor!”
“Well, I guess it will stop raining to-night,” said Tom, comfortingly.
“And in a day or two you’ll be playing baseball—or trying to!”
“A day or two!” Alf’s book slipped from his knees and fell to the
floor with an insulted rustling of leaves. With some difficulty he
dropped one foot from the window-seat and kicked it venomously. “A
day or two! Gee, I’ll be a doddering idiot before that.”
“You are now. Shut up and let me study.”
“What’s the good of studying?” growled Alf.
“Well, I understand,” replied the other, calmly, “that before they
allow you to graduate from Yardley Hall, Mr. Loring, they hold what
is known as a final examination. And the examination is due to begin
in just three months. Having survived the recent one by a hair’s
breadth, I thought I’d like to make sure of getting through the next.
I’m very fond of this place, Alf, but I’ll be switched if I want to stay
here another year.”
 “I think it would be rather good fun myself,” said Alf, with a faint
show of animation. “Think of the sport you could have. You wouldn’t
have to study much, you see, and life would be just one long loaf.”
“To hear you, any one would think you were the original lazy-
bones. Dry up for another ten minutes and just let me get this silly
stuff, will you?”
“All right.” Alf yawned and turned his attention again to the outer
world. He was a good-looking youth of eighteen, with a jolly, care-
free countenance, upon which his present expression of irritability
looked much out of place. Even hunched as he was into a faint
resemblance to a letter W, it was plain to be seen that he had all the
height that his age warranted. He was well-built, slim, and powerful,
with more muscle than flesh, and the Yardley Hall Football Team
under his leadership had in November last completed a successful
season by defeating Broadwood Academy, Yardley’s hated rival. Alf
was the best quarter-back that the school had known for many
years.
His roommate, Tom Dyer, was big, rangy, and sufficiently homely
of face to be attractive. He was ordinarily rather sleepy looking, and
was seldom given to chatter. He had very nice gray eyes, a pleasant,
whole-hearted smile, and was one of the best-liked fellows in school.
In age Tom was nineteen, having recently celebrated a birthday. He
had been basket-ball captain, but his principal athletic honors had
been won with shot and hammer in the dual meets with Broadwood.
Both boys were members of the First Class, and were due to leave
Yardley at the end of the next term.
The room in which they sat, Number 7 Dudley Hall, was shabbily
cozy and comfortable, combining study and bedroom. It was on the
first floor, with two windows looking on to the Yard, as the space
loosely enclosed by the school buildings was known, and so
possessed the merit of being doubly accessible; that is to say, one
might enter by the door or, if faculty was not looking, by the
window. The latter mode was a very popular one, inasmuch as it
was strictly prohibited, and the windows of Number 7 were in full
view of some four studies inhabited by instructors.
Alf looked at his watch, holding it close in the waning light. It was
a quarter past five. He slipped it back into his pocket with a sigh.
There was a good three-quarters of an hour to be lived through
before supper-time. At that moment his glance, wandering to the
Yard, descried a slim figure approaching along the path from Merle,
slopping carelessly through puddles and paying no heed to the rain.
Alf looked a moment and then smiled.
“Guess you’ll have to call it off now, Tom,” he announced. “Here
comes Gerald, and it’s a safe bet he’s headed for our humble
domicile.”
Tom groaned. “That kid will be the death of me if Maury doesn’t
call the track candidates pretty soon. Gerald asks me every time I
see him when we’re going to begin work, and whether I think he will
make the squad.”
Alf chuckled. “I thought when he got his Y at hockey last month
he wouldn’t be so keen about making the Track Team. He’s a funny
kid.”
“He’s a rather nice one, though,” said Tom. “Here he comes. Bet
you he will ask about track work before he’s been here two minutes.”
Footsteps sounded along the hall, and then there came a modest
knock on the door.
“Come in, Gerald,” called Tom.
The boy who entered was not large for his fifteen years, and
seemed at first glance a bit too slender and delicate to hope to
distinguish himself on the cinders. But his slenderness held a
litheness that spoke well for his muscles, and the apparent delicacy
was largely a matter of coloring, for Gerald Pennimore had the
fairest of pink and white skins, the bluest of blue eyes, and hair that
only barely escaped being yellow. He was a nice-looking youngster,
though, with an eager, expressive face, and an easy grace of
carriage that was good to see. He greeted his hosts, closed the door
behind him, and went over to the grate, where a little coal fire
glowed ruddily.
