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Seminars in Cell & Developmental Biology 20 (2009) 910–919

Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology

journa l homepage: www.e lsev ier .com/ locate /semcdb

eview

tudying intracellular transport using high-pressure freezing and Correlativeight Electron Microscopy

dward Browna, Judith Mantell a,b, Debbie Carterb, Gini Tillyb, Paul Verkadea,b,c,∗

Department of Biochemistry, School of Medical Sciences, University Walk, Bristol, BS8 1TD, United KingdomWolfson Bioimaging Facility, School of Medical Sciences, University Walk, Bristol, BS8 1TD, United KingdomDepartment of Physiology and Pharmacology, School of Medical Sciences, University Walk, Bristol, BS8 1TD, United Kingdom

r t i c l e i n f o

rticle history:vailable online 4 August 2009

eywords:orrelative Light Electron MicroscopyLEMigh-pressure freezinglectron microscopy

a b s t r a c t

Correlative Light Electron Microscopy (CLEM) aims at combining the best of light and electron microscopyin one experiment. Light microscopy (LM) is especially suited for providing a general overview withdata from lots of different cells and by using live cell imaging it can show the history or sequence ofevents between or inside cells. Electron microscopy (EM) on the other hand can provide a much higherresolution image of a particular event and provide additional spatial information, the so-called referencespace. 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

ntracellular trafficking 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 asthe electron microscope. For electron microscopy this currently means that the sample will have to becryo-fixed instead of the standard chemical fixation. In this paper we will discuss the rationale for usingcryofixation for CLEM experiments. In particular we will highlight a CLEM technique using high-pressurefreezing 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.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9101.1. Correlative Light Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 911

2. Ultrastructural preservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9113. Live cell imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9144. Markers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9155. Visualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 917

5.1. Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 918Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 918References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 918

. Introduction

The introduction of Green Fluorescent Protein (GFP) technology

dynamic cells really are, but also made us aware that some struc-tures also thought to be very dynamic are actually mostly stable (i.e.caveolae [2]). However, partially due to the emission wavelength

s a clonable fluorescent tag onto proteins [1] has revolutionizedife science research. It made it possible to do time-resolved imag-ng of proteins in living cells and organisms and is an essential tooln modern day research. It has led to new insights of how motile and

∗ Corresponding author at: School of Medical Sciences, University Walk, Bristol,S8 1TD, United Kingdom. Tel.: +44 117 3312179; fax: +44 117 3312168.

E-mail address: p.verkade@bristol.ac.uk (P. Verkade).

084-9521/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcdb.2009.07.006

of the fluorophores (in the range of 100 s of nm) the resolution ofstandard 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. Hencethere has been an increasing effort to get higher resolution from

the fluorescence light microscope. This eventually led to break-ing Abbe’s law of refraction/resolution. Nowadays there are severalsystems such as STED [3], SSIM [4], STORM [5], and PALM [6] avail-able that are able to get a resolution below 100 nm (see the paperby Patterson in this issue). In addition most of these systems are

evelop

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E. Brown et al. / Seminars in Cell & D

ow also able to do live cell imaging. There is one important aspecthat images from an electron microscope have that is not obtainedrom a fluorescence-based light microscopic image. Whereas inhe light microscope only one or more fluorophores light up inhese high-resolution systems, the electron microscope does notnly visualize the electron dense marker but also gives a lot ofnformation about the surrounding material. This so-called refer-nce space can give us information on whether a marker is insidehe lumen of a membrane-bound carrier or attached to the out-ide of the carrier. It may also tell us whether that same carrier isttached to a microtubule or to the actin cytoskeleton, etc. It is onef the aspects that is often overlooked when high-resolution lighticroscopy is compared to electron microscopy. But as we hope

o show in this paper it is the combination of both microscopyechniques that is more than just the sum of the parts. This isot only true for the combination of EM with “standard” LM tech-iques but also with the high-resolution LM techniques as was aptlyointed out by Jennifer Lippincott-Schwartz when high-resolution

ight microscopy was named technique of the year 2008 by Natureethods [7].

