Archives of Biochemistry and Biophysics 581 (2015) 98–110

Contents lists available at ScienceDirect

Archives of Biochemistry and Biophysics

journal homepage: www.elsevier.com/locate/yabbi

Review

Correlative microscopy
Céline Loussert Fonta 1, Bruno M. Humbel ⇑
Electron Microscopy Facility, University of Lausanne, Biophore, 1015 Lausanne, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: In recent years correlative microscopy, combining the power and advantages of different imaging system,
Received 9 March 2015 e.g., light, electrons, X-ray, NMR, etc., has become an important tool for biomedical research. Among all
and in revised form 26 May 2015 the possible combinations of techniques, light and electron microscopy, have made an especially big step
Available online 10 June 2015
forward and are being implemented in more and more research labs.
Electron microscopy profits from the high spatial resolution, the direct recognition of the cellular ultra-
Keywords: structure and identification of the organelles. It, however, has two severe limitations: the restricted field
Correlative microscopy
of view and the fact that no live imaging can be done. On the other hand light microscopy has the advan-
Light and Electron microscopy
STEM scanning transmission electron
tage of live imaging, following a fluorescently tagged molecule in real time and at lower magnifications
microscopy the large field of view facilitates the identification and location of sparse individual cells in a large con-
Mouse brain text, e.g., tissue. The combination of these two imaging techniques appears to be a valuable approach to
GFP green fluorescent protein dissect biological events at a submicrometer level.
Epoxy Light microscopy can be used to follow a labelled protein of interest, or a visible organelle such as mito-
Methacrylate chondria, in time, then the sample is fixed and the exactly same region is investigated by electron micros-
Cryo-fixation and Freeze-substitution copy. The time resolution is dependent on the speed of penetration and fixation when chemical fixatives
Tokuyasu cryo-sectioning
are used and on the reaction time of the operator for cryo-fixation. Light microscopy can also be used to
Immuno-(gold) labelling
identify cells of interest, e.g., a special cell type in tissue or cells that have been modified by either trans-
fections or RNAi, in a large population of non-modified cells. A further application is to find fluorescence
labels in cells on a large section to reduce searching time in the electron microscope. Multiple fluores-
cence labelling of a series of sections can be correlated with the ultrastructure of the individual sections
to get 3D information of the distribution of the marked proteins: array tomography. More and more
efforts are put in either converting a fluorescence label into an electron dense product or preserving
the fluorescence throughout preparation for the electron microscopy.
Here, we will review successful protocols and where possible try to extract common features to better
understand the importance of the individual steps in the preparation. Further the new instruments and
software, intended to ease correlative light and electron microscopy, are discussed. Last but not least we
will detail the approach we have chosen for correlative microscopy.
Ó 2015 Elsevier Inc. All rights reserved.

Introduction
For many years light and electron microscopy were two sepa-
rate imaging techniques. Either the researcher was a light micros-
copist or an electron microscopist. Also sample preparation
methods differed a lot from formalin fixation or acetone/methanol
precipitation to highly sophisticated cryo-preparation schemes.
Even today the division exists and talking about it is not always
well received. Fortunately, the two groups start to realise the
⇑ Corresponding author. Fax: +41 21 6925055.
E-mail addresses: Celine.Loussert@rdls.nestle.com (C. Loussert Fonta), Bruno.
Humbel@unil.ch (B.M. Humbel).
1
Present address: Nestle Research Center, Vers-chez-les-Blanc, Case postale 44,
1000 Lausanne 26, Switzerland.
advantages of both techniques and also how useful, yet important
it is to combine them to complete the picture in biological
research. The first successful correlative light and electron micros-
copy data of a study of microtubules in PtK2 cells were published
in 1978 [1,2].
Electron microscopy profits from the high spatial resolution and
the direct recognition of the cellular components. It, however, has
two severe limitations: the restricted field of view and the fact that
no live imaging can be done. Light microscopy has the advantage of
live imaging, following a tagged molecule in real time. Through
super-resolution the localisation of fluorescent molecules have
greatly improved [3–6] but there is no high-resolution information
of the total cellular ultrastructure as in electron microscopy. On
electron micrographs the reference structures can easily be

http://dx.doi.org/10.1016/j.abb.2015.05.017
0003-9861/Ó 2015 Elsevier Inc. All rights reserved.
 C. Loussert Fonta, B.M. Humbel / Archives of Biochemistry and Biophysics 581 (2015) 98–110 99

identified. The immuno-gold labelling of sparse antigens, however,

can be frustrating. What may still give a decent fluorescence signal
can be only a few gold particles on the section leading to no or at
best very doubtful results. Here, the direct combination of the two
information, localisation by (super-resolution) light microscopy
and high resolution morphology by electron microscopy is the
method of choice.
Combining the data provided by these two optical methods
enables live cell imaging, finding a single event in a large popula-
tion of cells and identifying the local surrounding of the event at
high resolution. In the last two decades correlating light and elec-
tron microscopy data has become popular and essential in address-
ing specific questions in cell biology [7]. New preparation methods
but also instruments and software dedicated to correlative micros-
copy have been developed.
In this review we address the current state of successful prepa-
Fig. 1. Fluorescence map. The light micrograph serves the operator as a guide to
ration schemes for the different approaches for correlative micros- find the marked cells in the electron microscope. Using the shape of the section and
copy. Special attention is given to the protocols that preserve the local surrounding of the cell of interest it can be identified in the electron
fluorescence throughout the preparation protocol for electron microscope. In more complicated tissue, the searching process can be very time
microscopy. Here, common denominators are searched and their consuming and sometimes even unsuccessful.
importance is discussed. Further, the newly developed light–elec-
tron microscopes are described and put in the perspective of their
applications. Last but not least we are describing in detail the
approach chosen in our lab, using the Tokuyasu cryo-sectioning
technique in combination with the Maps software.

