Seminars in Cell & Developmental Biology 20 (2009) 910–919
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Seminars in Cell & Developmental Biology
journal homepage: www.elsevier.com/locate/semcdb
Review
Studying intracellular transport using high-pressure freezing and Correlative
Light Electron Microscopy
Edward Brown a , Judith Mantell a,b , Debbie Carter b , Gini Tilly b , Paul Verkade a,b,c,∗
a
Department of Biochemistry, School of Medical Sciences, University Walk, Bristol, BS8 1TD, United Kingdom
Wolfson Bioimaging Facility, School of Medical Sciences, University Walk, Bristol, BS8 1TD, United Kingdom
c
Department of Physiology and Pharmacology, School of Medical Sciences, University Walk, Bristol, BS8 1TD, United Kingdom
b
a r t i c l e
i n f o
Article history:
Available online 4 August 2009
Keywords:
Correlative Light Electron Microscopy
CLEM
High-pressure freezing
Electron microscopy
Intracellular trafficking
a b s t r a c t
Correlative Light Electron Microscopy (CLEM) aims at combining the best of light and electron microscopy
in one experiment. Light microscopy (LM) is especially suited for providing a general overview with
data from lots of different cells and by using live cell imaging it can show the history or sequence of
events between or inside cells. Electron microscopy (EM) on the other hand can provide a much higher
resolution image of a particular event and provide additional spatial information, the so-called reference
space. CLEM thus has certain strengths over the application of both LM and EM techniques separately.
But combining both modalities however generally also means making compromises in one or both of the
techniques. Most often the preservation of ultrastructure for the electron microscopy part is sacrificed.
Ideally samples should be visualized in its most native state both in the light microscope as well as
the electron microscope. For electron microscopy this currently means that the sample will have to be
cryo-fixed instead of the standard chemical fixation. In this paper we will discuss the rationale for using
cryofixation for CLEM experiments. In particular we will highlight a CLEM technique using high-pressure
freezing in combination with live cell imaging. In addition we examine some of the EM analysis tools that
may be useful in combination with CLEM techniques.
© 2009 Elsevier Ltd. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
Correlative Light Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ultrastructural preservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Live cell imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Visualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
The introduction of Green Fluorescent Protein (GFP) technology
as a clonable fluorescent tag onto proteins [1] has revolutionized
life science research. It made it possible to do time-resolved imaging of proteins in living cells and organisms and is an essential tool
in modern day research. It has led to new insights of how motile and
∗ Corresponding author at: School of Medical Sciences, University Walk, Bristol,
BS8 1TD, United Kingdom. Tel.: +44 117 3312179; fax: +44 117 3312168.
E-mail address: [email protected] (P. Verkade).
1084-9521/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.semcdb.2009.07.006
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dynamic cells really are, but also made us aware that some structures also thought to be very dynamic are actually mostly stable (i.e.
caveolae [2]). However, partially due to the emission wavelength
of the fluorophores (in the range of 100 s of nm) the resolution of
standard light microscopes is also within the order of 100 s of nm.
Structures within a cell are mostly in the range of 10 s of nm. Hence
there has been an increasing effort to get higher resolution from
the fluorescence light microscope. This eventually led to breaking Abbe’s law of refraction/resolution. Nowadays there are several
systems such as STED [3], SSIM [4], STORM [5], and PALM [6] available that are able to get a resolution below 100 nm (see the paper
by Patterson in this issue). In addition most of these systems are
E. Brown et al. / Seminars in Cell & Developmental Biology 20 (2009) 910–919
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now also able to do live cell imaging. There is one important aspect
that images from an electron microscope have that is not obtained
from a fluorescence-based light microscopic image. Whereas in
the light microscope only one or more fluorophores light up in
these high-resolution systems, the electron microscope does not
only visualize the electron dense marker but also gives a lot of
information about the surrounding material. This so-called reference space can give us information on whether a marker is inside
the lumen of a membrane-bound carrier or attached to the outside of the carrier. It may also tell us whether that same carrier is
attached to a microtubule or to the actin cytoskeleton, etc. It is one
of the aspects that is often overlooked when high-resolution light
microscopy is compared to electron microscopy. But as we hope
to show in this paper it is the combination of both microscopy
techniques that is more than just the sum of the parts. This is
not only true for the combination of EM with “standard” LM techniques but also with the high-resolution LM techniques as was aptly
pointed out by Jennifer Lippincott-Schwartz when high-resolution
light microscopy was named technique of the year 2008 by Nature
Methods [7].