.1. Correlative Light Electron Microscopy

When one is thus studying structures that are below the resolu-ion of the light microscope it is in most cases impossible to assign aabel to a specific domain within these structures. For these issueslectron microscopy (EM) has been the technique of choice. Theajor limitation here is that living samples cannot be observed and

t needs a series of experiments to recapitulate the sequence of anvent. Combining the advantages of live cell imaging and the highesolution of the EM is the way to overcome with this issue. Correl-tive Light Electron Microscopy (CLEM) techniques try to do exactlyhat. CLEM techniques have been around for decades already (seeor instance the book edited by Hayat [8]) but have recently under-one a renaissance and the interest is still growing. Whereas inarlier days the correlation between the light and electron micro-copical image would mainly be made on different samples, currentechniques focus on visualizing the exact same structure or eventoth in the LM and the EM (Fig. 1). Using such an approach theistory of events can be reconciled at lower resolution with lighticroscopy but the event of interest can be studied at high reso-

ution. This then allows us for example to draw conclusions on theype of membrane structure that is formed. Using either live cellmaging or electron microscopy alone will never allow us to do thissee e.g. Fig. 1F). As was described in one of the reviews of the specialImaging in Cell Biology” series (Nat. Rev. Mol. Cell Biol. 2003, sept.upp.), CLEM is one of the hot topics within the imaging field [9].everal methods have appeared in recent years making significantmprovements to the technique for Cell Biological studies. It is notur intent to provide a review on all the existing CLEM techniquesut rather we will limit ourselves to highlighting some examplesnd we will discuss those and a few more examples further in theMarkers” paragraph.

The new era of CLEM more or less started with the paper byolishchuk et al. [10] in which they combined GFP LM imagingith immuno-labeling the GFP for EM. The antibody to GFP was

isualized either by performing a DAB precipitation reaction [10]r by immuno-gold staining [11]. This group was to our knowl-dge also the first to introduce the term CLEM for Correlativeight Electron Microscopy [12]. It should be noted that in theicroscopy field the term CLEM is also used for Controlled Light

xposure Microscopy [13]. The groups of Nilsson [14] and latern also Ellinger [15] succeeded in directly photo converting theFP signal into an electron dense DAB precipitate, avoiding anti-ody labeling that is very dependent on epitope availability. Asn alternative for GFP labeling the FlAsh/ReAsh compounds [16]

mental Biology 20 (2009) 910–919 911

and quantum dots [17,18] were developed. On the instrumentationside, the concepts of high-throughput analysis and high-resolutionLM imaging [19] were introduced for CLEM. Advances in fixationtechniques were described [20,21] and the full integration of alight microscopy into the electron microscope was accomplished[22,23].

When we set out to perform CLEM experiments on intracellulartransport we set ourselves a set of criteria that a CLEM experimentwould have to fulfill.

These were (more or less in order of importance):

1. Retain the best possible ultrastructure for the analysis in theelectron 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.

2. Ultrastructural preservation

Artifacts introduced during live cell imaging will generally bebelow the resolution of the light microscope. It is only when cellsare processed for electron microscopy that the preservation ofultrastructure becomes an issue. In a standard electron microscop-ical preparation experiment there are 2 steps that most criticallyinfluence the preservation of the structure. The first is the fixa-tion of the sample and the second is the dehydration step wherecellular water is replaced by a solvent and eventually for an embed-ding resin. There are 2 possible ways to fix or immobilize a sample;chemical or physical cryofixation. The speed of fixation and thedegree of cross-linking are the most important factors influenc-ing whether a sample is fixed well (resembling its natural state).The mechanism of conventional room temperature chemical fixa-tion is based on diffusion of aldehydes such as paraformaldehydeand glutaraldehyde, and for cell cultures fixation is achieved inthe range of seconds. Aldehydes mainly cross-link proteins andit is important to note that lipids are not cross-linked or immo-bilized at all in this initial step. It is only in later steps involvingfor instance osmium tetroxide where lipids are cross-linked/fixedas well. Although probably not always inducing artifacts, chemicalfixation, including the subsequent processing at room temperature,has been shown to induce alterations in morphology [24,25]. Some-times it is the dehydration step rather than the fixation itself that isconsidered the main source of artifacts [26]. Murk and coworkers[27] however clearly demonstrated that the first chemical fixationstep alone can lead to substantial shrinkage of various intracellularendocytic membrane structures when compared to cryofixation. Incontrast to chemical fixation, cryofixation of samples takes placein the millisecond range [28] and fixes everything. The principlebehind cryofixation (or immobilization as it is sometimes pre-ferred) is to remove the thermal energy (heat) from the systemas fast as possible, thus leaving no energy for proteins to moveanymore. Besides the extraction of material and the accompanyingshrinkage of organelles there is also a difference in the contrast ofthe 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 fixationshould be cryofixation. Cryofixation can be done by slam or impact

freezing, plunge freezing, or by high-pressure freezing (HPF). HPFnot only reaches a greater depth at which the sample is well frozen,it is generally more reproducible [28–30]. Cryofixation by HPF low-ers the melting point of water by 20 ◦C [31]. Ice crystal formationtherefore starts later than with other cryofixation techniques, and

