CHAPTER

Correlation of the Same

Fields Imaged in the TEM,
Confocal, LM, and MicroCT
by Image Registration: From
18
Specimen Preparation
to Displaying a Final
Composite Image
Douglas R. Keene*, Sara F. Tufa*, Melissa H. Wong{, Nicholas R. Smith{,
Lynn Y. Sakai*, William A. Horton*
*Research Center, Shriners Hospital for Children, Portland, Oregon, USA
{
Department of Cell and Developmental Biology, Oregon Health Sciences University,
Portland, Oregon, USA

CHAPTER OUTLINE
Introduction ............................................................................................................ 392
18.1 Methods ........................................................................................................399
18.1.1 General Discussion .................................................................... 399
18.1.2 Sample Preparation ................................................................... 403
18.1.3 En Bloc GFP Localization in Cultured Cells and Tissues................ 404
18.1.4 Embedding Cultures and Tissues for Postembedding,
Section-Surface Labeling ........................................................... 406
18.1.5 Immunolabeling the Surface of Ultrathin Sections........................ 407
18.1.6 Ultramicrotomy and Observation of Semi-Thin and Ultrathin
Sections Using Confocal Or Epifluorescent Imaging...................... 407
18.1.7 Registration of LM and EM Images ............................................. 408
18.1.8 Workflow for Overlaying Images Using Fiji (ImageJ Version 1.48o). 409
18.1.8.1 Materials .......................................................................... 413
References ............................................................................................................. 414
Further Reading ...................................................................................................... 417

Methods in Cell Biology, Volume 124, ISSN 0091-679X, http://dx.doi.org/10.1016/B978-0-12-801075-4.00018-5

© 2014 Elsevier Inc. All rights reserved.
391
392 CHAPTER 18 Registration of Image Fields for Correlative Microscopy

Abstract
Correlated imaging is the process of imaging a specimen with two complementary modalities
and then registering and overlaying the fields obtained in each modality to create a composite
view. One of the images is made somewhat transparent, allowing detail in the underlying image
to be visible and assisting in the registration of the two images. As an example, an image local-
izing a specific tissue component by fluorescence may be overlaid atop a TEM image of the
same field. The resulting composite image would demonstrate specific ultrastructural features
in the high-resolution TEM field, which are colorized in the overlay. Other examples include
composites from MicroCT or soft X-ray images overlaid atop light microscopy or TEM images.
Automated image registration may be facilitated by a variety of sophisticated computer pro-
grams utilized by high-throughput laboratories. This chapter is meant for the more occasional
user wishing to align images manually. ImageJ is a public domain, image processing program
developed at the National Institutes of Health and is available to anyone as a free download.
ImageJ performs marvelously well for the purpose of image registration; therefore, step-by-step
instructions are included here. Specimen handling, including fixation and choice of embedding
media, is not straightforward for correlative imaging. A step-by-step description of the protocols
which work in our laboratory is included for simultaneous localization in LM, EM and micro-
CT, as well as maintaining GFP emission in tissue embedded for TEM.

INTRODUCTION
No single imaging technology can reveal all the structural details within a biological
specimen, although the resolution gap between two of the more common methods,
light microscopy (LM) and electron microscopy, is certainly narrowing with the in-
troduction of “super resolution” microscopes. Built upon a platform of conventional
fluorescent microscopy, these technologies allow the positional mapping of light
emitted from a sample, but with nanometer resolution. Still, the images produced
by these methods lack structural detail; it is only by correlation with additional im-
aging methods that a specific region emitting fluorescence may be identified. This
correlation is dependent on recognizing common features in images collected with
each instrument. The aim of this chapter is to suggest specimen preparation and im-
aging tools aiding in the registration of two or more image fields to produce a single
aligned composite image.
The need to correlate LM images with higher magnification transmission electron
microscopy (TEM) images has long been recognized by microscopists. Deemed
“CLEM” (correlative light and electron microscopy), a brief PubMed search yields
many examples of the methodology. From the standpoint of aligning images and pro-
ducing an image overlay from two different modalities, correlating images from the
same section observed by fluorescence microscopy (FM) and TEM is straight for-
ward. Sims and Hardin (2005) presented a method to overlay laser scanning confocal
microscopy (LSCM) images of a GFP construct localizing AJM-1 to the apical junc-
tions of Caenorhabditis elegans embryos with TEM images collected from the same
ultrathin sections. Using Adobe Photoshop, the resulting images were aligned and
 Introduction 393

FIGURE 18.1
Caenorhabditis elegans embryo expressing GFP fusion protein. An LSCM image is
overlaid with a TEM image collected from the same grid to produce an integrated image.
Reprinted with permission from Sims and Hardin (2007).

overlaid to produce a “fluorescence-integrated TEM” image (Fig. 18.1). Describing

further refinements in methodology, Sims and Hardin (2007) published a step-by-
step sequence of their protocol. Building upon this work, our laboratory (Keene,
Tufa, Lunstrum, Holden, & Horton, 2008) developed an alternative protocol for sta-
bilizing GFP- and YFP-expressing constructs in cell culture systems. We stabilized
the cells in ice-cold 4% paraformaldehyde/1% glutaraldehyde for 30 min followed
by progressive lowering temperature (PLT) dehydration (Newman & Hobot, 1987)
to 90% EtOH, followed by embedding in London Resin White (LR White) media.
Adequately bright images were obtained in our Leica SPS SP2 laser scanning con-
focal system from 0.5-mm sections; however, GFP and YFP emission was not of suf-
ficient intensity to image in ultrathin sections. We therefore collected fluorescent
images from 0.5-mm thick sections and TEM images from adjacent ultrathin sections.
The registration of images was complicated by the fact that the sectioning artifacts in
0.5-mm sections mounted on glass are not consistent with sectioning artifacts in
80-nm sections mounted on formvar-coated grids; therefore, the two image sets
could not be registered without the need to “skew” or distort one image relative
to the other. Despite these challenges, the resulting composite images clearly dem-
onstrated localization of GFP and YFP constructs at the resolution of the electron
microscope (Fig. 18.2). As an aid in initial field alignment, we routinely adjusted
the brightness and contrast of the FM image to visualize cell silhouettes as these were
easily identified in low-magnification TEM images. This important step allowed
394 CHAPTER 18 Registration of Image Fields for Correlative Microscopy