and quantum dots [17,18] were developed. On the instrumentation
side, the concepts of high-throughput analysis and high-resolution
LM imaging [19] were introduced for CLEM. Advances in fixation
techniques were described [20,21] and the full integration of a
light microscopy into the electron microscope was accomplished
[22,23].
When we set out to perform CLEM experiments on intracellular
transport we set ourselves a set of criteria that a CLEM experiment
would have to fulfill.
These were (more or less in order of importance):
1.1. Correlative Light Electron Microscopy
2. Ultrastructural preservation
When one is thus studying structures that are below the resolution of the light microscope it is in most cases impossible to assign a
label to a specific domain within these structures. For these issues
electron microscopy (EM) has been the technique of choice. The
major limitation here is that living samples cannot be observed and
it needs a series of experiments to recapitulate the sequence of an
event. Combining the advantages of live cell imaging and the high
resolution of the EM is the way to overcome with this issue. Correlative Light Electron Microscopy (CLEM) techniques try to do exactly
that. CLEM techniques have been around for decades already (see
for instance the book edited by Hayat [8]) but have recently undergone a renaissance and the interest is still growing. Whereas in
earlier days the correlation between the light and electron microscopical image would mainly be made on different samples, current
techniques focus on visualizing the exact same structure or event
both in the LM and the EM (Fig. 1). Using such an approach the
history of events can be reconciled at lower resolution with light
microscopy but the event of interest can be studied at high resolution. This then allows us for example to draw conclusions on the
type of membrane structure that is formed. Using either live cell
imaging or electron microscopy alone will never allow us to do this
(see e.g. Fig. 1F). As was described in one of the reviews of the special
“Imaging in Cell Biology” series (Nat. Rev. Mol. Cell Biol. 2003, sept.
supp.), CLEM is one of the hot topics within the imaging field [9].
Several methods have appeared in recent years making significant
improvements to the technique for Cell Biological studies. It is not
our intent to provide a review on all the existing CLEM techniques
but rather we will limit ourselves to highlighting some examples
and we will discuss those and a few more examples further in the
“Markers” paragraph.
The new era of CLEM more or less started with the paper by
Polishchuk et al. [10] in which they combined GFP LM imaging
with immuno-labeling the GFP for EM. The antibody to GFP was
visualized either by performing a DAB precipitation reaction [10]
or by immuno-gold staining [11]. This group was to our knowledge also the first to introduce the term CLEM for Correlative
Light Electron Microscopy [12]. It should be noted that in the
microscopy field the term CLEM is also used for Controlled Light
Exposure Microscopy [13]. The groups of Nilsson [14] and later
on also Ellinger [15] succeeded in directly photo converting the
GFP signal into an electron dense DAB precipitate, avoiding antibody labeling that is very dependent on epitope availability. As
an alternative for GFP labeling the FlAsh/ReAsh compounds [16]
Artifacts introduced during live cell imaging will generally be
below the resolution of the light microscope. It is only when cells
are processed for electron microscopy that the preservation of
ultrastructure becomes an issue. In a standard electron microscopical preparation experiment there are 2 steps that most critically
influence the preservation of the structure. The first is the fixation of the sample and the second is the dehydration step where
cellular water is replaced by a solvent and eventually for an embedding resin. There are 2 possible ways to fix or immobilize a sample;
chemical or physical cryofixation. The speed of fixation and the
degree of cross-linking are the most important factors influencing whether a sample is fixed well (resembling its natural state).