912 E. Brown et al. / Seminars in Cell & Developmental Biology 20 (2009) 910–919

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 processare 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,l essed( gationw ince ww rotrusm ucture

ia2wrvwac(aaCwiapdasosar

ike in this case segregation of the 2 fluorophores (C) the sample is fixed and procD). Subsequently we can analyze the sample at higher resolution (E) and the segree are studying a segregation event at high resolution in the electron microscope shere an endocytic structure was observed in the electron microscope with some pembrane extensions are fusing with or segregating from the central endocytic str

ce crystal growth is also slower. In HPF a sample is contained inclosed carrier. The carrier is put under high pressure (around

050 bar) and is immediately thereafter sprayed with LN2. In thisay the water in the sample is frozen so fast that it becomes vit-

ified and it does not get a chance to form ice crystals. Whereasitrified water takes up as much space as liquid water, crystallineater (ice crystals) will take up more space than the cellular water

nd push proteins aside to form aggregates. After the freezing pro-ess, samples ideally would be processed for native cryosectioningalso known as frozen hydrated sectioning or CEMOVIS [32]) andnalysis in a cryo microscope to avoid any addition of chemical fix-tives or stains. We are currently technically not able to perform aLEM experiment (with HPF) in this way. In most cases the samplesill be processed for freeze substitution. In this process the water

s replaced by a solvent and chemically fixed at low temperaturend subsequently the sample is infiltrated with a resin for furtherrocessing at room temperature [20,30]. By performing the dehy-ration and chemical fixation steps at low temperature we limit thertifacts induced by the standard room temperature processing to

uch extent that the artifacts will be below the detection limit ofur resolution requirements. Obviously, this process is not alwaysuccessful. This may be dependent on a lot of different factors, suchs the thickness of the sample, the water content, the medium sur-ounding the samples, etc. but also by the handling of the samples

for electron microscopy. The same cell is first retraced in the electron microscopeevent can now be studied in more detail. It is important to note that we are sure

e have the live imaging data (history). This can be nicely demonstrated in image Fions attached to it. From this single image it is impossible to deduct whether these.

[30]. We think it is important to show what the artifacts associatedwith cryofixation and the subsequent processing look like in orderfor researchers to be able to identify them (Fig. 3).

So far there are only a few papers that have performed CLEMexperiments using HPF. The first published results used the intro-duction of tissue specific LM dyes into the sample [33,34]. AfterHPF and freeze substitution the block was sectioned and the blockface analyzed under the light microscope. Some dyes are still flu-orescent when processed this way. This helped the researchers tozoom into a particular cell type for further study in the EM. Müller-Reichert and colleagues used GFP expressing C. elegans worms tofollow mitotic processes [35]. Once a specific stage was observedthey could freeze the worm or embryo and study the processedsample knowing they had captured a specific cell cycle stage. Arecent paper by Jones et al. [36] used a similar approach to cap-ture hatching stages of Schistosoma embryos. They used standardbright field imaging video microscopy to stage their animals andsubsequently performed high-pressure freezing and freeze substi-tution on the samples. In the paper by Müller-Reichert there was

no further need to visualize the GFP after the freezing and freezesubstitution steps. This is a problem in general as after freeze sub-stitution GFP is generally not fluorescent anymore. Sims and Hardindeveloped a HPF and freeze substitution protocol that retains thestructure of the GFP and hence GFP is still fluorescent in the pro-

E. Brown et al. / Seminars in Cell & Developmental Biology 20 (2009) 910–919 913

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).T ferentm m is mi xed sah

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he overview images (A and C) show the difference in contrast obtained by the 2 difuch lighter (note, the background in both samples is equal). This is because osmiu

n to the Golgi area within the cell it is apparent that the membranes in chemically fiave straighter membranes that still appear to be under tension (D).