FIGURE 18.2
An LSCM image from a 0.5-mm section was overlaid with a TEM image from a serial
ultrathin section to produce this composite overlay of a human chondrocyte expressing
YFP::mCOMP.
From Keene et al. (2008).

rotational alignment so that the two images were in the same axial plane. Additional
features common to both imaging modalities could be recognized at progressively
higher magnifications.
As an initial evaluation of fluorescence emission intensity, imaging the block
face of an embedded sample is often a valued starting point. As an example, only
a fraction of the cultured cells shown in Fig. 18.2 expressed the YFP::mCOMP con-
struct. Areas within intact samples could be selected by placing the block surface
facedown on a large cover slip and imaged by FM or LSCM using the 20
objective
lens of an inverted microscope (Fig. 18.3). Similarly, using a 63
water dipping ob-
jective lens, Bell, Mitchell, Paultre, Posch, and Oparka (2013) were able to detect
fluorescent proteins in optical sections up to 40 mm below the surface of the block
face in LR White-embedded plant tissue. With the knowledge that a feature of inter-
est could be mapped via optical coordinates within a “z” stack relative to the block
face, the position within the block from which 0.5-mm sections would be cut and lat-
ter overlaid with TEM images could be determined with precision and correlated
back to the intact sample.
Correlation between other imaging modalities, such as LM or TEM with micro-
computed X-ray tomography (micro-CT), may be similarly accomplished. Micro-CT
is conventionally used to generate high-resolution 3D images for the nondestructive
evaluation of mineralized tissues such as insect exoskeletons, shells, and skeletal tis-
sues (Neues & Epple, 2008; Oest, Jones, Hatfield, & Prater, 2008). It was not until the
utilization of stains such as inorganic iodine, phosphotungstic acid, and osmium
(compared by Metscher, 2009a) that micro-CT use became widespread for the
 Introduction 395

FIGURE 18.3
The “faced” block of an intact sample may be imaged to select a field for further sampling.

evaluation of soft tissues. The critical elements in these stains have high atomic num-
bers (Z) with correspondingly high X-ray attenuations (Henke, Gullikson, & Davis,
1993) favorable to micro-CT imaging (iodine Z ¼ 53; osmium Z ¼ 76; lead Z ¼ 82,
and uranium Z ¼ 92). With the exception of iodine, these elements have been long
favored as contrasting stains in TEM. Their use as contrast agents for micro-CT al-
lows high-volume images of intact organisms. Brown fat and red blood cells are par-
ticularly well contrasted with iodine allowing analysis of blood-containing vascular
anatomy. Muscle fiber architecture may also be evaluated nondestructively with io-
dine staining (Jeffery, Stephenson, Gallagher, Jarvis, & Cox, 2010). Osmium is rec-
ognized to differentially stain lipids within white and brown adipose tissue, also
contrasting the lipoproteins of most bilayered cell and organelle membranes
(Hayat, 1970). This broad-staining characteristic allows detailed imaging of both ex-
ternal and internal regions of the embryos and organ systems (Johnson, Hansen, Wu,
Healy, & Johnson, 2006). Using this technology, we (Sengle, Tufa, Sakai,
Zulliger, & Keene, 2013) proposed to evaluate the fat content of mice harboring a
mutation in the connective tissue component fibrillin-2. We prepared intact mouse
limbs as if for TEM and then mapped small fat deposits in x, y, and z relative to the
block surface by micro-CT, without physically cutting into the sample. Following
identification of fat deposits in the micro-CT, we used an ultramicrotome to accu-
rately remove tissue sections so that the deposits might be precisely approached.
Similar to the method of overlaying fluorescent and TEM images, micro-CT images
were registered and overlaid atop LM images from half-micron sections (Fig. 18.4).
To determine if the deposits were white or brown fat, micro-CT images were overlaid
onto TEM images collected from ultrathin sections (Fig. 18.5). Further exemplifying
micro-CT as a complementary method to LM and TEM, Handschuh, Baeumler,
Schwaha, & Ruthensteiner (2013) imaged the fixed and embedded mollusk Mytilus
396 CHAPTER 18 Registration of Image Fields for Correlative Microscopy

FIGURE 18.4
A virtual slice of a region of high X-ray attenuation (B) is selected from the micro-CT data set,
then registered with an LM image (A) mapped to the same region. The regions of high
X-ray attenuation correlate to adipose tissue (C).
From Sengle et al. (2013).
 Introduction 397

FIGURE 18.5
A virtual slice within a micro-CT data set is overlaid with a TEM image of the same region.
From Sengle et al. (2013).

galloprovincialis by micro-CT. Serial 0.5-mm LM sections (n ¼ 585) were then cut

through the entire organism. The micro-CT dataset was visualized by volume ren-
dering using AMIRA. The renopericardial system, the nervous system, the digestive
system, muscles, and the gill vessels were segmented. Coregistration of the micro-
CT and LM images was accomplished by “affine registration” in AMIRA. The ca-
pabilities of this method are impressive, as any plane within the volume of the or-
ganism may be segmented out, with corresponding LM images selected within
AMIRA. Any one of the sections collected for LM can be remounted and cut for
TEM, allowing visualization of virtually any chosen region within the organism
by high-resolution microscopy. The method is particularly valuable, as it may be ap-
plied to the evaluation of archived TEM sample blocks. Similarly, Smith et al. (2014)
correlated structural data from soft X-ray tomography of intact, high-pressure frozen
Schizosaccharomyces pombe cells with internal localization of a GFP construct col-
lected using a cryorotation stage fitted within an LSCM system.
The recognition of the same structures in the LM and EM is often not trivial, and
one must rely on additional markers to coregister image sets. As demonstrated by
Micheva, Busse, Weiler, O’Rourke, and Smith (2010), DAPI images collected in
the epifluorescent microscope may be aligned with condensed heterochromatin im-
aged in the TEM. Yuan, Li, Hong, and Hong (2014) aligned IF and TEM images to
localize a GFP::mitochondrial fusion protein on the surface of semi-thin sections pre-
pared from high-pressure frozen, Lowicryl HM20 embedded tissue samples.
A secondary antibody conjugate with both a fluorescent and an ultrasmall
(0.6-nm) gold component (nanogold) was utilized. The registered overlays of IF
and TEM images demonstrated that localization was within fields of mitochondria;
398 CHAPTER 18 Registration of Image Fields for Correlative Microscopy