Methods of correlation

On-section immuno-labelling

There are many methods to prepare a sample for correlative

microscopy depending on the ‘cultural’ background of the lab
and the specific needs. The easiest and most straightforward way
is the labelling of thin sections. The specimen is prepared with a
protocol suitable for immuno labelling (electron) microscopy.
There is a wealth of methods based on chemical fixation and meth-
acrylate embedding [8–17], on cryo-fixation, freeze-substitution
and low-temperature embedding [18–21] or on Tokuyasu
cryo-sections [22–27]. From such prepared material sections of
200 nm but also ultrathin sections can be cut and mounted on glass
coverslips and immuno labelled with fluorescent antibodies [28–
34] for light microscopy investigations and consecutive sections
on grids, labelled with colloidal gold for electron microscopy.
Alternatively, sections can also be mounted directly on electron
microscopy grids or ITO coverslips (coverslips that are coated with
Indium-Tin-Oxide to make the surface electrically conductive,
Optics Balzers AG, Balzers, Liechtenstein) [35] and labelled with
fluorescent and gold conjugates for analysis with light microscopy
and thereafter in a TEM2 [36] or SEM [30].
After imaging in a fluorescence microscope the resulting micro-
graphs are used as a map, either lying next to the electron micro-
scope (Fig. 1) or overlaid with the electron micrograph using a
sophisticated software [37–40], to ease the localisation of the cells
of interest in the electron microscope (Fig. 2).
Often, however, it is desired to observe the fluorescent target in
the living cells before fixation to catch a special event [41–47] or to Fig. 2. Overlay of a fluorescence image with an electron micrograph. The fluores-
prepare a whole sample to find the place of interest, indicated by cence image was loaded into the correlation software Maps (see below) and
the fluorescence label, in a three-dimensional environment matched with the electron micrograph of the same section mounted on an ITO
coverslip. Groups of cells of interest were searched on the fluorescence image, the
[38,48–50]. In the following we describe different approaches that
magnification and position of the microscope were adjusted that the group of cells
was within the recording frame (pink frame in A) and then an electron micrograph
(B) was recorded (adapted from [30]). Bars represent 3 lm (A) and 2 lm (B). (For
2
Abbreviations used: LM, light microscope; EM, electron microscope; TEM, trans- interpretation of the references to colour in this figure legend, the reader is referred
mission electron microscopy; SEM, scanning electron microscopy; STEM, scanning to the web version of this article.)
transmission electron microscopy; ILEM, integrated laser electron microscope; GFP,
green fluorescent protein; miniSOG, mini singlet oxygen generator; RNAi, RNA
interference; ITO, indium tin oxide; CEMOVIS, cryo-electron microscopy of vitreous allow the identification of fluorescently labelled proteins in whole
sections; DAB, 3,30 -diaminobenzidine; GMA, glycol methacrylate. cells by light and electron microscopy.
100 C. Loussert Fonta, B.M. Humbel / Archives of Biochemistry and Biophysics 581 (2015) 98–110
Pre-embedding immuno-labelling (photo-oxidation and particulate
markers)
There are many possibilities to label a specific protein in a cell.
The classical approach is, after chemical fixation with aldehydes, to
permeabilise and immunolabel with a first antibody against the
protein of interest. The permeabilisation step can be omitted when
surface antigens are labelled [47] or the label is taken up by a nat-
ural process such as endocytosis [42]. In a second step the primary
antibody is detected with a fluorescent secondary antibody [51], an
antibody that fluoresces and has a particulate marker like fluoro-
nanogold (Nanoprobes, Inc., Yaphank, NY, USA) [52],
streptavidin-Alexa488-10 nm gold [42] or quantum dots (Life
Technologies, Grand Island, NY, USA) [53], which fluoresce and
are electron dense.
The cells of interest are identified by fluorescence microscopy,
then the sample is thoroughly fixed with higher concentrations
of glutaraldehyde and the fluorescence signal is transferred into a
diaminobenzidine precipitate [51,54]. 5 nm-sized and larger colloi-
dal gold particles or quantum dots are electron dense and directly
visible in the electron microscope. The ultra-small gold particles
generally need to be silver-enhanced [52]. Small quantum dots
are sometimes not easily seen in an electron dense cellular envi-
ronment this can be remedied by silver-enhancement [55]. After
the different markers are made sufficiently electron dense, the
sample is post-fixed with osmium tetroxide and embedded into
an epoxy resin. Caveat, quantum dots can be routed into a different
pathway in living cells [53] and osmium tetroxide is not always
compatible with silver-enhancement [56]. Alternatively, to classi-
cal embedding, cryo-fixation and freeze-substitution can be
applied [42].
More elegantly, clonable markers [57–61] are used to fluores-
cently tag the protein of interest. After imaging in a light micro-
scope the fluorescence signal is used to polymerise
diaminobenzidine into an insoluble osmophilic precipitate as done
with horseradish peroxidase [62]. This method uses the fact that
during fluorescence in combination of externally added oxygen,
the singlet oxygen molecules are generated that are needed for
the diaminobenzidine reaction. Hence, the precipitate is closely
associated with the fluorescence marker. There are methods
described to convert GFP [43,63–65], ReAsh [58] and an improved
system, the miniSOG, mini singlet oxygen generator [57] into the
osmophilic DAB precipitate.
As an additional option, areas or volumes of interest can be
marked during light microscopy investigation with a
near-infrared laser, the so called infrared (IR) branding. The mark-
ers are autofluorescent and can be photooxidised to an electron
dense signal for their re-location in the electron microscope [66].
Interestingly, although these methods are very potent for cor-
relative microscopy they have not yet found routine application
and the wish remains to preserve the fluorescence signal through-
out the preparation for electron microscopy imaging.
Resins
For electron microscopy sample preparation, two different
types of resins are available: Epoxies (Epon [67], Araldite [68],
Spurr’s low viscosity resin [69], Durcupan [70,71]) and methacy-
lates (London Resins [13], Lowicryls [9,11], Unicryl (Bioacryl)
[15], glycol methacrylate (GMA) [72]).
Epoxy resins can co-polymerise with the biological components
[73–75], meaning they cross-link with biomolecules [76–78]. The
epoxy group is very reactive and capable of ring opening reactions
to many organic moieties [74], hence also to fluorochromes and
thus it often reduces or even abolishes fluorescence. There is, how-
ever, a report of successful use of epoxy resin [48].
Methacrylates (comprehensively reviewed in [79]), however,
polymerise by radical chain reaction meaning they can only
cross-link with themselves, leaving the biomolecules untouched
[73–75]. All of the published recipes of successful preservation
of GFP fluorescence use methacrylates as resin. The different
methacrylate formulations have different polarities, where
Lowicryl K4M is the most hydrophilic, followed by Lowicryl
K11M, then by LR white, LR gold and Unicryl, and then by
Lowicryl HM20 and the least hydrophilic Lowicryl HM23 [79].
Even HM20 is not absolutely apolar, it can tolerate 0.5%
(w/w) of water, whereas the hydrophilic K4M tolerates up to
10% (w/w) of water [9,79]. No systematic experiments have
been done to investigate the influence of resin polarity on
the preservation of the fluorescence of GFP or other
fluorochromes.
Successful preparation protocols
In the following we will summarise a few of the successful
preparation protocols to preserve fluorescence with the aim to find
the common denominator.
Chemical fixation and resin embedding
Two protocols are based on chemical fixation. (a) Zebrafish was
fixed with 4% formaldehyde, 0.1% glutaraldehyde, 3% sucrose in
PBS, pH 7.4, then dehydrated with ethanol and embedded in LR
white [80]. (b) Tobacco petioles were fixed with 4% formaldehyde,
2% glutaraldehyde, 1 mM CaCl2 in 50 mM PIPES, (pH not men-
tioned), dehydrated in 90% ethanol/water and embedded in LR
white [81].
Cryo-fixation, freeze-substitution and resin embedding
The other protocols rely on cryo-fixation by high-pressure
freezing [82–86], freeze-substitution [87] and low-temperature
embedding [18,20,88–90]. It is, however, not clear if, next to the
improved structural preservation, the cryo-fixation–freeze-substi
tution approach is also beneficial for the preservation of the fluo-
rescence of GFP and other fluorescent dyes.
All freeze-substitution media are based on acetone without
[48,91] or with 1–5% added water [92–96]. Water addition
can improve membrane contrast [97,98], it might also help
to preserve the fluorescence signal. Except for one [95], they
all contain uranyl acetate as a fixative and stain. The concen-
tration varies from a saturated solution (0.1%) to 0.2%. The
higher concentration is added from a 20% methanol stock solu-
tion [92,94], which results in the addition of 4% methanol!
Interestingly, nobody of those using water tried to add the
uranyl acetate as an aqueous solution to avoid the negative
effects of methanol on the cellular ultrastructure [99]. In one
case the best results in terms of ultrastructure and preserva-
tion of the fluorochromes were obtained with 0.001% osmium
tetroxide and 0.1% potassium permanganate as fixatives
[95,96].
The freeze-substitution scheme varies only slightly, 30–58 h at
90 °C (85 °C), then warming with or without intermediate stops
to 50 °C to 20 °C. Usually the substitution medium remains
unchanged during the whole freeze-substitution programme
except in one protocol where the osmium tetroxide and potassium
permanganate is replaced at 50 °C for 0.1% uranyl acetate [96].
After the washing steps with acetone or ethanol, embedding takes
place into Lowicryl HM20 [48,91,92,94] Lowicryl K4M [94], LR
white [94] or GMA [95,96]. Watanabe et al. [95,96] noticed that
some batches of LR white are very acidic and therefore could inter-
fere with the fluorochromes that optimally fluoresce at higher pH
around 8.0–8.5 [100,101]. Peddie et al. [94] use the fast substitu-
tion protocol [102] but they do not comment if the fast protocol
 C. Loussert Fonta, B.M. Humbel / Archives of Biochemistry and Biophysics 581 (2015) 98–110
Fig. 3. Finding the area of interest. The whole 300 lm thick slice is imaged to find
the fluorescent neuron. The area containing the neuron is cut out and infiltrated
with sucrose. Bars represent 200 lm.
Fig. 4. CorrSight light microscope. In (A), the top of the light microscope is visible, the screen with the open software, depicting the fluorescence signal of a section, the lamp
in the cap for bright field imaging and the setup of the microscope slide holder. The large part with the handles but also the inner part (C) can easily be removed and replaced
by other setups. (B) The special holder for the pin of the microtome with the container to be filled with ice to keep the sample cold and in a moist atmosphere during
recording. (C) Mounting of the special setup.
101
has any influence on the stability and emission efficiency of the
fluorochromes.
As an additional note: uranyl acetate binds to phosphate groups
and can thus cross-link phospholipids and nucleic acids [103].
Osmium tetroxide at high concentration and room temperature
can act as a protease [104,105]. At low concentrations, e.g.,
0.02%, antigenicity is not completely abolished but reduced by
50–75% for a particular epitope (1987, unpublished observation),
used for chemical fixation during freeze-substitution osmium
tetroxide works more like a fixative [106,107] and the effect of
1–2% of osmium tetroxide on the antigenicity is comparable to
1–2% of glutaraldehyde [108].
Summarising the observations, we can conclude that the cellu-
lar ultrastructure is better preserved by cryo-fixation and
freeze-substitution; methacrylates are the preferred resins, how-
ever, LR white might be too acidic to retain high fluorescence effi-
ciency. 0.1–0.2% uranyl acetate or low concentrations of osmium
tetroxide and potassium permanganate do not abolish fluorescence
of GFP, mCherry and mTomato. The results give no clue on the
importance of added water during the substitution process and
washing steps, although an aqueous environment seems to be
important for maintaining GFP fluorescence [59]. It is advised to
store the resin blocks in the cold and dark to preserve the GFP sig-
nal and the sections need to be imaged as fast as possible. There is
one report on a reduction of fluorescence from living cells to resin
embedded cells of up to 70% [109].
Tokuyasu cryo-sectioning
An aqueous environment, at least a certain number of water
molecules close to the chromophore, seems to be important to
maintain fluorescence of GFP [59]. Therefore we decided to
setup a correlative approach based on the cryo-sectioning
method developed by Kyoteru Tokuyasu [22–24,110] and
refined by the group of Slot and Geuze [26,27]. During the
whole preparation process, until the final drying step, for imag-
ing in the electron microscope, the proteins are kept in an
aqueous environment.
102 C. Loussert Fonta, B.M. Humbel / Archives of Biochemistry and Biophysics 581 (2015) 98–110