essed block [37]. Although this development is very important, ast would be ideal to be able to have fluorescent GFP in an EM block,ur feeling is that for this particular protocol there have been tooany sacrifices made to the preservation of the ultrastructure and

he advantages of using HPF have been partially lost. Recently theroup of Parton [38] did succeed in retaining both GFP fluorescencend excellent ultrastructure by high-pressure freezing and freezeubstitution of Zebrafish. The critical step in this process was tomit osmium tetroxide from the freeze substitution protocol andmbedding in Lowicryl HM20. We have been experimenting withowicryl embedding after HPF as well for immuno-labeling pur-oses (see later) but have not been able to retain GFP fluorescence

n culture-grown cells yet. It will require further studies to deter-ine if the protocol developed by Nixon et al. will be applicable toore types of specimen.As outlined, samples would ideally be analyzed in the EM with-

ut any further processing. Especially the group of Baumeister

methods. Chemically fixed samples are generally dark and HPF processed samplesuch more reactive at room temperature than at sub-zero temperatures. If we zoommples are wrinkled because the structure has shrunk (B) whereas the HPF samples

has promoted the idea that the EM part of a CLEM experimentshould be done in a cryo electron microscope [21,39]. Their mainpurpose for using fluorescence light microscopy in a CLEM experi-ment is to find the location of fluorescent cells/structures. They canthen be easily retraced in the cryo electron microscope, avoidingbeam damage/heating of the sample normally induced by scan-ning the sample for the right structure in the electron microscope.To this end they [40] and others [41] have developed a cryo flu-orescence stage for the light microscope. A thin frozen sectionon an EM finder grid can be analyzed for fluorescence at LN2temperatures and the right coordinates of the finder grid usedfor 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 lim-ited to screening purposes. One important aspect that is lackingfor the use of these stages (other then screening and retracing)in intracellular membrane transport studies is that it is impos-sible to do live cell imaging or a fast transfer from the light

914 E. Brown et al. / Seminars in Cell & Developmental Biology 20 (2009) 910–919

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 materialquickly 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 excellentmorphology. 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 appearsto 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 havebeen sectioned perpendicular. In the transition zone (C) the first signs of protein aggregation appear inside the nucleus (white arrowhead). Although the membranes and thec nes dt age anc trast tm d 2 �m

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ytoplasm still have an appearance that could be called well frozen, certain membrahe protein aggregates inside the nucleus are already apparent in the overview imytoplasm also has a much more empty appearance and appears to have more conaterial that contains ice damage and that is well frozen. Scale bar = 5 �m for A, an

icroscope to a freezing device. These issues will be discussedext.

. Live cell imaging

In a review by Koster and Klumperman some years ago [9] itas stated that “High-pressure freezing is extremely rapid, but the

ime interval between cell selection under the light microscopend transfer to the high-pressure-freezing apparatus takes 15–20 s,hich is too slow to fix rapid intracellular movements at the exact

ime of interest. Methods that solve this problem would providenother powerful tool for correlative live-cell immunoEM/EM.” Athe same time we had already set out to develop such a tool. In col-aboration with Leica Microsystems we created an attachment to a

igh-pressure freezer called the Rapid Transfer System (RTS [20]).

n combination with a LM stage insert this attachment allows uso transfer a sample from under the light microscope into a high-ressure freezer within 4 s. Using this new system we have beenble to capture endocytic fusion events [20] where it is important

ig. 4. Model improvements to HPF-CLEM system.n the commercially available system there is a large distance between the cells and the oddition the medium only covers the carrier as a drop. Suggested improvements to the syhe cells very close to the coverslip (B). Also bathing the whole system in medium would

o not appear as sharp as they were in the zone above. Even deeper inside the tissue,d are also clearly visible in the cytoplasm in the zoom (D, black arrowhead). The

han the well-preserved zone. This can be considered a typical difference betweenfor B–D.

to note that we could only conclude it was a fusion event becausewe had the live cell imaging data. A time resolution of 4 s is cur-rently the fastest possible for a live cell imaging CLEM experimentin combination with cryofixation. This will allow only capturingcertain events that are in the second order time range but some-thing like synaptic vesicle fusion will be impossible. It thereforeneeds further development of freezing instrumentation to reducethe time between LM imaging and cryofixation. Besides the timeresolution there are also some caveats related to live cell imag-ing with the current system. Most important is the distance ofthe sample from the objective. The build up of the current sys-tem is shown in a model in Fig. 4A. There is a lot of medium and afinder grid between the objective and the cells. This can lead to animaging distance of over 200 �m. Even using a long-working dis-

tance 63× glycerol objective this hardly provides proper live cellimaging conditions. We would need a dry lens with a low NA tospan that distance with certainty, thus lowering our LM resolu-tion. In addition, imaging is done in a drop of medium. Especiallyon a microscope with a heated stage or chamber this droplet dries

bjective (A). This space is filled with a glass coverslip, medium and a finder grid. Instem can be modeled by removing the finder grid and thinning the carrier, bringingbe an improvement towards a more physiological imaging condition.