The mechanism of conventional room temperature chemical fixation is based on diffusion of aldehydes such as paraformaldehyde
and glutaraldehyde, and for cell cultures fixation is achieved in
the range of seconds. Aldehydes mainly cross-link proteins and
it is important to note that lipids are not cross-linked or immobilized at all in this initial step. It is only in later steps involving
for instance osmium tetroxide where lipids are cross-linked/fixed
as well. Although probably not always inducing artifacts, chemical
fixation, including the subsequent processing at room temperature,
has been shown to induce alterations in morphology [24,25]. Sometimes it is the dehydration step rather than the fixation itself that is
considered the main source of artifacts [26]. Murk and coworkers
[27] however clearly demonstrated that the first chemical fixation
step alone can lead to substantial shrinkage of various intracellular
endocytic membrane structures when compared to cryofixation. In
contrast to chemical fixation, cryofixation of samples takes place
in the millisecond range [28] and fixes everything. The principle
behind cryofixation (or immobilization as it is sometimes preferred) is to remove the thermal energy (heat) from the system
as fast as possible, thus leaving no energy for proteins to move
anymore. Besides the extraction of material and the accompanying
shrinkage of organelles there is also a difference in the contrast of
the samples (Fig. 2).
If preserving ultrastructure in its most native state is the goal,
it is now generally accepted that the method of choice for fixation
should be cryofixation. Cryofixation can be done by slam or impact
freezing, plunge freezing, or by high-pressure freezing (HPF). HPF
not only reaches a greater depth at which the sample is well frozen,
it is generally more reproducible [28–30]. Cryofixation by HPF lowers the melting point of water by 20 ◦ C [31]. Ice crystal formation
therefore starts later than with other cryofixation techniques, and
1. Retain the best possible ultrastructure for the analysis in the
electron microscope.
2. Possibility to do live cell imaging.
3. Use markers that are both fluorescent and electron dense, or that
the markers need as little as possible processing for visualization.
4. “Easy” retracing/visualization of the region/event of interest.
We will go through these steps in the above order.
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Fig. 1. The principle of CLEM.
Model describing how a CLEM experiment could be performed. We will do this using the internalization of 2 fluorescent proteins as an example but the steps in the process
are the same for most CLEM experiments. The process of internalization would be followed live at the light microscopical level (A–C). When an interesting event is observed,
like in this case segregation of the 2 fluorophores (C) the sample is fixed and processed for electron microscopy. The same cell is first retraced in the electron microscope
(D). Subsequently we can analyze the sample at higher resolution (E) and the segregation event can now be studied in more detail. It is important to note that we are sure
we are studying a segregation event at high resolution in the electron microscope since we have the live imaging data (history). This can be nicely demonstrated in image F
where an endocytic structure was observed in the electron microscope with some protrusions attached to it. From this single image it is impossible to deduct whether these
membrane extensions are fusing with or segregating from the central endocytic structure.
ice crystal growth is also slower. In HPF a sample is contained in
a closed carrier. The carrier is put under high pressure (around
2050 bar) and is immediately thereafter sprayed with LN2. In this
way the water in the sample is frozen so fast that it becomes vitrified and it does not get a chance to form ice crystals. Whereas
vitrified water takes up as much space as liquid water, crystalline
water (ice crystals) will take up more space than the cellular water
and push proteins aside to form aggregates. After the freezing process, samples ideally would be processed for native cryosectioning
(also known as frozen hydrated sectioning or CEMOVIS [32]) and
analysis in a cryo microscope to avoid any addition of chemical fixatives or stains. We are currently technically not able to perform a
CLEM experiment (with HPF) in this way. In most cases the samples
will be processed for freeze substitution. In this process the water
is replaced by a solvent and chemically fixed at low temperature
and subsequently the sample is infiltrated with a resin for further
processing at room temperature [20,30]. By performing the dehydration and chemical fixation steps at low temperature we limit the
artifacts induced by the standard room temperature processing to
such extent that the artifacts will be below the detection limit of
our resolution requirements. Obviously, this process is not always
successful. This may be dependent on a lot of different factors, such
as the thickness of the sample, the water content, the medium surrounding the samples, etc. but also by the handling of the samples
[30]. We think it is important to show what the artifacts associated
with cryofixation and the subsequent processing look like in order
for researchers to be able to identify them (Fig. 3).
