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Microarrays in Clinical Diagnostics 1st Edition Yilmaz
Niyaz Digital Instant Download
Author(s): Yilmaz Niyaz, Monika Stich, Bernd Sägmüller (auth.), Thomas
O. Joos PhD, Paolo Fortina MD, PhD (eds.)
ISBN(s): 9781592599233, 1592599230
Edition: 1
File Details: PDF, 5.09 MB
Year: 2005
Language: english
Microarrays in Clinical Diagnostics
M E T H O D S I N M O L E C U L A R M E D I C I N E™

John M. Walker, SERIES EDITOR

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M E T H O D S I N M O L E C U L A R M E D I C I N E™

Microarrays
in Clinical Diagnostics

Edited by

Thomas O. Joos, P hD
Biochemistry Department
NMI Natural and Medical Sciences Institute
at the University of Tuebingen, Reutlingen, Germany

Paolo Fortina, MD, PhD

Center for Translational Medicine, Department of Medicine
Jefferson Medical College, Philadelphia, PA
© 2005 Humana Press Inc.
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Cover design by Patricia F. Cleary.
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and Color Plate 7).
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eISBN 1-59259-923-0
ISSN 1543-1894

Library of Congress Cataloging-in-Publication Data

Microarrays in clinical diagnostics / edited by Thomas O. Joos, Paolo Fortina.
p. ; cm. -- (Methods in molecular medicine ; 114)
Includes bibliographical references and index.
ISBN 1-58829-394-7 (alk. paper)
1. DNA microarrays--Diagnostic use.
[DNLM: 1. Oligonucleotide Array Sequence Analysis. 2. Diagnostic
Techniques and Procedures. QZ 52 M6262 2005] I. Joos, Thomas. II. Fortina,
Paolo. III. Series.
RB43.8.D62M53 2005
616.07'58--dc22
2004026935
Preface
Within the last decade, microarray technology has evolved from an emerging
technology developed and used by a few laboratories into a well-established
technology used in laboratories all over the world. In fact, the need to
characterize genetic alterations is one of the highest priorities for the future of
medicine and the clinical management of disease. This technology allows the
rapid detection of point mutations, insertions or deletions, loss of hetero-
zygosity, and gene amplification, which constitute the major nucleic acid
variations associated with human disease. Additional disease-causing changes
may involve DNA methylation and microsatellite instability for which
automatable methods to detect instability in as few as 100 cells at multiple loci
are required. Furthermore, in some instances it may be necessary to detect one
tumor cell among a large number of normal cells, as well as to profile
differentially expressed genes. Eventually, the ultimate goals are to characterize
the entire genome rapidly and inexpensively, ideally using a single cell in order
to survey the whole genome for any nucleic acid variation.
Within the field of proteome research, microarray technology has been
adapted to the protein arena. Although DNA microarrays are quite popular and
in vogue, proteins (not genes) are the targets for drugs; therefore, there is an
increasing need to develop protein chips. Specifically, tools and methods are
needed for the identification and quantification of proteins, and for the study of
protein–protein interactions, enzyme–substrate interactions, and small-
molecule interactions. Enormous efforts have been undertaken to transfer
standard sandwich immunoassays in miniaturized and parallel formats to
analyze simultaneously the expression of a large number of proteins, e.g.,
serum or tumor biomarkers.
It is becoming clear that microarray technology is capable of fulfilling these
needs. Although some technologies are still confined to research laboratories,
such as those aimed at performing resequencing of known genes and protein
identification, rapid and robust methods are becoming available to address each
of these needs. However, some general goals for diagnostics including
sensitivity, specificity, high throughput, cost effectiveness, and turnaround time
still need improvement.
Finally, it is also clear that new tools in the nanoscale format are on the
horizon: quantum dots, nanoparticles, carbon nanotubes, and atomic force
microscopes are now being used to directly probe DNA structure. These

v
vi Preface

technologies represent an emerging approach promising increased throughput,

sensitivity, and sample processing, as well as facilitating single-cell and single-
molecule detection.
Microarrays in Clinical Diagnostics offers an overview of the world of
microarray technology. Because it is not clear which technology will eventually
prevail, we have tried to assemble a comprehensive survey of the varied
technologies now in use and to provide detailed methods sections in order to
support scientists who design and perform microarray experiments.
Thomas O. Joos, PhD
Paolo Fortina, MD, PhD
Contents