“Yes,” said Alf, “I should think you’d want to dry your shoes,
Gerald. You walked into every puddle in the Yard.”
“They’re not very wet,” responded Gerald, amiably.
“They’re soaking! It’s a mighty good thing for you that Dan isn’t
here.”
“I’m not afraid of him,” laughed Gerald.
“You’d better be,” said Tom. “He will tan your hide for you, son, if
he catches you doing stunts like that. Where is he to-day?”
“I don’t know. I expected to find him here.”
“I haven’t seen him since dinner,” said Alf.
“Pull a chair up there, Gerald, and get those shoes dry. Beastly
weather, isn’t it?”
“Ye-es, but I’m rather glad to see the rain, aren’t you? It will take
the snow off. I guess the track will be clean by to-morrow, won’t it,
Tom?”
Tom shot an amused glance at Alf. “I guess so, but it will take
some time to get it dried out and rolled down.”
“Will it? Do you know when Captain Maury is going to call the
candidates, Tom?”
“Yes, I saw him this morning, and he told me he was going to get
them together Monday,” answered Tom, patiently.
“Going to try the mile, Gerald?” inquired Alf, innocently.
“I want to. Do you think I’d stand any show of getting on the
team, Alf?”
“I guess so. What’s your best time for the mile, Gerald?”
 “I don’t quite know. Andy said he thought I did it once in about
five minutes in the cross country, but that was on a dirt road, of
course. I guess I could do a lot better than that on the cinders.”
“Rather! Besides, any chap can do better in warm weather. Even if
you shouldn’t make the team this spring, Gerald, you’d get a lot of
fun out of it, and it would do you good besides. It’s a bit
unfortunate, though, that Maury runs the mile himself. It’s awfully
hard to crowd the captain off the team.”
“Oh, I wasn’t expecting to do that,” Gerald replied, with amusing
naïveté. “I just thought maybe I could get a place. Has Broadwood
got good mile runners?”
“How about that, Tom?”
“Yes, I think so. Usually she’s better on the distances than
anything else. But we beat her in the cross country, and maybe our
men are as good as hers this year. I suppose Goodyear and Norcross
will both enter for the mile.”
“Are you going to be on the team this year, Alf?” Gerald asked.
“No, I guess not; not unless I’m pretty badly needed. What’s the
use? Both Rand and Bufford can beat me in the sprints.”
“You might crowd a Broadwood man out in the trials, though,”
said Tom. “And you wouldn’t have to train much; your baseball work
would keep you in trim.”
“Wouldn’t it be fine,” asked Gerald, enthusiastically, as he felt of
his damp shoes, “if we won the baseball and the track meet, too,
this year? That would be a clean sweep, wouldn’t it? Football, cross
country, hockey——”
“We won’t,” said Alf. “We never have in the school’s history. We’re
bound to drop either track or baseball. Personally, I hope it will be
track. Even then, though, we’d be doing ourselves proud, what?”
“We’ll be lucky if Broadwood doesn’t get track and baseball,” said
Tom, piling his books up.
 “Why? I thought we were pretty certain of the Duals,” said Alf.
Tom shrugged his shoulders.
“Don’t see why. Just because we ran away with Broadwood last
spring doesn’t mean that we’ve got an easy thing this year. She will
work a whole lot harder, I guess. And we haven’t the men we had
then. We’ve lost Wass in the hurdles, Bird in the quarter, Johnson
and Fyles in the high jump, and two or three second-string fellows
who might have made good this year. I guess we’ve got the sprints
cinched without a doubt, but I’m not very easy in my mind regarding
the field events.”
“Well, we know who will get first in the hammer,” laughed Alf.
“Meaning me? Perhaps; but if Broadwood gets enough seconds
and thirds she may fool us.”
Gerald turned, listened, and then retired hurriedly from the grate.
“There’s Dan,” he said. There was a knock and the door swung
open, admitting a disreputable figure in a dripping raincoat and a felt
hat, from the down-turned brim of which drops of water trickled.