Fig. 5. 3D acquisition of a neuron. In the wide-field microscope fluorescence images are recorded while stepping through the sample block at 10 lm per step in Z. The neuron
is in focus from image 8 to image 24, i.e., the fluorescent neuron begins at 80 lm below the block surface and ends at 240 lm.

Cryo-fixation and imaging of the frozen-hydrated sections Instrumentation

Another option is to keep the sample in its native water sur-
rounding. As water will evaporate in high vacuum it needs to
be kept frozen. Already in the early 80s biological material was
immobilised by cryo-fixation, sectioned and imaged at very low
temperatures [111–114]. This preparation method has been
improved considerably by the group of Prof. Jacques Dubochet
[115–117] and today with modern equipment CEMOVIS
(cryo-electron microscopy of vitreous sections) has become less
exclusive. The next step was to develop a cryo-stage for a light
microscope to image the fluorescence within the
frozen-hydrated section and record the X, Y positions of the area
of interest for high-resolution analysis in the electron microscope
[39,118]. A further step forward was the development of a com-
bined light–electron microscope [37,119] where the fluorescence
can directly be imaged in the TEM with an attached light micro-
scope, before imaging with electrons, thus omitting one
cryo-transfer step. As an additional benefit it has been noted that
the fluorescence is more stable in vacuum, probably due to the
absence of singlet oxygen molecules, and under cryogenic condi-
tions [120]. With this approaches correlative light and electron
microscopy has entered the realm of true cryo-microscopy
[40,121–124].
Correlative light and electron microscopy can be done with any
fluorescence and electron microscope. The fluorescence image is
used as a guide during electron microscopy to find the correspond-
ing cell (Fig. 1). To ease and speed-up the correlation between light
and electron microscopy new instruments and software packages
have been and are being developed and are discussed in the
following.
Light and electron microscopes as one instrument
The idea of using a combined light and electron microscope is
not new, already in 1982 an instrument was built for easy diagnos-
tics in routine surgical pathology [125]. This instrument, however,
was never commercialised and got forgotten.
The idea was picked-up by the groups of Prof. Arie Verkleij and
Prof. Hans Gerritsen of Utrecht University and a laser light micro-
scope (ILEM) was constructed. The ILEM could be attached, inte-
grated, at a side-entry port, 90° to the rod of the sample holder,
into the twin lens of a Tecnai electron microscope (FEI Company,
Eindhoven, The Netherlands) [37]. Fluorescently labelled sections
 C. Loussert Fonta, B.M. Humbel / Archives of Biochemistry and Biophysics 581 (2015) 98–110
Fig. 6. Observation of the area of interest in the ultramicrotome. The area of
interest, here the Lucifer yellow injected neuron (arrow head), is always visible
during microtomy and the block can be precisely trimmed. Then ultrathin sections
containing the neuron can be cut.
with or without additional gold label on grids are loaded into the
electron microscope, tilted 90° to the electron beam and the fluo-
rescence is imaged in vacuum with the ILEM. The coordinates of
the interesting cells are stored, the holder is tilted back into the
electron beam and the positions are recalled to image the same
areas by electron microscopy [36,37,126]. The fluorescence needs
to be imaged before the section is observed in the electron beam
as after a certain dose of electrons the observed area starts to auto-
fluoresce and the specific fluorescence on the section can no longer
be observed. A commercial version of this instrument, however,
with wide-field illumination, iCorr™, is available from FEI
Company, Eindhoven, The Netherlands.
The group of Prof. Kuniaki Nagayama had the same idea. In their
construction they keep the sample in the imaging position of the
electron microscope and added a mirror at 45° with a central hole
for the electrons to pass and image the fluorescence also with the
side-mounted light microscope [119,127]. This instrument is mar-
keted by JEOL Ltd., Tokyo, Japan.
There are 4 descriptions of correlative microscopes that use a
scanning electron microscope for the high-resolution imaging
along with a light microscope.
Firstly, the airSEM™ that can image samples kept under atmo-
spheric conditions [128]. The microscope consists of a field emis-
sion scanning electron microscope that is vacuum-sealed with a
membrane at the pole piece. The sample is 50–200 lm below the
103
membrane that serves as the final electron lens. Depending on
the nature of the detectors, they are mounted in the vacuum of
the microscope, e.g., the BSE detector, or outside under atmo-
spheric conditions. The airSEM can be coupled with any light
microscopy system for easy shuttling of the sample between the
imaging units [128].
A similar approach has been chosen by JEOL Ltd for their
ClairScope™ [129,130]. The scanning electron microscope with a
tungsten emitter is mounted up-side-down under a table. The col-
umn is sealed with a SiN window, 250 lm  250 lm and 100 nm
thick. The BSE detector is mounted in the vacuum of the SEM col-
umn. The sample is cultured and manipulated in the dish with the
SiN window above the pole piece of the electron column. Opposite
the electron beam a light microscope is attached to image fluores-
cence or cathodoluminescence [129,130].
Thirdly, there is also an inverted version of the ClairScope setup
described [131]. The handling of the cells with this instrument
seems a bit more tricky as they are hanging up-side down under
the SiN window and cannot be manipulated during imaging.
Fourthly, there is an inverted light microscope, the
Simultaneous Correlative Light–Electron Microscope (SCLEM), that
can be installed in any scanning electron microscope as an attach-
ment, [132,133]. The objective of the light microscope is mounted
in the vacuum chamber of the scanning electron microscope
directly under the electron beam. The light source and CCD camera
are outside. The sample is mounted on a coverslip above the light
optics. The advantage of this system is that high-numeric aperture
immersion-oil objectives, up to 1.4 NA, can be used; the electron
beam can be aligned with the photon beam at very high accuracy,
within 10 nm; and last but not least any light microscope system,
e.g., PALM, can be attached and fed into the light path [132]. The
SCLEM is produced and marketed by DELMIC BV, Delft, The
Netherlands.
The published electron micrographs of all those SEM systems,
however, are of minor quality [128–130,132,133] they do not make
use of the resolution power of the SEM. Effort in designing accurate
sample treatment and preparation is needed to be able to fully
profit from the theoretical resolution of the instruments. Using
thin sections of fluorescently labelled cells, comparable to the
TEM setups, the DELMIC system can provide stunning results
[134]. In addition this system has the potential to automate array
tomography [135,136].
Next to dedicated microscopes there are special holders, shut-
tles, developed to ease the transfer of the samples between a ded-
icated light to a dedicated electron microscope. Using individual
systems has the advantage that no compromises need to be done
in the preparation of the sample. The fluorescence signal can be
kept under optimal conditions and high-numeric aperture objec-
tives can be used. Further any light microscopy imaging mode from
wide-field to confocal to super-resolution can be applied. After the
fluorescence micrographs are taken, the sample can be further pro-
cessed, e.g., by immuno-gold labelling, and contrasted with heavy
metal solutions for optimal imaging conditions in the electron
microscope.
Calibrated transfer shuttles
Carl Zeiss Microscopy GmbH has developed the ‘Shuttle & Find’
system that uses coverslips with 3 fiducial markers to calibrate the
positions. Images are taken with any kind of light microscope, e.g.,
the super-resolution light microscope ZEISS ELYRA PS.1, using a
1.46 NA objective and the coordinates of the images are stored in
relation to the fiducials of the coverslip. After preparation for elec-
tron microscopy the samples are transferred to a scanning electron
microscope and the location of the fluorescence images is automat-
ically retrieved by the ‘Shuttle & Find’ system [49,137,138]. A similar
104 C. Loussert Fonta, B.M. Humbel / Archives of Biochemistry and Biophysics 581 (2015) 98–110