So far there are only a few papers that have performed CLEM
experiments using HPF. The first published results used the introduction of tissue specific LM dyes into the sample [33,34]. After
HPF and freeze substitution the block was sectioned and the block
face analyzed under the light microscope. Some dyes are still fluorescent when processed this way. This helped the researchers to
zoom into a particular cell type for further study in the EM. MüllerReichert and colleagues used GFP expressing C. elegans worms to
follow mitotic processes [35]. Once a specific stage was observed
they could freeze the worm or embryo and study the processed
sample knowing they had captured a specific cell cycle stage. A
recent paper by Jones et al. [36] used a similar approach to capture hatching stages of Schistosoma embryos. They used standard
bright field imaging video microscopy to stage their animals and
subsequently performed high-pressure freezing and freeze substitution on the samples. In the paper by Müller-Reichert there was
no further need to visualize the GFP after the freezing and freeze
substitution steps. This is a problem in general as after freeze substitution GFP is generally not fluorescent anymore. Sims and Hardin
developed a HPF and freeze substitution protocol that retains the
structure of the GFP and hence GFP is still fluorescent in the pro-
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Fig. 2. Chemical fixation vs. high-pressure freezing.
HELA cells were processed via conventional room temperature fixation and embedding (A + B) or via HPF and subsequent low-temperature fixation and embedding (C + D).
The overview images (A and C) show the difference in contrast obtained by the 2 different methods. Chemically fixed samples are generally dark and HPF processed samples
much lighter (note, the background in both samples is equal). This is because osmium is much more reactive at room temperature than at sub-zero temperatures. If we zoom
in to the Golgi area within the cell it is apparent that the membranes in chemically fixed samples are wrinkled because the structure has shrunk (B) whereas the HPF samples
have straighter membranes that still appear to be under tension (D).
cessed block [37]. Although this development is very important, as
it would be ideal to be able to have fluorescent GFP in an EM block,
our feeling is that for this particular protocol there have been too
many sacrifices made to the preservation of the ultrastructure and
the advantages of using HPF have been partially lost. Recently the
group of Parton [38] did succeed in retaining both GFP fluorescence
and excellent ultrastructure by high-pressure freezing and freeze
substitution of Zebrafish. The critical step in this process was to
omit osmium tetroxide from the freeze substitution protocol and
embedding in Lowicryl HM20. We have been experimenting with
Lowicryl embedding after HPF as well for immuno-labeling purposes (see later) but have not been able to retain GFP fluorescence
in culture-grown cells yet. It will require further studies to determine if the protocol developed by Nixon et al. will be applicable to
more types of specimen.
As outlined, samples would ideally be analyzed in the EM without any further processing. Especially the group of Baumeister
has promoted the idea that the EM part of a CLEM experiment
should be done in a cryo electron microscope [21,39]. Their main
purpose for using fluorescence light microscopy in a CLEM experiment is to find the location of fluorescent cells/structures. They can
then be easily retraced in the cryo electron microscope, avoiding
beam damage/heating of the sample normally induced by scanning the sample for the right structure in the electron microscope.
To this end they [40] and others [41] have developed a cryo fluorescence stage for the light microscope. A thin frozen section
on an EM finder grid can be analyzed for fluorescence at LN2
temperatures and the right coordinates of the finder grid used
for retracing in the EM. These stages have a limited NA of 0.55,
resulting in resolution of just over 1 ␮m [40] and are so far limited to screening purposes. One important aspect that is lacking
for the use of these stages (other then screening and retracing)
in intracellular membrane transport studies is that it is impossible to do live cell imaging or a fast transfer from the light
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Fig. 3. Ice crystal formation artifacts in HPF.