Preface .................................................................................................. v
Contributors ......................................................................................... ix
Color Plates ........................................................................................ xiii
1 Noncontact Laser Microdissection and Pressure Catapulting:
Sample Preparation for Genomic, Transcriptomic,
and Proteomic Analysis
Yilmaz Niyaz, Monika Stich, Bernd Sägmüller, Renate
Burgemeister, Gabriele Friedemann, Ulrich Sauer, Rainer
Gangnus, and Karin Schütze .................................................... 1
2 Applications of the Universal DNA Microarray
in Molecular Medicine
Reyna Favis, Norman P. Gerry, Yu-Wei Cheng,
and Francis Barany ................................................................. 25
3 Sensitive Detection of SARS Coronavirus RNA by a Novel
Asymmetric Multiplex Nested RT-PCR Amplification
Coupled With Oligonucleotide Microarray Hybridization
Zhi-wei Zhang, Yi-ming Zhou, Yan Zhang, Yong Guo,
Sheng-ce Tao, Ze Li, Qiong Zhang, and Jing Cheng ............. 59
4 Genotyping Single-Nucleotide Polymorphisms
by Minisequencing Using Tag Arrays
Lovisa Lovmar and Ann-Christine Syvänen .............................. 79
5 Single-Nucleotide Polymorphism and Mutation Identification
by the Nanogen Microelectronic Chip Technology
Maurizio Ferrari, Laura Cremonesi, Pierangelo Bonini,
Barbara Foglieni, and Stefania Stenirri ................................. 93
6 Molecular Diagnostic Testing for Inherited Thrombophilia
Using Invader®
Margaret A. Keller................................................................... 107
7 Controlled Agitation During Hybridization: Surface Acoustic
Waves Are Shaking Up Microarray Technology
Achim Wixforth ...................................................................... 121

vii
viii Contents

8 Rapid Screening for 31 Mutations and Polymorphisms

in the Cystic Fibrosis Transmembrane Regulator Gene
by Luminex® xMAP™ Suspension Array
Sherry A. Dunbar and James W. Jacobson ............................. 147
9 Protein Microarray-Based Screening of Antibody Specificity
Rhonda Bangham, Gregory A. Michaud, Barry Schweitzer,
and Paul F. Predki ............................................................... 173
10 Multiplexed Protein Analysis Using Antibody Microarrays
and Label-Based Detection
Brian B. Haab .......................................................................... 183
11 Allergen Microarrays
Tito Bacarese-Hamilton, Julian Gray, Andrea Ardizzoni,
and Andrea Crisanti ............................................................ 195
12 Enhanced Protein Profiling Arrays for Quantitative
Measurement of Protein Expression in Multiple Samples
Ruochun Huang, Qian Shi, Weimin Yang,
and Ruo-Pan Huang ............................................................ 209
13 Multiplexed Cytokine Sandwich Immunoassays:
Clinical Applications
Uma Prabhakar, Edward Eirikis, Bruce E. Miller,
and Hugh M. Davis ............................................................. 223
14 Validation and Quality Control of Protein Microarray-Based
Analytical Methods
Larry J. Kricka and Stephen R. Master .................................... 233
15 Tissue Microarrays
Ronald Simon, Martina Mirlacher, and Guido Sauter ............ 257
Index ................................................................................................. 269
Contributors

ANDREA ARDIZZONI • Department of Biological Sciences, Imperial College

London, London, UK
TITO BACARESE-HAMILTON • Department of Biological Sciences, Imperial
College London, London, UK
RHONDA BANGHAM • Research and Development, Protometrix Inc.,
Branford, CT
FRANCIS BARANY • Department of Microbiology and Immunology, Weill
Medical College of Cornell University, New York, NY
PIERANGELO BONINI • Diagnostica e Ricerca San Raffaele S.p.A., Università
Vita-Salute S. Raffaele, Milan, Italy
RENATE BURGEMEISTER • P.A.L.M. Microlaser Technologies AG, Bernried,
Germany
JING CHENG • State Key Laboratory of Biomembrane and Membrane
Biotechnology, Department of Biological Sciences and Biotechnology,
Tsinghua University; National Engineering Research Center for Beijing
Biochip Technology; CapitalBio Corporation, Beijing, China
YU-WEI CHENG • Department of Microbiology and Immunology, Weill
Medical College of Cornell University, New York, NY
LAURA CREMONESI • Unit of Genomics for Diagnosis of Human Pathologies,
Istituto di Ricovero e Cura a Carattere Scientifico Ospedale San
Raffaele, Milan, Italy
ANDREA CRISANTI • Department of Biological Sciences, Imperial College
London, London, UK
HUGH M. DAVIS • Department of Clinical Pharmacology, Centocor Inc.,
Malvern, PA
SHERRY A. DUNBAR • Research and Development, Luminex Corporation,
Austin, TX
EDWARD EIRIKIS • Department of Clinical Pharmacology, Centocor Inc.,
Malvern, PA
REYNA FAVIS • Department of Microbiology and Immunology, Weill Medical
College of Cornell University, New York, NY
MAURIZIO FERRARI • Unit of Genomics for Diagnosis of Human Pathologies,
Istituto di Ricovero e Cura a Carattere Scientifico Ospedale San
Raffaele; Diagnostica e Ricerca San Raffaele S.p.A, Milan, Italy