Fig. 7. A serial section of mouse brain tissue with eGFP labelled astrocytes. Serial Tokuyasu cryo-sections were collected on a Formvar-carbon coated copper finder grid and
imaged, sandwiched between a coverslip and microscope slide, using oil immersion objectives. The numbers indicate the areas imaged afterwards by electron microscopy.
The arrows indicate the cell of interest in the consecutive section that was not imaged as it is lying on a grid bar. Bar represents 500 lm.

Fig. 8. Bright field and fluorescence images in CorrSight. Bright field and fluorescence images at different magnification are recorded with Maps in the CorrSight light
microscope. All images are already linked to each other. (A) is a montage of a bright field overview of the whole grid and the fluorescence image. Note the central hole with
the arrow and the number 1 in the rim of the grid. (B) A higher resolution fluorescence image of the Tokuyasu serial section is shown. Bars represent 1 mm (A) and 300 lm
(B).

approach has been developed by JEOL and Nikon, the MiXcroscopy
correlative imaging solution. The same specimen holder is used for
both light microscopy (Nikon) and SEM (JEOL). The specimen regis-
tration is fully controlled by the MiXcroscopy software.
For correlative cryo-microscopy several cryo-stages for light
microscopy were developed. The first one by the Max-Planck
Institute in Munich [39,118], that is now marketed by FEI
Company. There is also a version from the University of Colorado
at Boulder [139] and the stage of Linkham Inc., was engineered
at the University of Leiden [140]. After imaging by cryo-light
microscopy the calibrated reference data are fed into the correla-
tive software controlling the TEM.
 C. Loussert Fonta, B.M. Humbel / Archives of Biochemistry and Biophysics 581 (2015) 98–110 105

Fig. 9. Alignment and correlation of the light and electron micrographs. A secondary electron image of the central hole and the number 1 is recorded and the light micrograph
is loaded into the Maps software. With 2 points, e.g., the very centre of the hole (green) and one corner of the number 1 (blue), the light micrograph is rotated and stretched to
match the electron micrograph. Bars represent 50 lm (A), 30 lm (B) and 400 lm (C).

Fig. 10. Result of coarse alignment. The precision of the coarse alignment of the fluorescence and electron micrograph is within 1.5–5 lm. In the example shown, the
fluorescently labelled cell is displaced by 4.97 lm (green line) in respect to the electron micrograph. Bar represents 7 lm.

Correlation and imaging software
The Maps software (FEI Company) is a specially designed soft-
ware to correlate light and electron microscopy for FEI CorrSight
light microscopes and scanning electron microscope systems
[30,38]. In addition, Maps software can read any type of images
obtained by any type of light microscope. A similar software,
CORAL (correlative microscopy for life science) was developed by
TESCAN Orsay holding. With CORAL any image can be correlated
with TESCAN SEM systems.
In this section we describe our approach for correlative electron
microscopy, the preparation scheme and in detail the use of the
Maps software.
The examples given are from brain tissue: one that expresses
eGFP in astrocytes [141] and one where one neuron was stained
with Lucifer yellow. Fresh brain tissue was cut with a vibratome
106 C. Loussert Fonta, B.M. Humbel / Archives of Biochemistry and Biophysics 581 (2015) 98–110

Fig. 11. Fine alignment. With the radio buttons in the lower left corner the light
micrograph can be shifted and rotated to perfectly match the electron micrograph.
Due to local distortion this alignment might be repeated for distant structures. Bar
represents 4 lm.
Fig. 12. Fluorescence to identify the cell of interest. The neuron was labelled by
Lucifer yellow during physiological experiments. Correlating the fluorescence with
the electron micrograph identifies the cell that has been studied. Note: the intenser
fluorescence signal over the crease. Bar represents 9 lm.

Fig. 13. Tileset setup. For large overview at high resolution the area of interest is covered with a set of tiles defined by the pixel resolution and the frame size. There is an
overlap of 10% for subsequent stitching. At 3 points the focus is fixed (white circles, WD Points 1–3) to calculate the focus for each tile and the set is recorded. Bar represents
20 lm.
 C. Loussert Fonta, B.M. Humbel / Archives of Biochemistry and Biophysics 581 (2015) 98–110 107