A piece of spleen tissue was high-pressure frozen and freeze substituted to Epon. The outside of the sample is to the left in overview A. After a certain “boundary” the material
quickly shows signs of ice damage. White arrows point to zooms of the region (B–D). B shows a zoom of the region that is well preserved and all structures show excellent
morphology. The chromatin inside the nucleus (N) is visible as dark amorphous material and nuclear pores can be identified where the chromatin is not as dense and appears
to make way for the pore. Inside the cytoplasm the appearance is “crowded” without empty spaces and intracellular membranes appear as sharp dark lines when they have
been sectioned perpendicular. In the transition zone (C) the first signs of protein aggregation appear inside the nucleus (white arrowhead). Although the membranes and the
cytoplasm still have an appearance that could be called well frozen, certain membranes do not appear as sharp as they were in the zone above. Even deeper inside the tissue,
the protein aggregates inside the nucleus are already apparent in the overview image and are also clearly visible in the cytoplasm in the zoom (D, black arrowhead). The
cytoplasm also has a much more empty appearance and appears to have more contrast than the well-preserved zone. This can be considered a typical difference between
material that contains ice damage and that is well frozen. Scale bar = 5 ␮m for A, and 2 ␮m for B–D.
microscope to a freezing device. These issues will be discussed
next.
3. Live cell imaging
In a review by Koster and Klumperman some years ago [9] it
was stated that “High-pressure freezing is extremely rapid, but the
time interval between cell selection under the light microscope
and transfer to the high-pressure-freezing apparatus takes 15–20 s,
which is too slow to fix rapid intracellular movements at the exact
time of interest. Methods that solve this problem would provide
another powerful tool for correlative live-cell immunoEM/EM.” At
the same time we had already set out to develop such a tool. In collaboration with Leica Microsystems we created an attachment to a
high-pressure freezer called the Rapid Transfer System (RTS [20]).
In combination with a LM stage insert this attachment allows us
to transfer a sample from under the light microscope into a highpressure freezer within 4 s. Using this new system we have been
able to capture endocytic fusion events [20] where it is important
to note that we could only conclude it was a fusion event because
we had the live cell imaging data. A time resolution of 4 s is currently the fastest possible for a live cell imaging CLEM experiment
in combination with cryofixation. This will allow only capturing
certain events that are in the second order time range but something like synaptic vesicle fusion will be impossible. It therefore
needs further development of freezing instrumentation to reduce
the time between LM imaging and cryofixation. Besides the time
resolution there are also some caveats related to live cell imaging with the current system. Most important is the distance of
the sample from the objective. The build up of the current system is shown in a model in Fig. 4A. There is a lot of medium and a
finder grid between the objective and the cells. This can lead to an
imaging distance of over 200 ␮m. Even using a long-working distance 63× glycerol objective this hardly provides proper live cell
imaging conditions. We would need a dry lens with a low NA to
span that distance with certainty, thus lowering our LM resolution. In addition, imaging is done in a drop of medium. Especially
on a microscope with a heated stage or chamber this droplet dries
Fig. 4. Model improvements to HPF-CLEM system.
In the commercially available system there is a large distance between the cells and the objective (A). This space is filled with a glass coverslip, medium and a finder grid. In
addition the medium only covers the carrier as a drop. Suggested improvements to the system can be modeled by removing the finder grid and thinning the carrier, bringing
the cells very close to the coverslip (B). Also bathing the whole system in medium would be an improvement towards a more physiological imaging condition.
E. Brown et al. / Seminars in Cell & Developmental Biology 20 (2009) 910–919
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4. Markers
One of the biggest areas of research in the CLEM field is the development of probes/markers. Markers are not the main subject of this
paper so we will be brief in this paragraph only highlighting a few
aspects of them. For specialized reviews on CLEM markers we refer
to papers by Ben Giepmans [42,43]. One important remark to make
though is that the ideal marker for CLEM would be fluorescent and
electron dense at the same time. Of course there are such markers
available, quantum dots for instance [42] but there is one additional
Fig. 5. The modified CLEM insert.