E. Brown et al. / Seminars in Cell & Develop

Fig. 5. The modified CLEM insert.The modified insert has a large opening to fit an imaging dish as is generally used forlive 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 oftim

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to papers by Ben Giepmans [42,43]. One important remark to makethough is that the ideal marker for CLEM would be fluorescent andelectron dense at the same time. Of course there are such markersavailable, quantum dots for instance [42] but there is one additional

Fig. 6. Improvements to the CLEM carrier system.The standard CLEM carrier (left in A) is relatively thick compared to a membranecarrier (right in A). This membrane carrier however has a solid bottom (B). A holeis drilled into the bottom to allow bright field imaging (C). After a sapphire disc is

he “fork” of the rapid loader has been removed (arrowhead in the insert) such thatt will bridge the distance between the insert and the dish, without drawing the

edium from the dish.

ut quite fast and live imaging is limited to a few minutes. Other-ise the cover slip used for imaging is “glued” to the rapid loader

Fig. 4A). To improve both the physiological and the imaging condi-ions, we initiated to make some changes to the system (Fig. 4B). Inhe new model the imaging distance would be drastically reducednd the imaging is done with the sample completely submersed inedium.How can one implement these changes? Live cell imaging is

enerally done in glass-bottomed dishes. It would be logical tory to implement those for our HPF system as well. For this pur-ose the hole in the stage insert, through which the objective is

maging and on which the sample is resting is widened so thatt fits a 3 cm imaging dish (Fig. 5). In addition we have mod-fied the (rapid) loader. We have taken out a piece of the flaturface of the rapid loader such that it can act as a bridge intohe imaging dish (Fig. 5, inset). Otherwise, by capillary force, itould drain medium from the dish. As a last modification weave removed part of the wall of the imaging dish. We only leavemm standing which is enough for the modified rapid loader toridge the space between the stage insert and the glass bottomf the imaging dish (see Fig. 5). We have recently learned thathere have been parallel developments in the laboratory of Kent

cDonald who also built a modified rapid loader based on theame bridge principle (to be published in Journal of Microscopy,009).

In the original system for CLEM using HPF as described in our008 paper [20], there is a 200 �m deep carrier in which a sapphireisc is placed. On top of that a finder grid is placed and pushed

nto the carrier to clamp the sapphire disc with cells into place.e have had very good results using the membrane carriers for

tandard HPF ([20], see Fig. 6). These carriers are much thinner thanhe original CLEM carriers and the insert for the sapphire disc islso shallower (100 �m, Fig. 6). They do however contain a solidembrane such that bright field microscopy would be impossible.

o solve this problem we decided to drill a hole in the carrier (Fig. 6).y using a shallower carrier it prevented us from using the finderrid as a retracing device. This is actually an advantage as using thatrid would have increased the imaging distance again. Followingiscussions with Professor Vic Small we decided to first glue theapphire disc in the modified membrane carrier and subsequentlyarbon coat the finder pattern on top of the sapphire disc (Fig. 6).

e can now use those new carriers to grow cells on and performur CLEM experiment. We would like to make a remark here thate cannot use glass coverslips for HPF experiments as they work

s insulators.

mental Biology 20 (2009) 910–919 915

4. Markers

One of the biggest areas of research in the CLEM field is the devel-opment of probes/markers. Markers are not the main subject of thispaper so we will be brief in this paragraph only highlighting a fewaspects of them. For specialized reviews on CLEM markers we refer

glued into the carrier, it is overlayed with a finder grid (D) and this is coated with athin layer of carbon (E). After removal of the finder grid, the finder pattern is visibleon the sapphire disc. After growing cells on these discs, the finder pattern is visiblein the light microscope (F) as well as in the first section of the electron microscopysample (G).

916 E. Brown et al. / Seminars in Cell & Developmental Biology 20 (2009) 910–919

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 oft elles wi e samS e obsh

cttOoaipbca[poAedTicccopih

he organelles (endosomes) that have taken up the marker (A). One of these organndicated in B) show that the organelle changes shape over time. At time point 0, thystem. After processing for EM, the cell of interest is retraced and the position of thigh resolution (arrowheads point to internalized quantum dots).