however, it was the immunogold that resolved the localization to the outer membrane
of the mitochondria. The method was complicated only by the need to “enhance” the
ultrasmall gold particles to be large enough to visualize in the TEM. Molecular
Probes now markets a variety of secondary antibody conjugates with fluorescent
and large (6 or 10 nm) gold components, eliminating the need for enhancement.
We have used these conjugates to successfully localize lysozyme to RER and Paneth
cell granules on the surface of sections from mouse small intestine embedded in LR
White (Fig. 18.6). A detailed protocol for specimen preparation, imaging, and align-
ment leading to this composite image is presented later in Section 18.1.
In our laboratory, an often employed strategy to aid in the initial recognition of
features in both the IF or LSCM and TEM relies on mounting ultrathin sections on
1
2-mm single-hole, formvar-coated grids so that the entire section fits within the
grid window. We shape the block face so that it is nonsymmetrical, allowing map-
ping of an ROI relative to a distinctive edge recognizable in both imaging modes.
Sections may also be mounted on “finder” grids. This initial mapping saves time
in locating a single cell among many others. Should the fate of an individual live
cultured cell be followed, for example after microinjection, the cells may be grown
on cover slips that are photoetched so that markings visible and recorded in the LM
remain in the ultrathin section (Reddick & Alto, 2012), or cells may be grown on
aclar films having recognizable patterns etched into them using the pulsed laser
of a microdissection microscope (Spiegelhalter et al., 2010).
A method which results in detail immediately apparent in both imaging modal-
ities involves the use of a “miniSOG,” a fluorescent protein which may be genetically
tagged to virtually any protein. When illuminated by blue light in fixed tissue, photo-
generated singlet oxygen locally polymerizes diaminobenzidine resulting in a

FIGURE 18.6
Molecular Probes Alexa 488/10-nm gold secondary conjugate allows simultaneous IF and
TEM localization of the same tissue component.
 18.1 Methods 399

precipitate that can be readily visualized in the electron microscope (Ellisman,

Deerinck, Shu, & Sosinsky, 2012; Shu et al., 2011). Image alignment between the
two modalities is complicated only since initial illumination is done prior to osmica-
tion and embedding, and EM is done on ultrathin sections collected from the bulkier
sample.
At higher magnifications, the use of dedicated correlation markers (fiducial
markers) visible in the different imaging modalities is particularly useful for image
registration. Schellenberger et al. (2014) registered cryo-FM and cryo-EM fields
according to finder-grid marks and gross sample features, then utilized TetraSpeck
(Invitrogen) 200-nm diameter multichannel fluorescent microspheres to monitor
shifts between multicolor fluorescent image channels. These particles were also vis-
ible in phase-contrast cryo-EM projection images and could be used to assign coor-
dinates between LM and EM to automatously record tomograms of structures
initially identified by cryo-FM. Kukulski et al. (2012) describe a similar method
in tissues prepared by HPF, freeze substitution, and embedding in Lowicryl.
Kopek, Shtengel, Grimm, Clayton, and Hess (2013) utilized photoactivated locali-
zation microscopy (PALM) and later interferometric PALM (iPALM) (Kopek,
Shtengel, Xu, Clayton, & Hess, 2012), to fantastically localize proteins within the
submitochondrial structure known as the nucleoid. iPalm localizes photoactivatable
fluorescent proteins in the image plain to approximately 20-nm resolution, and fur-
ther extracts the z position to approximately 10-nm resolution. Tokuyasu cryosec-
tions were prepared from fixed cell cultures supported on a cover glass coated
with 80-nm gold particles; then, the sections were then stained, covered with cyano-
acrylate, and imaged by FIB-SEM. The 80-nm gold particles were visible in both
image modalities, and together with a precise, automated registration program
(POLYWARP 1 image transformation from the IDL image processing language,
http://www.exclisvis.com/docs/POLYWARP.html), the average registration error
approximated only 5-nm (Fig. 18.7). The use of these 80-nm dedicated fiducial
markers was absolutely critical to the registration of images and to precise localiza-
tion within this mitochondrial domain.

18.1 METHODS
18.1.1 GENERAL DISCUSSION
For the preservation of native structure, the method of choice for the immobilization
of tissue components begins with cryostabilization by high-pressure freezing
(reviewed by McDonald, 2009). A significant advantage to freezing is that a biolog-
ical event can be followed using the live stage of an LM, then immobilized by fast
freezing within a few seconds of surveillance. Imaging cryopreparations at low
temperature may be accomplished using low-cost home-fabricated (Carlson &
Evans, 2011) or more sophisticated devices (McDonald, 2009; Schwartz, Sarbash,
Ataullakhanov, McIntosh, & Nicastro, 2007; van Driel, Valentijn, Valentijn,
Koning, & Koster, 2009; Verkade, 2008). As the problems associated with
400 CHAPTER 18 Registration of Image Fields for Correlative Microscopy

FIGURE 18.7
Panel (A) shows the PALM image of cells expressing TFAM-mEos2 and panel (B) shows
mitochondria (M) in a backscatter image collected by SEM of the same area. Panel (C) is the
integrated PALM/SEM image showing localization to the mitochondria nucleoloid.
Reprinted with permission from Kopek et al. (2013).
 18.1 Methods 401