The modified insert has a large opening to fit an imaging dish as is generally used for
live cell imaging experiments. The imaging dish has one part of the wall removed
(arrow) through which the rapid loader will enter the dish. In addition, a part of
the “fork” of the rapid loader has been removed (arrowhead in the insert) such that
it will bridge the distance between the insert and the dish, without drawing the
medium from the dish.
out quite fast and live imaging is limited to a few minutes. Otherwise the cover slip used for imaging is “glued” to the rapid loader
(Fig. 4A). To improve both the physiological and the imaging conditions, we initiated to make some changes to the system (Fig. 4B). In
the new model the imaging distance would be drastically reduced
and the imaging is done with the sample completely submersed in
medium.
How can one implement these changes? Live cell imaging is
generally done in glass-bottomed dishes. It would be logical to
try to implement those for our HPF system as well. For this purpose the hole in the stage insert, through which the objective is
imaging and on which the sample is resting is widened so that
it fits a 3 cm imaging dish (Fig. 5). In addition we have modified the (rapid) loader. We have taken out a piece of the flat
surface of the rapid loader such that it can act as a bridge into
the imaging dish (Fig. 5, inset). Otherwise, by capillary force, it
would drain medium from the dish. As a last modification we
have removed part of the wall of the imaging dish. We only leave
1 mm standing which is enough for the modified rapid loader to
bridge the space between the stage insert and the glass bottom
of the imaging dish (see Fig. 5). We have recently learned that
there have been parallel developments in the laboratory of Kent
McDonald who also built a modified rapid loader based on the
same bridge principle (to be published in Journal of Microscopy,
2009).
In the original system for CLEM using HPF as described in our
2008 paper [20], there is a 200 ␮m deep carrier in which a sapphire
disc is placed. On top of that a finder grid is placed and pushed
into the carrier to clamp the sapphire disc with cells into place.
We have had very good results using the membrane carriers for
standard HPF ([20], see Fig. 6). These carriers are much thinner than
the original CLEM carriers and the insert for the sapphire disc is
also shallower (100 ␮m, Fig. 6). They do however contain a solid
membrane such that bright field microscopy would be impossible.
To solve this problem we decided to drill a hole in the carrier (Fig. 6).
By using a shallower carrier it prevented us from using the finder
grid as a retracing device. This is actually an advantage as using that
grid would have increased the imaging distance again. Following
discussions with Professor Vic Small we decided to first glue the
sapphire disc in the modified membrane carrier and subsequently
carbon coat the finder pattern on top of the sapphire disc (Fig. 6).
We can now use those new carriers to grow cells on and perform
our CLEM experiment. We would like to make a remark here that
we cannot use glass coverslips for HPF experiments as they work
as insulators.
Fig. 6. Improvements to the CLEM carrier system.
The standard CLEM carrier (left in A) is relatively thick compared to a membrane
carrier (right in A). This membrane carrier however has a solid bottom (B). A hole
is drilled into the bottom to allow bright field imaging (C). After a sapphire disc is
glued into the carrier, it is overlayed with a finder grid (D) and this is coated with a
thin layer of carbon (E). After removal of the finder grid, the finder pattern is visible
on the sapphire disc. After growing cells on these discs, the finder pattern is visible
in the light microscope (F) as well as in the first section of the electron microscopy
sample (G).
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Fig. 7. A CLEM experiment.
EGF was coupled to quantum dots and internalized for 30 min into A431 cells. The overlay of the bright field DIC image with the fluorescence indicates the position of
the organelles (endosomes) that have taken up the marker (A). One of these organelles was followed by live cell imaging. Stills of the movie (seconds before end of movie
indicated in B) show that the organelle changes shape over time. At time point 0, the sample is transferred into the high-pressure freezer equipped with the Rapid Transfer
System. After processing for EM, the cell of interest is retraced and the position of the observed structure is indicated (boxed area in C). This organelle can now be studied at
high resolution (arrowheads point to internalized quantum dots).
criterion such a marker would have to fulfill. The probe would have
to be biosynthetic like GFP. Unfortunately cells cannot make quantum dots or gold particles so other methods had to be developed.