Fig. 14. Tileset of a brain section. (A) A part of a large stitched tileset of 44.9 lm by 41 lm with 1 nm pixel resolution. When gradually zooming in (B and C) the individual
gold particles indicating astrocytes become visible (C). Note that the cellular ultrastructure in the astrocyte is remarkably well preserved by the Tokuyasu preparation
method. Bar represents 2 lm.
108 C. Loussert Fonta, B.M. Humbel / Archives of Biochemistry and Biophysics 581 (2015) 98–110
into 300 lm slices then they where immediately fixed in 2% form-
aldehyde and 0.2% glutaraldehyde. In a first step, the fluorescently
labelled neuron is located (Fig. 3).
Tissue blocks fixed with aldehydes, were impregnated with
2.3 M sucrose and then mounted on a sample support pin of the
cryo-ultramicrotome. The block on the pin is kept humid and cool
either by a self-made support dish [38] for imaging with a Zeiss
fluorescence microscope (Imager.Z2, Carl Zeiss Microimaging
GmbH, Germany) or a special holder (Fig. 4) designed for the
CorrSight light microscope (former Till Photonics, now FEI
Company, Munich).
A 3D stack is recorded to define the Z-position of the area of
interest, e.g., eGFP expressing astrocytes or a fluorescently labelled
neuron (Fig. 5) in brain tissue with the Zeiss Axiovision rel.4.8 soft-
ware (Carl Zeiss) or the LA software (Live Acquisition, Till
Photonics) integrated into the Maps software (FEI Company).
After freezing, the pin is mounted in a cryo-ultramicrotome
equipped with a fluorescence stereo microscope [38] and the block
is trimmed until the area of interest, using the 3D information
obtained in the light microscope and the observation of the fluo-
rescence in the microtome (Fig. 6).
The sections, often serial sections (Fig. 7), are collected on
carbon-coated electron microscopy grids.
The grids are sandwiched between a microscope slide and a
coverslip in PBS and sealed with nail polish. With a light micro-
scope low resolution, 20 objective NA 0.5, the whole grid is
imaged using a montage option in bright field (Fig. 8A) and fluores-
cence and then fluorescence of the section at higher resolution,
40 oil objective NA 1.3 (Figs. 7 and 8B). These maps are then used
to find the same areas in the electron microscope. Any fluorescence
microscope can be used but the CorrSight has an added advantage
that it operates with the correlative Maps software and all light
micrographs of one particular project are stored with their respec-
tive coordinates and are automatically linked to each other.
After light microscopy imaging is completed, the project is
transferred on the SEM computer and opened in the Maps software
installed on that system. The grids are recovered from the light
microscopy setup and further treated, e.g., by immuno gold label-
ling, post-stained with uranyl acetate and embedded in methyl cel-
lulose before loading into the STEM holder of the scanning electron
microscope [38]. An electron micrograph is recorded with the
Everhart–Thornley secondary electron detector. We usually take
an image of the central hole with the arrow and the number 1 at
the border of the grid, but any two points that are clearly visible
on the light and electron micrograph can be selected (Fig. 9). In
the 2 points alignment scheme, we select the very centre of the
hole and a corner of the number 1. In case the images are mirrored,
the light micrograph can be flipped to match the orientation of the
grid in the electron microscope.
Then the software rotates and stretches the light micrograph to
match the 2 points of the electron micrograph, which aligns the
light micrograph (or the entire project when imaged in the
CorrSight) with the current grid position in the SEM. After the
coarse alignment there might be a displacement in the range of
1.5–5.5 lm (Fig. 10). To fine align an individual section to compen-
sate for small shifts and distortions that may occur during the dry-
ing process of the section, the alignment can be repeated at higher
magnification. The software also has a 1 point alignment option for
X, Y translational shifts or a 3 point alignment to better compen-
sate for distortions.
For fine alignment the layers can be shifted and rotated with the
radio buttons in the ‘fine alignment’ window (Fig. 11, lower left
corner) until precise overlap is achieved.
With the overlay of the fluorescence signal and the electron
micrograph the fluorescently labelled neuron could unequivocally
be identified without the need of an electron microscopy marker
(Fig. 12).
For large image acquisition, tilesets covering the entire area of
interest with the desired pixel resolution, typically 1 nm, are
drawn (Fig. 13). Our usual imaging conditions are: 30 keV,
800 pA, 5 mm working distance, 6144  4096 pixels per frame,
1–3 ls dwell time, using the STEM III detector (FEI Company) in
the high-angle annular-dark field mode [142,143]. Up to now the
3 point focus regime was the most reliable. At 3 points (Fig. 13,
white circles) close to the area of interest the section is focused
at higher magnification, then brightness and contrast are adjusted
at the same magnification and imaging conditions the tileset will
be recorded. After all these adjustments are done the recording
can be started. With the specified settings, typical recording time
is about 1 h for a tileset of 8  11 images. The overlapping zone
of the tiles often is seen as a brighter strip, which is rather disturb-
ing. To avoid this effect, the whole area of interest, when possible
the whole mesh is pre-irradiated with a high current, 13–26 nA;
large frame size, 6144  4096 pixels; and short dwell time,
50 ns; for about 30 min.
The tiles can be stitched by the software and exported as tiff
files or raw files when they become too big. An other useful option
is the export as an HD view file (http://research.microsoft.com/en-
us/um/redmond/groups/ivm/HDView/) that, under the Windows
operating system, allows viewing the whole recorded field and to
zooming into the individual areas of interest (Fig. 14).
Outlook
Correlative light and electron microscopy has become a stan-
dard technique for many investigations in biomedical research.
With it, the micro- and nanoscopic dimensions in imaging are cov-
ered. Light microscopy can image areas in millimetre in X, Y and