ix
x Contributors

BARBARA FOGLIENI • Unit of Genomics for Diagnosis of Human Pathologies,

Istituto di Ricovero e Cura a Carattere Scientifico Ospedale San
Raffaele, Milan, Italy
PAOLO FORTINA • Center for Translational Medicine, Department
of Medicine, Jefferson Medical College, Thomas Jefferson University,
Philadelphia, PA
GABRIELE FRIEDEMANN • P.A.L.M. Microlaser Technologies AG, Bernried,
Germany
RAINER GANGNUS • P.A.L.M. Microlaser Technologies AG, Bernried,
Germany
NORMAN P. GERRY • Department of Genetics and Genomics, Boston
University School of Medicine, Boston, MA
JULIAN GRAY • Department of Biological Sciences, Imperial College London,
London, UK
YONG GUO • State Key Laboratory of Biomembrane and Membrane
Biotechnology, Department of Biological Sciences and Biotechnology,
Tsinghua University, Beijing, China
BRIAN B. HAAB • Laboratory of Cancer Immunodiagnostics, Van Andel
Research Institute, Grand Rapids, MI
RUOCHUN HUANG • Department of Gynecology and Obstetrics, Emory
University School of Medicine, Atlanta, GA
RUO-PAN HUANG • Department of Gynecology and Obstetrics, Emory
University School of Medicine, Atlanta, GA
JAMES W. JACOBSON • Research and Development, Luminex Corporation,
Austin, TX
THOMAS O. JOOS • Head, Biochemistry Department, NMI Natural and Medical
Sciences Institute at the University of Tuebingen, Reutlingen, Germany
MARGARET A. KELLER • Cardeza Special Hemostasis Laboratory, Cardeza
Foundation for Hematologic Research and Division of Hematology,
Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA
LARRY J. KRICKA • Department of Pathology and Laboratory Medicine,
University of Pennsylvania, Philadelphia, PA
ZE LI • National Engineering Research Center for Beijing Biochip
Technology; CapitalBio Corporation, Beijing, China
LOVISA LOVMAR • Department of Medical Sciences, Uppsala University,
Uppsala, Sweden
STEPHEN R. MASTER • Department of Pathology and Laboratory Medicine,
University of Pennsylvania, Philadelphia, PA
GREGORY A. MICHAUD • Research and Development, Protometrix Inc.,
Branford, CT
Contributors xi

BRUCE E. MILLER • Department of Clinical Pharmacology, Centocor Inc.,

Malvern, PA
MARTINA MIRLACHER • Department of Pathology, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany
YILMAZ NIYAZ • P.A.L.M. Microlaser Technologies AG, Bernried, Germany
UMA PRABHAKAR • Department of Clinical Pharmacology, Centocor Inc.,
Malvern, PA
PAUL F. PREDKI • Research and Development, Protometrix Inc., Branford, CT
BERND SÄGMÜLLER • P.A.L.M. Microlaser Technologies AG, Bernried, Germany
ULRICH SAUER • P.A.L.M. Microlaser Technologies AG, Bernried, Germany
GUIDO SAUTER • Department of Pathology, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany
KARIN SCHÜTZE • P.A.L.M. Microlaser Technologies AG, Bernried, Germany
BARRY SCHWEITZER • Research and Development, Protometrix Inc.,
Branford, CT
QIAN SHI • Department of Gynecology and Obstetrics, Emory University
School of Medicine, Atlanta, GA
RONALD SIMON • Department of Pathology, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany
STEFANIA STENIRRI • Unit of Genomics for Diagnosis of Human Pathologies,
Istituto di Ricovero e Cura a Carattere Scientifico Ospedale San
Raffaele, Milan, Italy
MONIKA STICH • P.A.L.M. Microlaser Technologies AG, Bernried, Germany
ANN-CHRISTINE SYVÄNEN • Department of Medical Sciences, Uppsala
University, Uppsala, Sweden
SHENG-CE TAO • State Key Laboratory of Biomembrane and Membrane
Biotechnology, Department of Biological Sciences and Biotechnology,
Tsinghua University, Beijing, China
ACHIM WIXFORTH • Chair for Experimental Physics I, University of Augsburg,
Augsburg, Germany
WEIMIN YANG • Department of Gynecology and Obstetrics, Emory
University School of Medicine, Atlanta, GA
QIONG ZHANG • National Engineering Research Center for Beijing Biochip
Technology; CapitalBio Corporation, Beijing, China
YAN ZHANG • State Key Laboratory of Biomembrane and Membrane
Biotechnology, Department of Biological Sciences and Biotechnology,
Tsinghua University, Beijing, China
ZHI-WEI ZHANG • State Key Laboratory of Biomembrane and Membrane
Biotechnology, Department of Biological Sciences and Biotechnology,
Tsinghua University, Beijing, China
xii Contributors