One of the most interesting ones is FlAsh/ReAsh. This technique
only requires a small (approx. 10 amino acids including 4 cysteines)
addition to the protein of interest and this construct is expressed
in cells. After addition of the membrane-permeable FlAsh compound to the cells, this arsenic containing compound specifically
binds to the tetra-cystein motif and only then becomes fluorescent. The fluorescence of the red fluorescent ReAsh compound can
also be used to photo convert DAB to an electron dense precipitate
[16]. Although still interesting the technique has so far not completely lived up to its promise based on unpublished reports (and
our own experience) that the technique is not always successful.
As described earlier GFP can be visualized via different methods for
electron microscopy, and recently the group of Klumperman also
developed a CLEM technique to perform GFP immuno-labeling on
Tokuyasu cryo sections [44]. This technique combines the live cell
imaging capabilities of GFP with the high immuno-labeling efficiency of the Tokuyasu cryosectioning technique. These Tokuyasu
cryo sections are also a valuable tool for CLEM on tissue [45]. The
cryo sections are so thin (≈70 nm) that they do not suffer from
out of focus blur and hence can provide very sharp images, outperforming the conventional widefield and confocal microscopes
in z-resolution [46]. Unfortunately none of the above techniques
have been used in combination with HPF. They all only work in a
water-containing environment. After HPF and freeze substitution
the sample will be in an organic solvent and thus incompatible with
the aforementioned techniques. It will require inclusion of a rehydration protocol such as developed by Slot and coworkers [47]
for those techniques to work in combination with HPF.
For studying certain aspects of the endocytic process, the choice
of the marker is less problematic. It is best to have the fluorophore
and electron dense (gold) particle attached to the same molecule
but this may not always be possible. We have used EGF-biotin prebound to streptavidin quantum dots (Fig. 7) or streptavidin Alexa
488–10 nm gold (Invitrogen) [20] to visualize the trafficking of EGF
carriers. In this paper we are also using Transferrin (TF)–Alexa
594 (Invitrogen) that was custom coupled to 5 nm gold (Aurion)
as our marker protein (see Figs. 8 and 9). Attaching a gold particle close to a fluorophore may however quench the fluorescence
[48,49] and thus by using such probes one could still be studying different molecules. One where there is gold attached but no
fluorescence, the other where there is no gold attached to the fluorophore that will be fluorescent. Attachment of a gold particle close
to a fluorophore may however also lead to enhancement of the fluorescence. It was calculated that a distance of 5 nm between the gold
particle and the fluorophore would give optimal enhancement [50].
We are currently studying what effects we are experiencing with
the different probes we are using. We should note that we cannot
couple TF to quantum dots as we have found that this TF complex
will be routed to the degradative pathway rather than being recy-
E. Brown et al. / Seminars in Cell & Developmental Biology 20 (2009) 910–919
917
Fig. 8. Bright field TEM vs. STEM.
Both quantum dots (Fig. 7) as well as small 5 nm gold particles (arrow) are not easily visualized by conventional bright field TEM (A). Especially in HPF and freeze substituted
samples the material is very dense and obscures the small particles. STEM imaging specifically picks up those small particles made of heavy atoms (cadmium in quantum dots
or gold). They are now “lighting up” in comparison to the other material (C). Although the contrast in STEM is reversed this does explain the better visualization. Compare
images B and C, and A and D, where B is the contrast inversed image of A, and D is the contrast inversed image of C. Lowicryl embedding after HPF and freeze substitution
can result in excellent antibody labeling efficiency and ultrastructure as shown in figure E for a labeling against a nuclear protein.
cled (unpublished results). With these possible effects in mind we
are using those probes for our studies. This strategy can however
only be applied to compounds that are taken up from the outside
of cells and is not suitable for proteins residing on the cytoplasmic
side of cells. To fully understand the endocytic sorting mechanisms
one also needs to study the regulatory proteins that are associated
at the cytoplasmic side of the endosome. For those studies we will
need to be able to label those proteins either by one of the methods
described above, immuno-labeling techniques as described in the
following paragraph, or a completely new technique.