postembedding methods are avoided using cryofixation following the Tokuyasu cryo
immunogold labeling technique (Tokuyasu, 1973), the method appears to have the
best success for maintaining emission of GFP fusion proteins; however, subcellular
details are often obscured in cryosections due to poor tissue contrast.
Cryostabilization followed by freeze substitution and embedding may result in
improved morphology, but fluorescent proteins are quenched by the acidic, dehy-
drated, and oxidizing conditions required for fixation and polymer embedding of
the specimen. Hence, the emission intensities of many flourophores may not be ad-
equate following embedding. Reviewed by Campbell and Choy (2001), the pH of
wild-type GFP (wtGFP) is stable from pH 6 to 10, decreases at pH less than 6,
and increases from pH 10 to 12 (Haupts, Maiti, Schwille, & Webb, 1998). Different
GFP variants have different pH sensitivities, with current GFP mutants having pKas
ranging from 4.8 to 8.0. In the context of this paragraph, it is interesting to note that
the pH sensitivity of GFP variants may serve as molecular reporters of shifting pH
environments (Bizzarri, Serresi, Luin, & Beltram, 2009). The need to monitor and
adjust pH during processing of tissues carrying GFP constructs is essential, and
knowledge of the peculiarities of the GFP variant may be invaluable. In our hands,
YFP seems to endure fixation, dehydration, and embedding media somewhat better
than wtGFP. Brown, Fetter, Tkachuk, and Clayton (2010) used PALM microscopy to
localize mitochondrial proteins in HPF-cultured cells, which were subsequently
freeze substituted in 95% EtOH containing 1–2% glutaraldehyde, then infiltrated
in LR White with 95% EtOH at 20  C, then cold-polymerized at 20  C using ben-
zoyl peroxide-catalyzed resin. In a detailed protocol, the authors point out the impor-
tance of storing the catalyzed resin for no more than 1 month, and further discuss a
method for adjusting the pH of LR White to near-neutral using ethanolamine.
Watanabe et al. (2011) also used PALM and simulated emission depletion micros-
copy together with electron microscopy to localize fluorescently tagged proteins at
high resolution. This comprehensive study in methodology points out the need to
avoid absolute dehydration, the need to adjust the pH of LR White from the typical
5.5 to just under 7.0, and also the variability of LR White in polymerization. In our
experiments between 2005 and 2007, we repeatedly localized YFP and GFP con-
structs within several tissues using LR White without adjustment of pH. But within
the past few years, we have been much less successful in localizing stable GFP con-
structs in many of these same cells and tissues newly embedded in LR White.
A qualitative assessment of the emission intensity of GFP, observed throughout
the protocol, revealed that emission remained stable through fixation and Tris–
Glycine, then decreased slightly but progressively through graded dehydration to
90% EtOH. There was a marked decrease in emission during infiltration in 1:1,
1:2, and 1:3 of 90% EtOH:LR White, then emission strongly decreased during infil-
tration in 100% LR White. This is true despite having adjusted the pH of the media
and varying fixation and dehydration conditions. Many report that the GFP-
expressing system of their choice is sensitive to higher percentages of glutaraldehyde
and limit exposure to 0.1%; however, we have experimented with up to 1% glutar-
aldehyde with 4% paraformaldehyde without significant decrease in GFP emission.
402 CHAPTER 18 Registration of Image Fields for Correlative Microscopy

Despite failures with LR White, we have had success in parallel experiments using
LR Gold polymerized at 20  C. In our hands, LR Gold does not section as well as
LR White and the ultrastructure suffers considerably. McDonald, Sharp, and Rickoll
(2012) report good results using Lowicryl HM20 polymerized at low temperature.
From the varying reports in the literature and from our own experience, it is clear
that for each experimental system a compromise must be found between preservation
of GFP fluorescence and tissue morphology.
Methods of localization using primary and secondary antibody conjugates cir-
cumvent the issues of preserving GFP fluorescence en bloc. The size of primary an-
tibodies (about 19 nm in length) limits the ultimate resolution of localization
particularly when secondary antibody conjugates are also used, but the method
may be preferred when GFP-expressing systems are not available or when GFP is
not visible after embedding. As an example of the latter, sections cut from
GFP-expressing tissue may be labeled with primary antibody to GFP followed by
a secondary antibody conjugate. Several commercial antibodies do well in labeling
exposed GFP epitopes on the surface of lightly fixed, LR White-embedded tissue
including Abcam (#ab290) and Life Technologies (#A11122). Figure 18.8 demon-
strates localization of mCOMP::YFP to dilated RER in a cultured cell embedded in
LR White followed by a 6-nm gold secondary antibody conjugate. Although we
favor LR White for labeling antigens exposed at the surface of sections, others in-
cluding Fabig et al. (2012) report success using the methacrylate Lowicryl K4M
for immunolabeling nonfluorescing GFP domains. In theory, virtually any primary

FIGURE 18.8
Ultrathin sections cut from cells expressing mCOMP::YFP fusion protein were
immunolabeled with a primary antibody to GFP followed by a secondary 6 nm gold
conjugate, resulting in high-density labeling to dilated RER.
Reprinted from Keene et al. (2008).
 18.1 Methods 403

antibody can be used successfully to localize an antigen using a postembedding pro-

tocol. The screenshots shown in Workflow for Overlaying Images Using FIJI
presented in Section 18.1.8 are from mouse small intestine fixed in 4% paraformal-
dehyde/0.5% glutaraldehyde and embedded in LR White. Ultrathin sections were
immunolabeled using rabbit antibody to lysozyme. A 10-nm gold/Alexa 488 goat
anti-rabbit secondary conjugate facilitated imaging by FM or LSCM; then the same
grid was examined for gold particle distribution in the TEM. Secretory granules
within Paneth cells were intensely florescent, and corresponding structures could
be found easily in the TEM.

18.1.2 SAMPLE PREPARATION

Notes
• Cultured cells are grown on Lux “Thermanox” 13-mm diameter cover slips
which are “cell culture treated” on one side, sterile, and impervious to acetone,
propylene oxide and embedding media.
• Costar 24-well culture clusters may be used as both the growth and processing
chamber for the cover slips up to and including 90% ethanol. They are
manufactured from polystyrene and will not tolerate acetone, propylene oxide or
embedding media.
• Sarstedt 50-ml polypropylene centrifuge tubes may serve as processing chambers
for individual cover slips. Polypropylene will tolerate acetone, propylene oxide
and embedding media, and the round sides and conical bottom ensure that the
culture surface of the cover slips is protected from mechanical damage.
• Embedding is accomplished using polypropylene Wheaton “snap caps” which
have been baked a minimum of 24 h in a 60–70  C oven. Do not use paper labels.
Embed the discs with the culture surface facing up.
• LR White will not polymerize in the presence of atmospheric oxygen. For
thermal polymerization of LR White, include one or two small thermoses
of liquid nitrogen introduced into the oven just prior to the samples. The
oxygen-rich atmosphere in the oven will be displaced with an oxygen-depleted
atmosphere (Fig. 18.9).
• LR Gold also will not polymerize in the presence of atmospheric oxygen. For
polymerization at 20 to 35  C, we utilize the chamber of a Leica AFS
(automatic freeze substitution) device, which ensures a nitrogen-rich atmosphere
during polymerization using an UV light source. Alternatives to the AFS are
gelatin embedding capsules which have been warmed in a 70  C oven prior to use,
driving out excess moisture from the gelatin. The gelatin capsules are filled
with media, then capped with as little air as possible. Do not use Beem capsules
since the UV light will not penetrate these capsules. Polymerization can be
accomplished using a “black light” bulb over a foil-lined box placed in the freezer.
• For best results, use cryofixation (high-pressure freezing) followed by
low-temperature embedding in Lowicryl HM20. If this is not possible, the next
404 CHAPTER 18 Registration of Image Fields for Correlative Microscopy