YI-MING ZHOU • State Key Laboratory of Biomembrane and Membrane

Biotechnology, Department of Biological Sciences and Biotechnology,
Tsinghua University, Beijing, China
Color Plates

Color plates 1–14 follow p. 18 and plates 15–19 follow p. 178.

COLOR PLATE 1 The PALM® MicroBeam-HT (Chapter 1, Fig. 2, see pp. 3, 4).
COLOR PLATE 2 Scheme for laser ablation (Chapter 1, Fig. 3, see pp. 3, 4).
COLOR PLATE 3 Peak absorption wavelengths; absorption maxima of DNA
and proteins (Chapter 1, Fig. 4, see p. 5).
®
COLOR PLATE 4 PALM RoboMover and RoboSoftware (Chapter 1, Fig. 5,
see pp. 6, 7).
COLOR PLATE 5 MicroBeam-HT: automatic recognition of defined areas of
samples (Chapter 1, Fig. 6A, see pp. 8, 9).
COLOR PLATE 6 MicroBeam-HT: automatic detection of labeled samples
(Chapter 1, Fig. 6B, see pp. 8, 9).
COLOR PLATE 7 LMPC of cultured HepG2 cells in a PALM DuplexDish
(Chapter 1, Fig. 7, see pp. 16, 17).
COLOR PLATE 8 Overview of target amplification, probe amplification, sig-
nal amplification, and arrays used in molecular diagnostics
(Chapter 2, Fig. 1, see pp. 26–29).
COLOR PLATE 9 Schematic diagram and results for the detection of multiple
mutations using PCR/PCR/LDR/Universal Array (Chapter
2, Fig. 2, see pp. 29–31).
COLOR PLATE 10 Schematic diagram for the detection of multiple single-
nucleotide polymorphisms using LDR/PCR/Universal Ar-
ray (Chapter 2, Fig. 3, see pp. 33, 34).
COLOR PLATE 11 Electrophoretogram of 30-plex detection in genomic DNA
(Chapter 2, Fig. 4, see pp. 33, 35).
COLOR PLATE 12 Ligase-based multiplexed single-nucleotide polymorphism
assays combined with zip-code universal display (Chapter
2, Fig. 5, see pp. 36–38).
COLOR PLATE 13 Validation of arrays following spotting (Chapter 2, Fig. 6,
see pp. 41, 42).

xiii
xiv Color Plates

COLOR PLATE 14 Schematic of PCR-PCR/LDR assay (Chapter 2, Fig. 7, see

pp. 45, 46).
COLOR PLATE 15 Universal array images of methylation profiles of selected
promoter regions in normal and colorectal tumor cell line
genomic DNAs (Chapter 2, Fig. 8, see pp. 48, 49).
COLOR PLATE 16 Fluorescent image of the yeast ProtoArrayTM (Chapter 9,
Fig. 1, see p. 174).
COLOR PLATE 17 Fluorescent images of antibody probing of the yeast prote-
ome microarray (Chapter 9, Fig. 2, see p. 177).
COLOR PLATE 18 Schematic array format and fluorescent scan for allergen
investigation (Chapter 11, Fig. 1, see pp. 201, 202).
COLOR PLATE 19 Clustering analysis of angiogenic factors measured by
enhanced protein profiling arrays between normal subjects
and patients with gynecological diseases (Chapter 12,
Fig. 3, see p. 219).
C OLOR PLATE 20 Tissue microarray manufacturing and applications
(Chapter 15, Fig. 2, see pp. 264, 265).
1
Noncontact Laser Microdissection
and Pressure Catapulting
Sample Preparation for Genomic, Transcriptomic,
and Proteomic Analysis