5. Visualization
Since we do not add any fixatives and change the conformation
of proteins HPF is also very well suited for preservation of antigenicity. To achieve good immuno-labeling however critically depends
on the process steps taken after HPF. Immuno-labeling cannot be
done on frozen hydrated sections so again it usually involves freeze
substitution. It is a combination of the freeze substitution cocktail
with the resin that will ultimately determine the success of the
immuno-labeling. Whereas Epoxy resins are not very suited for
918
E. Brown et al. / Seminars in Cell & Developmental Biology 20 (2009) 910–919
Fig. 9. From an LM overview to high resolution Electron Tomography.
Bright field (BF) and combined BF and fluorescence overview images of Hela cells grown on sapphire discs (A and B, respectively). The sapphire discs were clamped in the
standard CLEM carriers and hence the bars of the finder grid are visible. The sample was high-pressure frozen and freeze substituted to Epon, and the cells were retraced for
EM (C). One of the fluorescent structures was further studied using Electron Tomography (D). Shown is one slice of the tomogram and the model of an endosome (green). Two
extensions can be observed extending from the endosome (arrows). Some other structures can be observed that are modeled in other colors that were potentially connected
to one of the extensions.
immuno-labeling because they cross-link the resin with the cellular components, methacrylic resins are much better suited since
they only cross-link the resin and leave the cellular components
“intact” [51]. Of those methacrylic resins we have mainly used
Lowicryl HM20. The sample is freeze-substituted from −90 ◦ C to
−50 ◦ C in the presence of acetone and 0.1% uranyl acetate and at
−50 ◦ C it is infiltrated with the Lowicryl resin and subsequently
UV polymerized. Since there is very little extraction of material
by the HPF–freeze substitution method, the material is very dense
and small gold particles are hard to be visualized even in Lowicryl
embedded material (Fig. 8A). One additional advantage of using
Lowicryl resin over Epon is that Lowicryl is more electron-light and
allows easier visualization of small gold probes. Only fairly recently
cell biologists have also started using Scanning TEM (STEM), and in
particular High Angle Annular Dark Field (HAADF), to better visualize small heavy particles in/on biological samples [52]. HAADF
imaging provides a better contrast and signal to noise imaging than
standard bright field TEM [53]. Indeed, visualization of internalized
small 5 nm gold particles is much easier in STEM than in standard TEM mode (Fig. 8A–D). Osmium tetroxide is generally omitted
from the freeze substitution medium and this probably is beneficial for the high immuno-labeling efficiency [38]. The combination
of STEM with Lowicryl immuno-labeling can provide high labeling
with excellent visualization of the ultrastructure (Fig. 8E).
Besides CLEM there is one other technique that is very fashionable in the cell biological EM field, which is Electron Tomography
(ET). This technique can provide a 3-dimensional view of an EM
slice instead of the usual 2-D projection (for reviews on ET, see
[54] and [55]). ET can be done both with resin-embedded samples
(Fig. 9) and cryo samples. The combinations of CLEM with room
temperature and cryo ET have been described (see e.g. [35] and
[39], respectively). Also the combination of STEM and ET is again
able to visualize small marker particles in a 3-D volume [56,52].
5.1. Future
Bringing together all the described techniques is the challenge
for the future. Capturing an endocytic fusion event (live cell imaging) using high-pressure freezing is one thing [20] but can we do
that with the identification of both the internalized cargo as well as
the peripherally associated regulatory proteins (Markers). Can we
acquire the event in 3D with the necessary resolution and recognition of the particles (visualization) and finally can we do this all in a
manner that best represents the native ultrastructure of the process
(preservation). It will be the combination of existing techniques,
development of new tools, and implementation of new equipment
that in the end will let us design the ultimate CLEM experiment.
Acknowledgements
The authors would like to thank Jana Smigova for her help with
the development of the Lowicryl freeze substitution protocol and
the sample preparation and Quintin de Robillard for his help with
the Electron Tomography and the modeling.
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