200–300 lm in thickness at about 200 nm lateral resolution and
electron microscopy in the micrometre range and thickness in
the 100 nm range down to subnanometre resolution.
In biology there are many more dimensions to be covered.
Further, the morphological information can be complemented with
chemical information. Presently, soft X-ray microscopy stands
between light an electron microscopy. The thickness of a sample
that can be analysed is about 15 lm and the achieved resolution
below 50 nm [144]. Soft X-ray microscopy is usually associated
with a synchrotron [144,145] but laboratory based models are
upcoming [146]. Soft X-ray imaging in addition can provide chem-
ical information [147]. An other exciting development is combin-
ing NMR chemical data with electron microscopy and localisation
of an isotope-labelled precursors by nanoSIMS technology [148].
13
C labelled glucose was fed to animals and the metabolites could
be identified by NMR. After high-resolution mapping using elec-
tron microscopy the distribution of the isotope was localised
within cells of brain and liver tissue to study the glycogen metab-
olism [148].
The ideal situation would be to detect a diseased area in the
body of a living person, e.g., by MRI or X-ray computed tomogra-
phy, take a biopsy and narrow down the area by light microscopy
until high resolution imaging by electron microscopy. The morpho-
logical data could be complemented by functional data gathered by
NMR, nanoSIMS or mass spectroscopy.
Acknowledgments
The authors would like to thank Dr. J.M. Mateos and Dr. J. Ster
for brain tissue with Lucifer yellow labelled neurons and Dr. A.
Volterra for brain tissue with GPF labelled astrocytes. W.
Blanchard provided the images in Figs. 1 and 4. We are especially
 C. Loussert Fonta, B.M. Humbel / Archives of Biochemistry and Biophysics 581 (2015) 98–110
indebted to B. Lich, C. Mathisen, Dr. M. Langhorst, Dr. A. de Marco
and Dr. L. Hekking, I. Gestmann, FEI Company, for the fruitful col-
laboration with the Maps software. Dr. H. Schwarz and Dr. C.
Wurm are acknowledged for their collaboration on on-section
labelling (Fig. 2) and M. van der Werff and S. Gerrits for their sup-
port during the writing of the article. We also thank P. Brey for crit-
ical reading the manuscript. The STEM work was made possible by
the financial support of the Faculty of Biology and Medicine of the
University of Lausanne, Switzerland, and the Swiss National
Science Foundation, R’Equip grant 316030_128692.
References
[1] M. Osborn, R.E. Webster, K. Weber, J. Cell Biol. 77 (1978) R27–R34.
[2] R.E. Webster, M. Osborn, K. Weber, Exp. Cell Res. 117 (1978) 47–61.
[3] S.W. Hell, M. Dyba, S. Jakobs, Curr. Opin. Neurobiol. 14 (2004) 599–609.
[4] R. Henriques, C. Griffiths, E.H. Rego, M.M. Mhlanga, Biopolymers 95 (2011)
322–331.
[5] E. Betzig, G.H. Patterson, R. Sougrat, O.W. Lindwasser, S. Olenych, J.S.
Bonifacino, M.W. Davidson, J. Lippincott-Schwartz, H.F. Hess, Science 313
(2006) 1642–1645.
[6] M.J. Rust, M. Bates, X. Zhuang, Nat. Methods 3 (2006) 793–796.
[7] S. Modla, K. Czymmek, Micron 42 (2011) 773–792.
[8] J. Roth, M. Bendayan, E. Carlemalm, W. Villiger, M. Garavito, J. Histochem.
Cytochem. 29 (1981) 663–671.
[9] E. Carlemalm, R.M. Garavito, W. Villiger, J. Microsc. 126 (1982) 123–143.
[10] B.L. Armbruster, E. Kellenberger, E. Carlemalm, W. Villiger, R.M. Garavito, J.A.
Hobot, R. Chiovetti, J.D. Acetarin, in: J.P. Revel, T. Barnard, G.H. Haggis (Eds.),
The Science of Biological Specimen Preparation 1983, SEM Inc., AMF O’Hare,
IL 60666, 1983, pp. 77–81.
[11] J.D. Acetarin, E. Carlemalm, W. Villiger, J. Microsc. 143 (1986) 81–88.
[12] B.G. Timms, Am. J. Anat. 175 (1986) 267–275.
[13] G.R. Newman, J.A. Hobot, J. Histochem. Cytochem. 35 (1987) 971–981.
[14] J.A. Hobot, G.R. Newman, Scanning Microsc. Suppl. 5 (1991) S27–S41.
[15] C. Scala, G. Cenacchi, C. Ferrari, G. Pasquinelli, P. Preda, G.C. Manara, J.
Histochem. Cytochem. 40 (1992) 1799–1804.
[16] J.J. Bogers, H.A. Nibbeling, A.M. Deelder, E.A. Van Marck, J. Histochem.
Cytochem. 44 (1996) 43–48.
[17] G. Goping, G.A.J. Kuijpers, R. Vinet, H.B. Pollard, J. Histochem. Cytochem. 44
(1996) 289–295.
[18] B. Humbel, T. Marti, M. Müller, Beitr. Elektronenmikroskop. Direktabb. Oberfl.
16 (1983) 585–594.
[19] B. Humbel, M. Müller, in: M. Müller, R.P. Becker, A. Boyde, J.J. Wolosewick
(Eds.), The Science of Biological Specimen Preparation 1985, SEM Inc., AMF
O’Hare, 1986, pp. 175–183.
[20] B.M. Humbel, H. Schwarz, in: A.J. Verkleij, J.L.M. Leunissen (Eds.), Immuno-
Gold Labeling in Cell Biology, CRC Press, Boca Raton, 1989, pp. 115–134.
[21] H. Schwarz, B.M. Humbel, Scanning Microsc. Suppl. 3 (1989) 57–64.
[22] K.T. Tokuyasu, J. Cell Biol. 57 (1973) 551–565.
[23] K.T. Tokuyasu, S.J. Singer, J. Cell Biol. 71 (1976) 894–906.
[24] K.T. Tokuyasu, Histochem. J. 12 (1980) 381–403.
[25] G. Griffiths, K. Simons, G. Warren, K.T. Tokuyasu, Methods Enzymol. 96 (1983)
466–485.
[26] W. Liou, H.J. Geuze, J.W. Slot, Histochem. Cell Biol. 106 (1996) 41–58.
[27] J.W. Slot, H.J. Geuze, Nat. Protoc. 2 (2007) 2480–2491.
[28] T. Kurth, H. Schwarz, S. Schneider, P. Hausen, Cell Tissue Res. 286 (1996) 1–
12.
[29] H. Schwarz, in: H.A. Calderón Benavides, M.J. Yacamán, L.F. Jiménez, J.B.
Kouri (Eds.), Electron Microscopy 1998, ICEM 14, Institute of Physics
Publishing, Bristol, Philadelphia, 1998, pp. 865–866.
[30] H. Schwarz, B.M. Humbel, Methods Mol. Biol. 1117 (2014) 559–592.
[31] J.M. Robinson, T. Takizawa, A. Pombo, P.R. Cook, J. Histochem. Cytochem. 49
(2001) 803–808.
[32] T. Takizawa, J.M. Robinson, Placenta 24 (2003) 557–565.
[33] T. Takizawa, J.M. Robinson, J. Nippon Med. Sch. 71 (2004) 306–307.
[34] G. Vicidomini, M.C. Gagliani, M. Canfora, K. Cortese, F. Frosi, C. Santangelo, P.P.
Di Fiore, P. Boccacci, A. Diaspro, C. Tacchetti, Traffic 9 (2008) 1828–1838.
[35] H. Pluk, D.J. Stokes, B. Lich, B. Wieringa, J.A.M. Fransen, J. Microsc. 233 (2009)
353–363.
[36] M.A. Karreman, A.V. Agronskaia, A.J. Verkleij, F.F.M. Cremers, H.C. Gerritsen,
B.M. Humbel, Biol. Cell 101 (2009) 287–299.
[37] A.V. Agronskaia, J.A. Valentijn, L.F. van Driel, C.T.W.M. Schneijdenberg, B.M.
Humbel, P.M.P. van Bergen en Henegouwen, A.J. Verkleij, A.J. Koster, H.C.
Gerritsen, J. Struct. Biol. 164 (2008) 183–189.