Yilmaz Niyaz, Monika Stich, Bernd Sägmüller, Renate Burgemeister,

Gabriele Friedemann, Ulrich Sauer, Rainer Gangnus, and Karin Schütze

Summary
The understanding of the molecular mechanisms of cellular metabolism and proliferation
necessitates accurate identification, isolation, and finally characterization of a specific cell or a
population of cells and subsequently their subsets of biomolecules.
For the simultaneous analysis of thousands of molecular parameters within a single experi-
ment, as realized by DNA, RNA, and protein microarray technologies, a defined number of
homogeneous cells derived from a distinct morphological origin is required. Sample preparation
is therefore a very crucial step for high-resolution downstream applications.
Laser microdissection and laser pressure catapulting (LMPC) enables such pure and homoge-
neous sample preparation, resulting in an eminent increase in the specificity of molecular analyses.
For microdissection, the force of focused laser light is used to excise selected cells or large tissue
areas from object slides or from living cell culture down to a resolution of individual single cells and
subcellular components like organelles or chromosomes, respectively. After microdissection this
sample is directly catapulted into an appropriate collection device. As the entire process works with-
out any mechanical contact, it enables pure sample retrieval from morphologically defined origin
without cross contamination. Wherever homogenous samples are required for subsequent analysis
of, e.g., cell areas, single cells, or chromosomes, the PALM® MicroBeam system is an indispensable
tool. The integration of image analysis platforms fully automates screening, identification, and
finally subsequent high-throughput sample handling. These samples can be directly linked into ver-
satile downstream applications, such as single-cell mRNA-extraction, different PCR methods,
microarray techniques, and many others. Acceleration in sample generation vastly increases the
throughput in molecular laboratories and leads to an increasing knowledge about differentially
regulated mRNAs and expressed proteins, providing new insights into cellular mechanisms and
therefore enabling the development of systems for tumor biomarker identification, early detection
of disease-causing alterations, therapeutic targeting and/or patient-tailored therapy.

From: Methods in Molecular Medicine, Vol. 144, Microarrays in Clinical Diagnostics

Edited by: T. Joos and P. Fortina © Humana Press Inc., Totowa, NJ

1
2 Niyaz et al.

Key Words: Laser microdissection and pressure catapulting; high-resolution sample preparation;
single cell analysis; microarray technology.

1. Introduction
Histopathological tissue sections are typically composed of a variety of
different cell types in a complex 3D architecture. Each of these cell types
shows a different expression pattern of mRNA and protein within their tissue
context. Modern biomedical research is usually interested in the molecular
expression profile of a distinct cell type within the specimen. Such studies
rely on pure sample preparation, as the cells of interest constitute only a small
proportion of the specimen and therefore have to be separated from other
compounds of the tissue. Among various options for achieving homogeneous
samples, only laser microdissection offers high-resolution control of sample
composition by selecting or rejecting individual cells and tissue areas of
interest, respectively. Therefore, this ability to manipulate cells individually
has made a great impact on our ongoing understanding of cellular physiology
and molecular pathology by means of genomic, transcriptomic, and pro-
teomic research (1–3). Recent technical advances have now opened new hori-
zons for cellular investigations.
The PALM® MicroBeam (P.A.L.M. Microlaser Technologies, Bernried,
Germany, http://www.palm-microlaser.com) allows completely noncontact
sample preparation by the combined applications of laser microdissection and
pressure catapulting (LMPC). In brief, a pulsed UV-A laser is coupled to a
microscope and focused on the sample plane. Thus the microscope, known as
an optoanalytical device, turns into a versatile micromanipulation tool: selected
specimens of different origins can first be laser microdissected and then ejected
against gravity. This patented laser pressure catapulting (LPC) technology
drives the sample from the objective plane toward a collection device, e.g., a
standard microfuge cap, solely by a laser-induced transportation process. Thus
the PALM micromanipulation system has neither physical nor mechanical con-
tact with the specimen, and the risk of contamination of the isolated samples is
minimized. Moreover, this technology is a paramount prerequisite for homoge-
neous sample capture as the extracted samples are traceable from a morpholog-
ically defined origin. These samples can be used in a variety of downstream
applications (Fig. 1 and Color Plate 1 following p.18.), e.g., genome-wide
expression profiling using cDNA or protein microarrays (4–7).
The unique combination of noncontact microsurgery and sample preparation
(from subcellular compounds to entire tissue areas) is performed in numerous
research institutes or industrial laboratories throughout the world (cf. http://
www.palm-microlaser.com/aboutus/PALM-Referenzen.pdf).
Noncontact Laser Microdissection and Pressure Catapulting 3