FIGURE 18.9
A thermos filled with liquid nitrogen is placed in the polymerization oven to insure an
oxygen-depleted atmosphere.

best approach is to use PLT (progressive lowering of temperature) dehydration,

followed by infiltration and embedding with Lowicryl HM20, LR Gold, or
LR White.

18.1.3 EN BLOC GFP LOCALIZATION IN CULTURED CELLS

AND TISSUES
• Tissues should be processed as quickly after sacrifice as possible. Cultured cells
are grown on 13-mm diameter Lux Permanox cover slips.
GFP emission may be monitored during the protocol by sacrificing a disc which is
placed cell side down on a cover glass, then imaged by FM or LSCM with an inverted
microscope.
• Within a few minutes of removing the culture from the incubator, rinse the cells in
warm, serum-free Dulbecco’s modified eagle media (SFM) twice, each for 5 min.
Using a stereoscope, scratch a mark onto the disc so that the cell side of the disc
can be recognized during embedding. Then transfer the processing chamber to an
ice bucket. Fresh tissue should be rinsed briefly in ice-cold SFM.
All further processing of cell cultures should be done using ice-cold solutions and the
samples kept on ice for the duration of the procedure (see PLT protocol for tissues).
• Fix cultured cells for 30 min in ice-cold 4% paraformaldehyde/1%
glutaraldehyde buffered with SFM. Tissue should be cut into pieces no larger than
1
2 mm and fixed 30–60 min, dependent on size.
• Rinse cultured cells two times over 5 min; rinse tissues three times over 15 min
in SFM.
 18.1 Methods 405

• Incubate cultures in 0.15 M Tris–HCl, pH 7.4, containing 0.05 M Glycine for

60 min on ice. Tissues should incubate 4 h overnight in ice-cold solution.
• Dehydrate cultures and tissues in ice-cold ethanol: 15 min each in 30%, 50%,
70%, and 90% ethanol in water.
• Infiltrate cultures and tissues for 30 min each in ice-cold 1:1, 1:2, and 1:3 of 90%
EtOH:LR Gold (including 0.5% benzoin methyl ether).
If possible for tissues, dehydrate at progressively lower temperature beginning on ice
with 30% ethanol then cooling to 10  C over 15 min, then at 10  C with 50%
ethanol; than in 70% ethanol at 10  C cooling to 20  C over 15 min, then con-
tinuing all additional steps to 100% embedding media at 20  C.
Achieving the low temperatures necessary for PLT dehydration and low-
temperature polymerization of embedding media may be accomplished using the
Leica AFS device. If this tool is not available, temperatures from 17 to 77 
may be achieved using a cooling bath consisting of dry ice with varying ratios of eth-
ylene glycol and ethanol. For example, a temperature of 28  may be reached with
ethylene glycol at 80% and 100% ethanol at 20% (Lee & Jensen, 2000). As suggested
by Dr. Kent McDonald (U.C. Berkeley), temperatures in this range may also be em-
pirically acquired by positioning a rack above dry ice (personal communication).
• Infiltrate cultures for 30 min each in three changes of ice-cold 100% LR Gold
(including 0.5% benzoin methyl ether) or HM20 monostep (requiring no
catalyst). Incubate tissues for 60 min each change. For tissues, the last change
may continue overnight. For cultured cells, avoid an overnight incubation in
media.
Note: LR Gold requires the addition of 0.50% benzoin methyl ether to polymerize at
low temperature. Mix the benzoin methyl ether into solution using nitrogen bubbles.
Use only glass or polypropylene pipettes when handling LR Gold or LR White.
• For polymerization of cultured cells in the Leica AFS device, cool the sample
chamber to 10  . Scratch an identifying mark onto the outer surface of the
Wheaton snap cap (marker ink solubilizes upon contact with media). Place these
empty snap caps into the chamber and fill half way with 100% media.
Quickly transfer tissues or cultured cell discs, cell side up and submerge into
the media. Hopefully, a recognizable mark (such as an “a,” “b,” “L,” but not an
“x”) was scratched onto the cell side of the disc; otherwise, the cells are
difficult to see and the cell side will need to be determined using a stereoscope
during which time condensation will form on the warming surfaces,
potentially causing problems with polymerization. After the samples are
introduced, fill the caps to the rim with media.
• Decrease the temperature of the chamber to the polymerization temperature
(20 to 35  ). This will cool the chamber and also fill it with a nitrogen-rich
atmosphere. Cover the sample chamber with glass then polymerize using the UV
light included with the system. Polymerize for 24 h and if necessary, continue
polymerization at 4  for another 4 h.
406 CHAPTER 18 Registration of Image Fields for Correlative Microscopy

FIGURE 18.10
A partial edge of a round thermanox cover slip embedded in LR White is delineated with
arrows; the remaining edge of the cover slip has been exposed by a trench cut using a Dremel
3000 variable speed rotary tool fitted with a Dremel #106 engraving cutter. The cover slip
will separate easily after the entire edge of the disc is exposed by the trench.

• After polymerization, there may be a thin film of liquid media at the surface of the
polymerized discs; wipe it away with 100% ethanol. As the samples are released
from the polypropylene snap caps, score an identifying mark into the
polymerized media; marking pens will not be permanent. To ensure easy release
of the culture discs from polymerized media, use a high-speed rotary tool
(Dremel) fitted with a Dremel 106 1/1600 Engraving Cutter, carving a trench
around the edge of the disc (Fig. 18.10), then the disc will separate easily. Use a
fine coping saw to cut out individual blocks from the culture.