[38] C. Loussert Fonta, A. Leis, C. Mathisen, D.S. Bouvier, W. Blanchard, A. Volterra,
B. Lich, B.M. Humbel, J. Struct. Biol. 189 (2015) 53–61.
[39] A. Sartori, R. Gatz, F. Beck, A. Rigort, W. Baumeister, J.M. Plitzko, J. Struct. Biol.
160 (2007) 135–145.
[40] F.G.A. Faas, M. Bárcena, A.V. Agronskaia, H.C. Gerritsen, K.B. Moscicka, A.A.
Diebolder, L.F. Van Driel, R.W.A.L. Limpens, E. Bos, R.B.G. Ravelli, R.I. Koning,
A.J. Koster, J. Struct. Biol. 181 (2013) 283–290.
109
[41] A. Manninen, P. Verkade, S. Le Lay, J. Torkko, M. Kasper, J. Füllekrug, K.
Simons, Mol. Cell. Biol. 25 (2005) 10087–10096.
[42] P. Verkade, J. Microsc. 230 (2008) 317–328.
[43] M. Grabenbauer, W.J.C. Geerts, J. Fernadez-Rodriguez, A. Hoenger, A.J. Koster,
T. Nilsson, Nat. Methods 2 (2005) 857–862.
[44] A.A. Mironov, R.S. Polishchuk, G.V. Beznoussenko, Methods Cell Biol. 88
(2008) 83–95.
[45] A.A. Mironov, G.V. Beznoussenko, J. Microsc. 235 (2009) 308–321.
[46] C.-L. Forestier, C. Machu, C. Loussert, P. Pescher, G.F. Späth, Cell Host Microbe
9 (2011) 319–330.
[47] C. Loussert, C.-L. Forestier, B.M. Humbel, Methods Cell Biol. 111 (2012) 59–73.
[48] S.S. Biel, K. Kawaschinski, K.-P. Wittern, U. Hintze, R. Wepf, J. Microsc. 212
(2003) 91–99.
[49] M.S. Lucas, M. Günthert, P. Gasser, F. Lucas, R. Wepf, Methods Cell Biol. 111
(2012) 325–356.
[50] K. Wilke, K. Wick, F.J. Keil, K.-P. Wittern, R. Wepf, S.S. Biel, Exp. Dermatol. 17
(2008) 73–80.
[51] T.J. Deerinck, M.E. Martone, V. Lev-Ram, D.P.L. Green, R.Y. Tsien, D.L. Spector,
S. Huang, M.H. Ellisman, J. Cell Biol. 126 (1994) 901–910.
[52] B.M. Humbel, M.D.M. de Jong, W.H. Müller, A.J. Verkleij, Microsc. Res. Tech. 42
(1998) 43–48.
[53] E. Brown, P. Verkade, Protoplasma 244 (2010) 91–97.
[54] R.E. Pagano, M.A. Sepanski, O.C. Martin, J. Cell Biol. 109 (1989) 2067–2079.
[55] Y.-D. Stierhof, in: A. Cavalier, D. Spehner, B.M. Humbel (Eds.), Handbook for
Cryo-Preparation Methods for Electron Microscopy, CRC Press Inc., Boca
Raton, USA, 2008, pp. 588–616.
[56] K. Pohl, Y.-D. Stierhof, Microsc. Res. Tech. 42 (1998) 59–65.
[57] X. Shu, V. Lev-Ram, T.J. Deerinck, Y. Qi, E.B. Ramko, M.W. Davidson, Y. Jin,
M.H. Ellisman, R.Y. Tsien, PLoS Biol. 9 (2011) e1001041.
[58] G. Gaietta, T.J. Deerinck, S.R. Adams, J. Bouwer, O. Tour, D.W. Laird, G.E.
Sosinsky, R.Y. Tsien, M.H. Ellisman, Science 296 (2002) 503–507.
[59] R.Y. Tsien, Annu. Rev. Biochem. 67 (1998) 509–544.
[60] R.Y. Tsien, Integr. Biol. 2 (2010) 77–93.
[61] M.G. Paez-Segala, M.G. Sun, G. Shtengel, S. Viswanathan, M.A. Baird, J.J.
Macklin, R. Patel, J.R. Allen, E.S. Howe, G. Piszczek, H.F. Hess, M.W. Davidson,
Y. Wang, L.L. Looger, Nat. Methods (2014), http://dx.doi.org/10.1038/
nmeth.3225.
[62] J. Stinchcombe, H. Nomoto, D. Cutler, C. Hopkins, J. Cell Biol. 131 (1995)
1387–1401.
[63] M. Grabenbauer, Methods Cell Biol. 111 (2012) 117–138.
[64] C. Meißlitzer-Ruppitsch, C. Röhrl, J. Neumüller, M. Pavelka, A. Ellinger, J.
Microsc. 235 (2009) 322–335.
[65] C. Meißlitzer-Ruppitsch, M. Vetterlein, H. Stangl, S. Maier, J. Neumüller, M.
Freissmuth, M. Pavelka, A. Ellinger, Histochem. Cell Biol. 130 (2008) 407–419.
[66] D. Bishop, I. Nikić, M. Brinkoetter, S. Knecht, S. Potz, M. Kerschensteiner, T.
Misgeld, Nat. Methods 8 (2011) 568–570.
[67] J.H. Luft, J. Biophys. Biochem. Cytol. 9 (1961) 409–414.
[68] A.M. Glauert, R.H. Glauert, J. Biophys. Biochem. Cytol. 4 (1958) 191–194.
[69] A.R. Spurr, J. Ultrastruct. Res. 26 (1969) 31–43.
[70] H. Kushida, J. Electron Microsc. 12 (1963) 71–73.
[71] W. Stäubli, J. Cell Biol. 16 (1963) 197–201.
[72] M. Rosenberg, P. Bartl, J. Leško, J. Ultrastruct. Res. 4 (1960) 298–303.
[73] B.E. Causton, Proc. RMS 16 (1981) 265–267.
[74] B.E. Causton, in: J.M. Polak, I.M. Varndell (Eds.), Immunolabelling for Electron
Microscopy, Elsevier Science Publishers, Amsterdam, 1984, pp. 29–36.
[75] B.E. Causton, in: M. Müller, R.P. Becker, A. Boyde, J.J. Wolosewick (Eds.), The
Science of Biological Specimen Preparation 1985, SEM Inc., AMF O’Hare, 1986,
pp. 209–214.
[76] I.R. Gibbons, Nature 184 (1959) 375–376.
[77] N. Matsko, M. Müller, J. Struct. Biol. 152 (2005) 92–103.
[78] L. Wang, B.M. Humbel, E.W. Roubos, Brain Res. Protoc. 15 (2005) 155–163.
[79] G.R. Newman, J.A. Hobot, Resin Microscopy and On-
Section Immunocytochemistry, Springer-Verlag, Berlin, Heidelberg, New
York, 2001.
[80] K. Luby-Phelps, G. Ning, J. Fogerty, J.C. Besharse, J. Histochem. Cytochem. 51
(2003) 271–274.
[81] K. Bell, S. Mitchell, D. Paultre, M. Posch, K. Oparka, Plant Physiol. 161 (2013)
1595–1603.
[82] U. Riehle, Über die Vitrifizierung verdünnter wässriger Lösungen, Federal
Institute of Technology (ETH), Zürich, 1968.
[83] M. Müller, H. Moor, in: J.P. Revel, T. Barnard, G.H. Haggis (Eds.), Science of
Biological Specimen Preparation 1983, SEM Inc., AMF O’Hare, 1984, pp. 131–
138.
[84] H. Moor, in: R.A. Steinbrecht, K. Zierold (Eds.), Cryotechniques in Biological
Electron Micoscopy, Springer-Verlag, Berlin, Heidelberg, 1987, pp. 175–191.
[85] D. Studer, M. Michel, M. Wohlwend, E.B. Hunziker, M.D. Buschmann, J.
Microsc. 179 (1995) 321–332.
[86] D. Studer, W. Graber, A. Al-Amoudi, P. Eggli, J. Microsc. 203 (2001) 285.
[87] A. Van Harreveld, J. Crowell, S.K. Malhotra, J. Cell Biol. 25 (1965) 117–137.
[88] H. Fernández-Morán, Science 129 (1959) 1284–1285.
[89] H. Fernández-Morán, Ann. N. Y. Acad. Sci. 85 (1960) 689–713.
[90] M. Müller, T. Marti, S. Kriz, in: P. Brederoo, W. de Priester (Eds.), Proc. 7th Eur.
Congr. Electron Microsc., The Hague, 1980, pp. 720–721.
[91] W. Kukulski, M. Schorb, S. Welsch, A. Picco, M. Kaksonen, J.A.G. Briggs,
Methods Cell Biol. 111 (2012) 235–257.
110 C. Loussert Fonta, B.M. Humbel / Archives of Biochemistry and Biophysics 581 (2015) 98–110
[92] S.J. Nixon, R.I. Webb, M. Floetenmeyer, N. Schieber, H.P. Lo, R.G. Parton, Traffic
10 (2009) 131–136.
[93] W. Kukulski, M. Schorb, S. Welsch, A. Picco, M. Kaksonen, J.A.G. Briggs, J. Cell
Biol. 192 (2011) 111–119.
[94] C.J. Peddie, K. Blight, E. Wilson, C. Melia, J. Marrison, R. Carzaniga, M.-C.
Domart, P. O’Toole, B. Larijani, L.M. Collinson, Ultramicroscopy (2014), http://
dx.doi.org/10.1016/j.ultramic.2014.1002.1001i.
[95] S. Watanabe, A. Punge, G. Hollopeter, K.I. Willig, R.J. Hobson, M.W. Davis, S.W.
Hell, E.M. Jorgensen, Nat. Methods 8 (2011) 80–84.
[96] S. Watanabe, E.M. Jorgensen, Methods Cell Biol. 111 (2012) 283–306.