“Hello, you chaps! Fine day, isn’t it?”
“Who’s your tramp friend, Tom?” asked Alf. “Isn’t he a sight?
Where’s the dog? Why, if it isn’t our old friend, Mr. Vinton! Ouch!”
The final remark was emphatic and spontaneous, for Dan’s wet
hat sailed across the room with beautiful precision, and landed fairly
against Alf’s face with a damp and dismal splash.
The others grinned enjoyably as Alf wiped the rain from his eyes
and looked about for a weapon. Finding nothing save the hat, and
doubting his ability to use that effectually, he had recourse to verbal
weapons.
“Canaille!” he hissed. “Dog of a Christian! Varlet!”
“Go it!” laughed Dan, shedding his raincoat. “It was a bully shot,
though, wasn’t it? What have you fellows been doing?”
 “Leading a quiet, studious, respectable existence until you broke
in with your low, rough-house manners,” responded Alf, severely.
“Dan, you’re a mucker.”
“Alf, you’re a gentleman.”
“That’s a lie,” answered Alf, with dignity, subsiding on the window-
seat again and hugging his knees. “Where have you been, you old
brute?”
“You’d never guess,” replied Dan, with a laugh, as he backed to
the fireplace and held his hands to the warmth.
“Taking tea with Old Toby,” hazarded Alf. (Old Toby was school
vernacular for Dr. Tobias Hewitt, Principal.)
“Not as bad as that, Alf. I’ve been sliding around the river in two
inches of slush on what Roeder calls his ice yacht. Seen it? It looks
like somebody’s front gate with a leg-of-mutton sail stuck up on it.”
“Must have been fun in this weather,” laughed Alf.
“It wasn’t so bad until we went into a hole up near Flat Island and
had to work for half an hour pulling the silly thing out. I wanted to
let it stay there; told him it would float down when the ice thawed;
but he insisted on rescuing it.”
“You’re a crazy chump,” said Alf, viewing him, however, with
evident affection. Dan Vinton was tall and lithe and long-limbed,
with a wide-awake, alert appearance and an almost disconcerting
ability to think quickly and act in the same way. In age he was just
over sixteen, and he was a trifle large for his years. He had steady
brown eyes, brown hair, a short, straight nose, and a pleasant,
good-tempered mouth. Dan was a Second-Class fellow and had been
chosen football captain in the fall.
“I’d give a dollar for a nice cup of hot chocolate,” he announced.
“I’m hungry as a bear. Got anything to eat, you fellows?”
“Not a thing,” replied Alf. “I can’t keep grub here; Tom eats it all
up. Anyhow, eating between meals,” he added, virtuously, “is very
bad for the health.”
“It’s good for the tummy, though,” said Dan, crossing over and
seating himself at the other end of the window-seat. “Well, what’s
new?”
“New! Nothing’s new. Nothing has happened in this dead-and-alive
hole since—since the hockey game. I detest this time of year, don’t
you?”
“It is a bit dull, but I guess we’ll be outdoors in a few days. Gee,
but I’ll be glad to feel a baseball again!”
“Me too. We’ve been discussing the Track Team’s chances. Now
that Gerald has decided to come out for the mile it looks like a pretty
sure thing for Yardley.”
“Oh, you can make all the fun you want,” said Gerald, cheerfully.
“I’ll bet, though, that I’ll win just as many points as you will, Alf.”
“That’s a good safe wager,” observed Tom, lazily. “Of course, I’m
not saying Alf might not win a third some time if he could keep his
feet. But he always takes a header just before the tape, and tears up
the track. Gets an idea, I suppose, that the quickest way to get
there is to slide. Shows his baseball training.”
“Oh, run away! I never fell but once, you old chump!”
“That’s all Adam fell,” said Dan, “and see what happened to him!
By the way, did I tell you what Tom calls his ice-boat? The Planked
Steak.”
“Go ahead,” said Alf, “what’s the joke?”
“I asked him why he called it that and he said it was made of
planks, and the mast was the stake. Not bad, what?”
Alf groaned. “It sounds like one of Tom’s jokes. His sense of
humor is decidedly heavy.”
“My sense of hunger is decidedly strong,” said Tom. “And it’s five
minutes of six. Let’s go over. Want to wash up here, you two?”