18.1.4 EMBEDDING CULTURES AND TISSUES FOR POSTEMBEDDING,

SECTION-SURFACE LABELING
Prepare cultured cells and tissues with the same protocol listed above for en bloc GFP
localization; however, the aldehyde concentration of the primary fixative may need
to be reduced to as little as 1.5% paraformaldehyde/0.1% glutaraldehyde. In the ini-
tial pilot experiment, try different aldehyde concentrations to determine the best
compromise between labeling density and ultrastructure. For reference, the tissue
shown in Fig. 18.6 was fixed in 4% paraformaldehyde/0.5% glutaraldehyde. Our best
results are with the embedding media LR White without pH adjustment. Others
(McDonald et al., 2012) report success with Lowicryl HM20. Swirl the contents
within the bottle containing media prior to use, and add a nitrogen cap when the bot-
tle is stored. Polymerization is accomplished in polypropylene snap caps in a 60 
oven with an opening (thermometer, vacuum vent valve), door held closed, contain-
ing one or two small thermoses of liquid nitrogen (Fig. 18.9). Two Styrofoam coffee
cups may be stacked together to contain liquid nitrogen. A large beaker of dry ice is
 18.1 Methods 407

an alternative. Do not open the oven for several hours to ensure an oxygen-depleted
atmosphere.

18.1.5 IMMUNOLABELING THE SURFACE OF ULTRATHIN SECTIONS

Protocols for postembedding labeling vary; McDonald et al. (2012) describe a
method which works well in their hands. We use the following protocol for virtually
all epitopes exposed at the surface of an ultrathin section.
• Sections are cut using a diamond knife with a low water level in the boat, just
enough to keep the diamond knife edge wet. An antistatic device is used to help
keep the block face dry. Sections are cut 60–90 nm thick and mounted onto
formvar-coated 1
2-mm nickel slot grids, recently made hydrophilic by glow-
discharge. We use a homemade device for discharge (after Aebi & Pollard,
1987). This step helps ensure that the section lies flat onto the formvar. Use
nonmagnetic forceps when handling nickel grids. Dry the grids thoroughly before
proceeding.
• Use a PTFE Immunostaining Pad (Pelco) with 8
5 depressions. You may also
use parafilm. Float the grids, section side down, onto the surface of each solution.
Place the immunostaining pad in a glass Petri dish with wet filter paper at the
bottom to ensure a humid environment. Move the grids from one solution to
another using nonmagnetic forceps or a wire loop. All incubations are at ambient
temperature. Incubate in 30-ml of each of the following solutions:
• 0.15 M Tris–HCl, pH 7.5, for 15 min
• 0.15 M Tris–HCl, pH 7.5, with 0.05 M glycine for 60 min
• 2% nonfat dry milk, 0.5% ovalbumin, and 0.5% fish gelatin in Tris–HCl for
30 min
• Quick rinse in 0.15 M Tris–HCl
• Primary antibody with appropriate dilution: typically, we dilute concentrated
antibody (1 mg/ml) 1:50 in Tris–HCl. This is the dilution we use for the Abcam
polyclonal antibody 290 recognizing GFP. Incubate for 2.5 h.
• Three drops Tris–HCl over 15 min
• Secondary antibody with appropriate dilution: typically, we dilute Aurion
secondary gold conjugate 1:10; we also dilute Molecular Probes Alexa 488/10nm
gold dual conjugate 1:10 in Tris–HCl. Incubate for 60 min.
• Three drops of Tris–HCl over 15 min
• Three drops of dH2O over 15 min
• Wick away excess water and dry the grids.

18.1.6 ULTRAMICROTOMY AND OBSERVATION OF SEMI-THIN AND

ULTRATHIN SECTIONS USING CONFOCAL OR EPIFLUORESCENT
IMAGING
• For CLEM, blocks are trimmed with a nonsymmetrical block shape. The block is
faced off with a glass or diamond knife; then it may be placed facedown on a
408 CHAPTER 18 Registration of Image Fields for Correlative Microscopy

cover slip and evaluated for positive fluorescent emission by either FM or LSCM
using an inverted microscope.
• Semi-thin (0.5–1.0 mm) sections are picked from the water surface of a glass or
diamond knife boat using an eyelash stick, then transferred to a small drop of
water on a 24
50-mm No.1 glass cover slip. The sections are air-dried and
examined unstained and without mounting media. Ultrathin sections are mounted
on single-hole 1
2-mm formvar-coated slot grids, then placed section side
down on the surface of a cover glass supported by the stage of an inverted
microscope. Images may be collected using dry objectives; when imaged using a
63
, water immersion 1.2 NA lens, a drop of water is added to the lens side
of the cover slip but not the grid side. Any aberrations in the imaging path
resulting from the tiny air interface under the grid are negligible.
• Initial assessment by IF or LSCM is done as quickly as possible to minimize
photo bleaching. Low-magnification, low-resolution images are collected to
determine a region of interest. These low-magnification images are invaluable as
“section maps,” as a first step in correlating LM imaging fields to EM fields.
It is also helpful to record on paper (draw) the relative position of a region of
interest within the space of the nonsymmetrical section. Image collection is a
compromise between photo bleaching and high-resolution recording.
• For the final image used in CLEM, using LSCM we typically collect a
2048
2048 image with a scan speed of 400 and line averaging ¼ 4 with no field
averaging. We collect an image of the ROI first with a Plan Apo 20
dry
objective (NA ¼ 0.8), then with a Plan Apo 63
water immersion objective
(NA ¼ 1.2). We are sure to wet the 63
lens prior to an imaging session so that
the section need not be moved to expose the lens to water. Aperture and other
beam settings are typically set to maximize resolution dependent on the objective
lens used. If the goal is to image the same ultrathin section by both FM and
TEM, it is important to image the section first by FM, since damage caused by the
electron beam will extinguish the fluorophore. If the goal is to correlate a semi-
thin section with a serial ultrathin section, we cut the ultrathin section first and
then cut the semi-thin section. This seems a reliable sequence for collecting an
acceptable ultrathin section.