[97] P. Walther, A. Ziegler, J. Microsc. 208 (2002) 3–10.
[98] C. Buser, P. Walther, J. Microsc. 230 (2008) 268–277.
[99] D. Studer, M. Chiquet, E.B. Hunziker, J. Struct. Biol. 117 (1996) 81–85.
[100] D.A. Lennette, Am. J. Clin. Pathol. 69 (1978) 647–648.
[101] J. Rodriguez, F. Deinhardt, Virology 12 (1960) 316–317.
[102] K.L. McDonald, R.I. Webb, J. Microsc. 243 (2011) 227–233.
[103] W. Bernhard, J. Ultrastruct. Res. 27 (1969) 250–265.
[104] E.J. Behrman, in: J.P. Revel, T. Barnard, G.H. Haggis (Eds.), The Science of
Biological Specimen Preparation 1983, SEM Inc., AMF O’Hare, IL 60666, 1984,
pp. 1–5.
[105] P. Maupin-Szamier, T.D. Pollard, J. Cell Biol. 77 (1978) 837–853.
[106] D.L. White, S.B. Andrews, J.W. Faller, R.J. Barrnett, Biochim. Biophys. Acta 436
(1976) 577–592.
[107] B.M. Humbel, in: A. Cavalier, D. Spehner, B.M. Humbel (Eds.), Handbook for
Cryo-Preparation Methods for Electron Microscopy, CRC Press Inc., Boca
Raton, USA, 2008, pp. 320–341.
[108] Y.-D. Stierhof, E. van Donselaar, H. Schwarz, B.M. Humbel, in: A. Cavalier, D.
Spehner, B.M. Humbel (Eds.), Handbook for Cryo-Preparation Methods for
Electron Microscopy, CRC Press Inc., Boca Raton, USA, 2008, pp. 343–365.
[109] M. Perkovic, M. Kunz, U. Endesfelder, S. Bunse, C. Wigge, Z. Yu, V.-V.
Hodirnau, M.P. Scheffer, A. Seybert, S. Malkusch, E.M. Schuman, M.
Heilemann, A.S. Frangakis, J. Struct. Biol. 186 (2014) 205–213.
[110] K.T. Tokuyasu, A.H. Dutton, B. Geiger, S.J. Singer, Proc. Natl. Acad. Sci. U.S.A. 78
(1981) 7619–7623.
[111] K. Zierold, Scanning Electron Microsc. (1982) 1205–1214.
[112] J. Dubochet, A.W. McDowall, B. Menge, E.N. Schmid, K.G. Lickfeld, J. Bacteriol.
155 (1983) 381–390.
[113] P. Frederik, W.M. Busing, W.M.A. Hax, J. Microsc. 126 (1982) RP1–RP2.
[114] M.F. Wendt-Gallitelli, P. Stohr, H. Wolburg, W. Schlote, Scanning Electron
Microsc. (1980) 499–509.
[115] A. Al-Amoudi, L.P.O. Norlen, J. Dubochet, J. Struct. Biol. 148 (2004) 131–135.
[116] B. Zuber, M. Haenni, T. Ribeiro, K. Minnig, F. Lopes, P. Moreillon, J. Dubochet, J.
Bacteriol. 188 (2006) 6652–6660.
[117] J. Dubochet, A. Al-Amoudi, C. Bouchet-Marquis, M. Eltsov, B. Zuber, in: A.
Cavalier, D. Spehner, B.M. Humbel (Eds.), Handbook for Cryo-Preparation
Methods for Electron Microscopy, CRC Press Inc., Boca Raton, USA, 2008, pp.
255–289.
[118] A. Sartori, R. Gatz, F. Beck, A. Kossel, A.P. Leis, W. Baumeister, J.M. Plitzko,
Microsc. Microanal. 11 (2005) 16–17.
[119] H. Iijima, Y. Fukuda, Y. Arai, S. Terakawa, N. Yamamoto, K. Nagayama, J.
Struct. Biol. 185 (2014) 107–115.
[120] W.E. Moerner, M. Orrit, Science 283 (1999) 1670–1676.
[121] M. Gruska, O. Medalia, W. Baumeister, A. Leis, J. Struct. Biol. 161 (2008) 384–
392.
[122] A. Rigort, F.J.B. Bäuerlein, A. Leis, M. Gruska, C. Hoffmann, T. Laugks, U. Böhm,
M. Eibauer, H. Gnaegi, W. Baumeister, J.M. Plitzko, J. Struct. Biol. 172 (2010)
169–179.
[123] C. Hagen, S. Werner, S. Carregal-Romero, A.N. Malhas, B.G. Klupp, P.
Guttmann, S. Rehbein, K. Henzler, T.C. Mettenleiter, D.J. Vaux, W.J. Parak, G.
Schneider, K. Grünewald, Ultramicroscopy 146 (2014) 46–54.
[124] Y. Fukuda, N. Schrod, M. Schaffer, L.R. Feng, W. Baumeister, V. Lucic,
Ultramicroscopy 143 (2014) 15–23.
[125] S. Jones, S.K. Chapman, P.R. Crocker, G. Carson, D.A. Levison, J. Clin. Pathol. 35
(1982) 425–429.
[126] M.A. Karreman, A.V. Agronskaia, E.G. van Donselaar, K. Vocking, F. Fereidouni,
B.M. Humbel, C.T. Verrips, A.J. Verkleij, H.C. Gerritsen, J. Struct. Biol. 180
(2012) 382–386.
[127] H. Iijima, H. Minoda, Y. Arai, Y. Kaneko, K. Nagayama, An electron-photon
hybrid microscope for on-line CLEM, Gordon Research Conference on 3DEM,
Il Ciocco, Italy, 2010.
[128] I. Solomonov, D. Talmi-Frank, Y. Milstein, S. Addadi, A. Aloshin, I. Sagi, Sci.
Rep. 4 (2014) 5987. 5910.1038/srep05987.
[129] T. Kinoshita, Y. Mori, K. Hirano, S. Sugimoto, K.-I. Okuda, S. Matsumoto, T.
Namiki, T. Ebihara, M. Kawata, H. Nishiyama, M. Sato, M. Suga, K.
Higashiyama, K. Sonomoto, Y. Mizunoe, S. Nishihara, C. Sato, Microsc.
Microanal. 20 (2014) 469–483.
[130] H. Nishiyama, M. Koizumi, K. Ogawa, S. Kitamura, Y. Konyuba, Y. Watanabe,
N. Ohbayashi, M. Fukuda, M. Suga, C. Sato, Ultramicroscopy 147 (2014) 86–
97.
[131] N. Liv, I. Lazić, P. Kruit, J.P. Hoogenboom, Ultramicroscopy 143 (2014) 93–99.
[132] N. Liv, A.C. Zonnevylle, A.C. Narvaez, A.P.J. Effting, P.W. Voorneveld, M.S.
Lucas, J.C. Hardwick, R.A. Wepf, P. Kruit, J.P. Hoogenboom, PLoS ONE 8 (2013)
e55707.
[133] A.C. Zonnevylle, R.F.C. van Tol, N. Liv, A.C. Narvaez, A.P.J. Effting, P. Kruit, J.P.
Hoogenboom, J. Microsc. 252 (2013) 48–70.
[134] C.J. Peddie, N. Liv, J.P. Hoogenboom, L.M. Collinson, Methods Cell Biol. 124
(2014) 363–389.
[135] K.D. Micheva, S.J. Smith, Neuron 55 (2007) 25–36.
[136] D. Oberti, M.A. Kirschmann, R.H.R. Hahnloser, Front. Neurosci. 5 (2011) 1–8.
[137] W.-J. Wang, H.G. Tay, R. Soni, G.S. Perumal, M.G. Goll, F.P. Macaluso, J.M.
Asara, J.D. Amack, M.-F.B. Tsou, Nat. Cell Biol. 15 (2013) 591–601.
[138] M. Husain, C. Thomas, M. Kirschmann, D. Oberti, R. Hahnloser, Microsc.
Microanal. 17 (2011) 242–243.
[139] C.L. Schwartz, V.I. Sarbash, F.I. Ataullakhanov, J.R. McIntosh, D. Nicastro, J.
Microsc. 227 (2007) 98–109.
[140] L.F. Van Driel, J.A. Valentijn, K.M. Valentijn, R.I. Koning, A.J. Koster, Eur. J. Cell
Biol. 88 (2009) 669–684.
[141] C. Nolte, M. Matyash, T. Pivneva, C.G. Schipke, C. Ohlemeyer, U.-K. Hanisch, F.
Kirchhoff, H. Kettenmann, Glia 33 (2001) 72–86.
[142] M.T. Otten, D.J. Stenzel, D.R. Cousens, B.M. Humbel, J.L.M. Leunissen, Y.-D.
Stierhof, W.M. Busing, Scanning 14 (1992) 282–289.
[143] A.E. Yakushevska, M.N. Lebbink, W.J.C. Geerts, L. Spek, E.G. van Donselaar,
K.A. Jansen, B.M. Humbel, J.A. Post, A.J. Verkleij, A.J. Koster, J. Struct. Biol. 159
(2007) 381–391.
[144] E.A. Smith, B.P. Cinquin, M. Do, G. McDermott, M.A. Le Gros, C.A. Larabell,
Ultramicroscopy 143 (2014) 33–40.
[145] R. Carzaniga, M.-C. Domart, E. Duke, L.M. Collinson, Methods Cell Biol. 124
(2014) 151–178.
[146] D.B. Carlson, J. Gelb, V. Palshin, J.E. Evans, Microsc. Microanal. 19 (2013) 22–
29.
[147] S. Roudeau, A. Carmona, L. Perrin, R. Ortega, Anal. Bioanal. Chem. 406 (2014)
6979–6991.
[148] Y. Takado, G. Knott, B.M. Humbel, S. Escrig, M. Mojgan, A. Meibom, A.
Comment, Nanomed. NBM 11 (2015) 239–245.