Fig. 1. Workflow for LMPC Applications. Abbreviations: FISH, fluorescence in

situ hybridization; HE, Hematoxylin & Eosin; LMPC, laser microdissection and pres-
sure catapulting; MALDI-TOF; MS, matrix-assisted laser desorption ionization time-of-
flight mass spectrometry; nLC-MS, non-flow liquid chromatography mass spectrometry
with ion trapping; PCR, quantitative polymerase chain reaction; RT, reverse transcreptase;
SELDI, surface-entranced laser desorption ionization. (See Color Plate 1 following p.18.)

1.1. The Force of Focal Light

1.1.1. Laser Microdissection
The PALM MicroBeam (Fig. 2; see Color Plate 2 following p.18.) is equipped
with a UV-A laser coupled through the epifluorescence path to an inverted
research microscope and focused on a micron-sized spot on the sample via the
objective lenses (Fig. 3; See Color Plate 3 following p.18.). The beam focus diam-
eter results mainly from the wavelength and beam quality of the laser, the magnifi-
cation and numerical aperture (NA) of the applied objective, and the specimen’s
absorbance behavior. Laser microdissection is possible with several objective
magnifications from 5 to 100×. Higher aperture objectives, e.g., a 100× oil immer-
sion objective (NA = 1.3), are necessary for a minimum cutting size of less than
700 nm (8), allowing microdissection and microsurgery of single nuclei, filaments,
chromosomes, or even chromosomal parts (9). For best focusing results, a laser of
high beam quality and an objective with an NA >1 is required.
4 Niyaz et al.

Fig. 2. The PALM® MicroBeam-HT. (See Color Plate 2 following p.18.)

Fig. 3. Scheme of laser ablation. A high-intensity beam of single wavelength radiation

(laser) is focused through the objective. Depending on the numerical aperture of the objec-
tive, laser focus diameters of less than 1 μm can be achieved. Within this spot, material
becomes ablated owing to a photofragmentation process. (See Color Plate 3 following p.18.)
Noncontact Laser Microdissection and Pressure Catapulting 5

Fig. 4. Peak absorption wavelengths. Absorption maxima of DNA and proteins. The
wavelengths of the laser used lie outside the local absorption maxima of these
biomolecules and thus affect neither biomolecular information nor the viability of the
microdissected specimen. (See Color Plate 3 following p.18.)

The pulsed UV-A laser beam used for LMPC is routinely focused to <1
μm, at which point it impacts the sample with high energy density (≤10
MW/cm2). This condensed radiation within the focal spot leads to the forma-
tion of a high energetic microplasma, in which the energy transfer is suffi-
cient to break the molecule bonds, resulting in photofragmentation of the
radiated matter without any mechanical contact. Thus, at the focal point,
unwanted material is disintegrated into atoms and small molecules, a not
fully understood phenomenon called ablative photo-decomposition. How-
ever, as this cutting is a fast photochemical process devoid of heat transfer,
adjacent material out of the focus is not affected (10,11). The nonfocused laser
light next to the focus becomes scattered and travels through adjacent areas
without any impact on the specimen. Because of the reduced photon density
and because it is out of range of the peak absorption wavelengths for DNA,
RNA, or proteins (Fig. 4; See Color Plate 3 following p.18.), this radiation
does not interfere with biological material (12,13). Therefore biomolecules
can routinely be isolated from the specimen for downstream analyses and
applications (14), and even living cells can be captured (15,16). The viability
of the treated cells is not constrained, as they readily enter the cell cycle and
6 Niyaz et al.

Fig. 5. PALM® RoboMover PALM® RoboSoftware. The RoboMover and the

graphical user interface of the RoboSoftware. (See Color Plate 4 following p.18.)

proliferate after laser treatment (16,17). As the effective laser energy is con-
centrated at the focal spot only, it is even possible to perform laser micro-
surgery or microinjection within living specimens. Numerous publications in
the field of cell and developmental biology or from assisted human fertiliza-
tion procedures have proved the safety of laser-based microsurgery and
microdissection (18–27). This technology has also been established in medical
laser surgery as the only nonheating surgical process.
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