18.1.7 REGISTRATION OF LM AND EM IMAGES

• Correlation of LM and EM imaging fields is typically accomplished in several
steps, “macro to micro.” LM images are collected prior to the EM imaging
session.
• A 20
(or lower magnification) image collected in the LM is displayed on a
monitor while in the EM suite; with the brightness and contrast adjusted so that
cell profiles are imaged. Open the LM image using a program that allows not
only the control of brightness and contrast but also rotational alignment and
flipping of the image (i.e., Adobe Photoshop, Microsoft Office 2010 Picture
Manager, IrfanView, QuickTime).
 18.1 Methods 409

• During initial stages of observation in the EM, locate the approximate area from
which the LM images were collected by observing the entire section at low
magnification. Find the region relative to the shape of the section noted during
LM observation. Control the computer displaying the LM image with a
cordless mouse from the TEM work surface so that the image may be easily
manipulated.
• Once a corresponding region of interest is recognized on both monitors, rotate the
LM image field so that it matches the live-field imaged in the EM. It may be
that the EM grid is upside down relative to the section imaged in the LM. If
so, “flip” the LM image using the imaging software to avoid handing the grid any
more than needed. Once the region imaged in the LM is recognized in the EM,
collect progressively higher magnification images of the region of interest.
Progressively higher magnifications will ensure no beam damage in the lower
magnification images. These TEM images will be used as the background on
which LM images are overlaid when composing the registered composite images.
• You may wish to make initial EM observations on unstained sections. If the
region photographed in the LM is found to be defective in the EM (dirt, wrinkles),
you may then return to the LM to take additional images. Should your EM
imaging system have the ability to collect image montages, these can be
enormously useful in observing LM data overlaid atop large image fields
tolerating substantial digital enlargement.
• LR White, LR Gold, and Lowicryl HM20 may be stained for EM using uranyl
acetate in 50% EtOH for 5 min, 5 min in water, then stain in quarter-strength
Reynold’s lead citrate for 15 s before a final 5-minute water wash. The use of
“grid sticks” allows the staining of multiple grids simultaneously.

18.1.8 WORKFLOW FOR OVERLAYING IMAGES USING FIJI (IMAGEJ

VERSION 1.48O)
Download Fiji (http://fiji.sc/wiki/index.php/Downloads). Fiji is a distribution of
ImageJ together with Java, Java 3D, and several plugins organized to assist research
in life sciences, targeting image registration, stitching, segmentation, feature extrac-
tion, and 3D visualization, among others. After installing Fiji, check to be sure it is
the latest version (Fiji/Help/Update Fiji).
• Open Fiji
• Open the images to be overlaid. The images should be in .tiff format. Open the
background image (TEM) first and keep it open, then open the image to be
overlaid (LM) (File/Open/double click on image). Images may also be dragged
into the work bar of Fiji to open them.
• Manipulate one of the images so that fields are approximately oriented with
respect to one another. This may require that one image be flipped (Image/
Transform/Flip horizontally or vertically) and/or rotated (Image/Transform/
Rotate).
410 CHAPTER 18 Registration of Image Fields for Correlative Microscopy

• Crop the images so that almost the same region remains in each image (click on
one image, select the Rectangular selection on the far left of the tool bar, crop
the image (Image/Crop); then do the same for the other image.
• Adjust images so that they are both 8 bit (Image/Type/8bit). It is not important if
color remains in the image to be overlaid.
• Open the TurboReg Plugin (Plugins/Registration/TurboReg).
TurboReg (Thévenaz, Ruttimann, & Unser, 1998) is used to register one image
(Source) to a second image (Target); Fiji is then used to overlay the registered image
atop the Target image.
• Within the TurboReg window, confirm that the Source is the image to be overlaid
(LM) and that the Target is the image on which the overlaid image is placed
(TEM). If each image was opened in the order suggested above, this will be
correct. Change them if necessary. Check the boxes for “Bilinear,” “Save on
Exit,” and “Accurate.”
• Each image will now have a green cross, a magenta cross, a blue cross, and a
yellow cross. Place the green cross on a feature at the upper left quadrant of one
image. Looking to the second image, move the green cross to the corresponding
feature. If you wish to magnify either image, you may select the magnifying tool
in the Fiji tool bar but remember to go back to the select tool when placing a cross.
You may also adjust the contrast and brightness of either image (Image/Adjust/
Brightness/Contrast). Move the magenta, blue, and yellow crosses to common
features on each image in the other three quadrants (these crosses are difficult to
see in the screenshot below) (Fig. 18.11; colored arrows point to the smaller
crosses).
• Select the TurboReg window again; press the “Manual” button. A new 32-bit
grayscale image will appear which is automatically named “Output.”
• If you wish, save the “landmarks” selected in the previous window, giving it a
file name.
• The Output image produced in TurboReg may now be manipulated in Fiji.
• Select the “Output” image. There are actually two images here; each image is
accessible via the slider. Move the slider to the black image (Mask) and delete it
(Image/Stacks/Delete Slice); now only the Data image remains. Match the bit
size of the Output file to the Target image (Image/Type/8bit).
• Next, merge the Output with the Target image (Image/Color/Merge Channels).
Choose the color you prefer for the data contained in the overlaid image. If
you would like your overlaid image to be blue on top of a grayscale (EM) image,
then set red to “none,” green to “none,” blue to “output,” gray to the file
name of the background EM image, and cyan, magenta, and yellow to “none.”
Check the boxes “create composite” and “keep source images,” then press
OK (Fig. 18.12).
When choosing a color for your overlay, consider that approximately 8% of
males and 0.4% of females are red–green color blind (Albrecht, 2010, also see
http://en.wikipedia.org/wiki/Color_blindness). Individuals with red–green color
 18.1 Methods 411

FIGURE 18.11
In this screenshot, both a confocal image (on the right) and a TEM image of the same region
(on the left) were opened in FIJI. The TurboReg plugin was initialized and “Bilinear,” “Save
on Exit,” and “Accurate” were selected. The green, pink, blue, and yellow crosses were
moved to the same features on each image. Next, “Manual” will be selected to form an
aligned “Output” image (Fig. 18.12).