 “Yes,” Dan answered, “though I feel as though I was pretty well
washed already. I’ll bet there isn’t a really dry spot about me.
Where’d you get this villainous soap, Tom?”
“Don’t ask me; that’s some of Alf’s. Doesn’t it smell fierce?”
“Awful! Where’d you find it, Alf?”
“That soap,” responded Alf, haughtily, “is the best made, and
extremely expensive. The delicious perfume which you mention and
can’t appreciate is lilac. That soap costs me two and a half cents a
cake, at Wallace’s.”
“Well, then, Wallace has at last got even for the glasses you broke
there once,” laughed Dan. “I’ve noticed an unpleasant atmosphere
about you for some time. Now it’s explained. All ready? Come on,
then; let’s eat!”
 CHAPTER II
THE S. P. M.

While our four friends are satisfying four very healthy appetites,
let’s look about us a little. The place is Wissining, Connecticut, and
Wissining, in case you happen not to be acquainted with it, is on the
Sound, about equidistant from New Haven and Newport. Perhaps
you can locate Greenburg better, for Greenburg is quite a city in a
small way, and something of a manufacturing town. Wissining lies
just across the river from Greenburg, and Yardley Hall School is
about a half-mile from the Wissining station. It may be that you
have never noticed it, even if you have traveled that way, for the
railroad passes through the Yardley property by way of a cut, and
the school buildings are not long in sight. But if you look sharp as
your train crosses the bridge over the little Wissining River, you will
see them describing a rough semicircle on the edge of a not distant
hill; Clarke, Whitson, Oxford, Merle, and the Kingdon Gymnasium.
Dudley you won’t see for the reason that it is situated back of the
other buildings and across the Yard. Oxford is a recitation hall; but,
besides class-rooms, it holds Dr. Hewitt’s apartments, the office, the
laboratories, the library, the assembly hall, and the rooms of the two
school societies, Oxford and Cambridge. The dining-room, or
commons, is in Whitson.
The school property consists of some forty acres of hill, woodland,
and meadow, and ascends gradually from the shore to the plateau
whereon the buildings are set, and then descends as gently to the
curving river at the back. Here are the tennis courts and the athletic
field, the golf links and the boat house; and here, near the river-
bank not long since, was the ice rink whereon Yardley defeated the
Broadwood hockey team and won the first leg of the Pennimore Cup,
the trophy presented by Gerald’s father.
Yardley usually holds two hundred and seventy students, their
ages ranging from twelve to twenty. There are five classes known as
First, Second, Third, Fourth, and Preparatory, and Yardley’s
graduates have a habit of going on to Yale for the rest of their
education, although there have been occasions when rash youths
have preferred Harvard. Broadwood, which is situated some four
miles distant as the crow flies, is a prominent feeder to Princeton,
and so rivalries begun at these schools are often nourished at
college. There have been other stories written about Yardley Hall,
and so if you want a more detailed description of the school you
have only to refer to a book called “Forward Pass,” though for my
part I think you already have heard enough about it to answer our
purpose. It’s a good school, is Yardley Hall; good in all ways; and,
which is more important, it turns out some fine fellows. If I had
space to set down a list of all the eminent government officials,
scientists, writers, jurists, diplomats and the like who have
graduated you would be vastly impressed. But I haven’t, and you
must just take my word for it. I might add that it has turned out a
large number of athletes who, if their renown has been more
fleeting, have won honor and acclaim.
There was a stereoptican lecture that night in Assembly Hall and,
after they had finished supper, Dan was all for hearing it. But Alf
refused to entertain the idea for a moment.
“It’s something about the Irish Lakes,” he said, “and no one cares
a fig for the Irish Lakes. It’s wet enough here to-night without
having to listen to a lot of drool about the Lake of Killarney and—and
the others. If the chap would lecture on Irish bulls I might go. No,
my soul craves excitement, Dan.”
“So does mine,” Dan laughed, “but I don’t know where to find it.
We might go up to Cambridge and watch Chambers and Rand play
backgammon.”
 “Awful thought! No, you come over to our room, Dan, and Tom
and I will entertain you. Bring little Geraldine along, if you like.”