blindness cannot discern between the two colors; both appear yellow–brown. To sim-
ulate how a color image is perceived by individuals with specific color insensitiv-
ities, open the image in FIJI (FIJI/Image/Color/Simulate Color Blindness). FIJI
may be used to simulate the perception of the full spectrum of colors. Importantly,
magenta is perceived as a hue of blue by color blind individuals; therefore Magenta/
Green images are perceived as Blue/Yellow. FIJI allows one-button conversion of a
Red/Green image to Magenta/Green (FIJI/Image/Color/Convert Red to Magenta)
may be changed using Adobe Photoshop/Image/Adjustment/Hue/Saturation.
• This creates a “Composite” File, composed of two images.
• Importantly, both the underlying and overlying images can be adjusted for
contrast and brightness (use the slider to select the layer/Image/Adjust/). This
adjustment affects the opacity of the overlaid image and is a critical adjustment.
Demonstrated by Fig. 18.14, when the overlaid CF image is made increasingly
opaque, only lysozyme labeling within the Paneth cell secretory granules is
realized. Making the overlaid CF image increasingly less opaque reveals labeling
not only to Paneth cell secretory granules but also to the RER within the
Paneth cells (Fig. 18.13).
• The images composing the Composite file may not be perfectly aligned. This
could be due to any number of reasons, including sectioning artifacts such as
wrinkles or compression. Images may be manually aligned relative to each other
by selecting one image of the Composite (usually the overlaid image), then
distorting this image relative to the background image (Fiji/Plugins/Transform/
412 CHAPTER 18 Registration of Image Fields for Correlative Microscopy

FIGURE 18.12
The initial “Output” image is composed of an aligned image and a black mask, selectable
using the slider. Select the black mask and delete it (Image/Stacks/Delete slice), leaving only
the aligned image as in this screenshot. Next, select the TEM image and match it to the bit
depth of the “Output” image (Image/Type/-bit). The images may now be merged (Image/
Color/Merge Channels). In this example, the “Output” image was assigned the blue channel
and the TEM image was assigned the gray channel, producing an aligned “Composite”
image.

FIGURE 18.13
The “Composite” image has two components, selectable using the slider, each of which may
be further adjusted for contrast/brightness. Careful adjustment of contrast and brightness
is critical. Improper adjustment of this image (left) would miss cytoplasmic localization (right)
verified by immunogold labeling (shown in Fig. 18.6).
 18.1 Methods 413

FIGURE 18.14
Should the alignment of two images composing the “Composite” image be unsatisfactory, one
of the images may be transformed, or skewed, relative to the other. In this screenshot,
the confocal portion of the image was manually dragged to match the TEM portion of the
image at each of the seven points selected.

Interactive Moving Least Squares). Choose Affine, choose interactive preview.

Left-click on more than three misaligned regions, then select (hold down left
mouse button) and drag to the correct alignment. You may select as many regions
as necessary to deform the image to a perfect alignment. On the keyboard,
pressing “u” will toggle in grid that can be used in determining the extent of
deformation throughout the entire image (Fig. 18.14).
• Save the Composite as a .tiff (File/Save As/Tiff. . .name the image); it can be
opened latter in Fiji to make further adjustments. To open the fully adjusted
image in other programs, convert the Composite to a single flattened RGB image
(Image/Type/RGB Color) and save is as a .tiff.

18.1.8.1 Materials
Fiducial markers: AU nanospheres: Microspheres-Nanospheres, Cold Spring,
NY (http://www.microspheres-nanospheres.com/Microspheres/Inorganic/
Metals/Gold.htm)
414 CHAPTER 18 Registration of Image Fields for Correlative Microscopy

Fluorescent polymer-coated Au gold particles: http://www.nanopartz.com/

fluorescence_gold_nanoparticles.asp
Embedding media: LR White resin kit, Ted Pella, Inc. (#18181); LR Gold resin
kit, Electron Microscopy Sciences (#14370); Lowicryl HM20 embedding media
monostep, Electron Microscopy Sciences (#14345)
Primary antibodies: Abcam GFP antibody, Abcam (#ab290); Life Technologies
GFP antibody, Life Technologies (#A11122)
Secondary antibodies: Aurion gold conjugates, Electron Microscopy Sciences;
goat anti-rabbit 6 nm, EM grade (#25103); goat anti-rabbit 10 nm, EM grade
(#25108); Dual fluorescent-gold conjugate (Molecular Probes Alexafluor 488
with goat anti-rabbit IgG 10-nm gold conjugate), Life Technologies (#31566).
Tools and materials: Lux Thermanox cover slips, 13 mm diameter, Electron
Microscopy Sciences (#72281); Corning Costar cell culture plates, Fisher
Scientific (#07-200-84); Sarstedt 50-mL conical tubes, Phenix Research
(#SS-7004); Wheaton snap caps, Fisher Scientific (#06-450-201); Gelatin
embedding capsules, Electron Microscopy Sciences (#71000); 2 mm
1 mm slot
grids, Electron Microscopy Sciences: copper-palladium (#G2010-CP special
order); nickel (#G2010-Ni); NRD Static master ionizer, Rice Lake Weighing
Systems (#76676); PELCO PTFE immunostaining pad, Ted Pella, Inc.
(#10526-1); 24 mm
50 mm cover glass, Fisher Scientific (#12-548-5M); Anti-
magnetic tweezers, Electron Microscopy Sciences (#72700-D); Grid stick,
Electron Microscopy Sciences (#71175); Sylvania Blacklight Blue F15T8/BLB
15W (#21625); Spiral saw blades, Electron Microscopy Sciences (#72015-02)
Fixatives: 10% aqueous formaldehyde, Tousimis (#1008C); 50% EM grade
aqueous glutaraldehyde, Electron Microscopy Sciences (#16316)
Chemicals: Dulbecco’s Modification of Eagle’s Medium (DMEM), Cellgro
(#10-013-CV); TRIS, Electron Microscopy Sciences (#11720); Glycine, Sigma-
Aldrich (#410225-50g); Carnation nonfat dry milk (grocery store); Chicken egg
albumin, Sigma-Aldrich (A5503); Cold water fish gelatin, Electron Microscopy
Sciences (#25560); Benzoin methyl ether, Electron Microscopy Sciences
(#11290).

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FURTHER READING
Metscher, B. D. (2009b). Micro-CT for developmental biology: A versatile tool for high-
contrast 3D imaging at histological resolutions. Developmental Dynamics, 238, 632–640.