“He’s gone off with Thompson. I’ll come over for awhile after the
lecture.”
“You won’t. You’ll be drowned in the Irish Lakes. Let the old
lecture go.” But Dan was obdurate. Alf called on Tom for aid.
“Tell him to come, Tom,” he said. “We’ll dance and sing and recite
poetry for him, won’t we?”
“Maybe you will,” was the calm response. “I’m going over to
Oxford for awhile. There’s a debate and a concert.”
Alf groaned.
“Another of your silly vaudevilles! All right, go ahead, both of you.
But you’ll be sorry when you come back and find that I’ve blown up
the building or assaulted a faculty from sheer boredom. You’ll wish
then that you’d been kind to me.”
They parted on the steps of Whitson, Dan and Tom scudding
across to Oxford, and Alf, hands in pockets and head drooping
dejectedly, walking off through the downpour toward Dudley. Dan
tried to persuade Tom to accompany him to the lecture, and Tom
strove to induce Dan to accept the hospitality of Oxford Society.
They argued it out at the head of each flight of stairs and consumed
some ten or fifteen minutes, and finally Tom tried to kidnap Dan by
main force in the upper corridor, and was severely reprimanded by
an usher for unseemly noise. The lecture was mildly interesting and
lasted the better part of an hour. At the back of the hall a group of
younger fellows, among whom was Gerald, found the darkened
room much to their liking and spent most of the time cutting-up. The
lecturer, a spare, nervous gentleman with a prominent Adam’s apple
and a very bald head, was visibly annoyed at times, and when one
of the pictures was thrown on the screen upside-down didn’t
discover the fact until the snickers of his audience appraised him
that something was wrong. After the entertainment was over Dan
met Gerald in the corridor and took him off to Alf’s room. They
scuttled over to Dudley through the rain and slush and found Alf
alone in his glory, his feet to the fire and a tablet and pencil in his
hands.
“Where’s Tom?” he asked. “I need him. Hello, Gerald. Fate, Mr.
Pennimore, has decreed that you be one of us. Your appearance, as
welcome as unexpected, decides the matter. I congratulate you.”
“What the dickens are you babbling about?” asked Dan, ruffling
Alf’s hair. “What’s the game?”
“You shall know in due time. I can’t explain it more than once, and
so we will await the arrival of Mr. Dyer, our respected colleague.
While you fellows have been wasting your valuable time in aimless
pleasures I have been working.” He held up a leaf from the tablet
scrawled upon on both sides.
“Is it poetry?” asked Gerald.
“Or an essay for The Scholiast?” suggested Dan.
“No, children, it is—But here comes Mr. Dyer. Welcome, Mr. Dyer.
Remove your coat and join our little home circle.”
“Alf’s got one of his silly fits,” said Dan. “Sit down, Tom, and let
him get it off his chest.”
Alf arose, turned his back to the fireplace, thrust one hand
between the buttons of his waistcoat and faced his audience
impressively. Dan and Tom cheered subduedly.
“Gentlemen,” began Alf. “(For the moment we will suppose that
you are gentlemen.) There is an adage which has it that Satan finds
some mischief still for idle hands to do. At this time of year when the
inclemency of the weather and—ah—lack of athletics deprive most
of us of occupation, leaving us little wherewith to interest ourselves
save degrading studies, it is especially desirable that our minds and
hands should be kept busy to the end that Satan shall not get in his
work with us. Let us keep out of mischief at all cost, say I.”
“Hooray!” applauded Dan. Alf bowed profoundly.
Welcome to our website – the perfect destination for book lovers and
knowledge seekers. We believe that every book holds a new world,
offering opportunities for learning, discovery, and personal growth.
That’s why we are dedicated to bringing you a diverse collection of
books, ranging from classic literature and specialized publications to
self-development guides and children's books.

More than just a book-buying platform, we strive to be a bridge

connecting you with timeless cultural and intellectual values. With an
elegant, user-friendly interface and a smart search system, you can
quickly find the books that best suit your interests. Additionally,
our special promotions and home delivery services help you save time
and fully enjoy the joy of reading.

Join us on a journey of knowledge exploration, passion nurturing, and

personal growth every day!

ebookbell.com