riterion such a marker would have to fulfill. The probe would haveo be biosynthetic like GFP. Unfortunately cells cannot make quan-um dots or gold particles so other methods had to be developed.ne of the most interesting ones is FlAsh/ReAsh. This techniquenly requires a small (approx. 10 amino acids including 4 cysteines)ddition to the protein of interest and this construct is expressedn cells. After addition of the membrane-permeable FlAsh com-ound to the cells, this arsenic containing compound specificallyinds to the tetra-cystein motif and only then becomes fluores-ent. The fluorescence of the red fluorescent ReAsh compound canlso be used to photo convert DAB to an electron dense precipitate16]. Although still interesting the technique has so far not com-letely lived up to its promise based on unpublished reports (andur own experience) that the technique is not always successful.s described earlier GFP can be visualized via different methods forlectron microscopy, and recently the group of Klumperman alsoeveloped a CLEM technique to perform GFP immuno-labeling onokuyasu cryo sections [44]. This technique combines the live cellmaging capabilities of GFP with the high immuno-labeling effi-iency of the Tokuyasu cryosectioning technique. These Tokuyasuryo sections are also a valuable tool for CLEM on tissue [45]. The

ryo sections are so thin (≈70 nm) that they do not suffer fromut of focus blur and hence can provide very sharp images, out-erforming the conventional widefield and confocal microscopes

n z-resolution [46]. Unfortunately none of the above techniquesave been used in combination with HPF. They all only work in a

as followed by live cell imaging. Stills of the movie (seconds before end of movieple is transferred into the high-pressure freezer equipped with the Rapid Transfererved structure is indicated (boxed area in C). This organelle can now be studied at

water-containing environment. After HPF and freeze substitutionthe sample will be in an organic solvent and thus incompatible withthe aforementioned techniques. It will require inclusion of a re-hydration 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 choiceof the marker is less problematic. It is best to have the fluorophoreand electron dense (gold) particle attached to the same moleculebut this may not always be possible. We have used EGF-biotin pre-bound to streptavidin quantum dots (Fig. 7) or streptavidin Alexa488–10 nm gold (Invitrogen) [20] to visualize the trafficking of EGFcarriers. In this paper we are also using Transferrin (TF)–Alexa594 (Invitrogen) that was custom coupled to 5 nm gold (Aurion)as our marker protein (see Figs. 8 and 9). Attaching a gold parti-cle close to a fluorophore may however quench the fluorescence[48,49] and thus by using such probes one could still be study-ing different molecules. One where there is gold attached but nofluorescence, the other where there is no gold attached to the fluo-rophore that will be fluorescent. Attachment of a gold particle closeto a fluorophore may however also lead to enhancement of the fluo-rescence. 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 withthe different probes we are using. We should note that we cannotcouple TF to quantum dots as we have found that this TF complexwill 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 substituteds pecifico ugh thi the coc gure E

caoosoandf

amples the material is very dense and obscures the small particles. STEM imaging sr gold). They are now “lighting up” in comparison to the other material (C). Althomages B and C, and A and D, where B is the contrast inversed image of A, and D isan result in excellent antibody labeling efficiency and ultrastructure as shown in fi

led (unpublished results). With these possible effects in mind were using those probes for our studies. This strategy can howevernly be applied to compounds that are taken up from the outsidef cells and is not suitable for proteins residing on the cytoplasmicide of cells. To fully understand the endocytic sorting mechanisms

ne also needs to study the regulatory proteins that are associatedt the cytoplasmic side of the endosome. For those studies we willeed to be able to label those proteins either by one of the methodsescribed above, immuno-labeling techniques as described in theollowing paragraph, or a completely new technique.

ally picks up those small particles made of heavy atoms (cadmium in quantum dotse contrast in STEM is reversed this does explain the better visualization. Comparentrast inversed image of C. Lowicryl embedding after HPF and freeze substitutionfor a labeling against a nuclear protein.

5. Visualization

Since we do not add any fixatives and change the conformationof proteins HPF is also very well suited for preservation of antigenic-ity. To achieve good immuno-labeling however critically depends

on the process steps taken after HPF. Immuno-labeling cannot bedone on frozen hydrated sections so again it usually involves freezesubstitution. It is a combination of the freeze substitution cocktailwith the resin that will ultimately determine the success of theimmuno-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 thes was hE aphy (e cturet

ilt“L−−UbaeLacpaissdfcow

a(s[(t[a

5

fi

tandard CLEM carriers and hence the bars of the finder grid are visible. The sampleM (C). One of the fluorescent structures was further studied using Electron Tomogrxtensions can be observed extending from the endosome (arrows). Some other struo one of the extensions.

mmuno-labeling because they cross-link the resin with the cel-ular components, methacrylic resins are much better suited sincehey only cross-link the resin and leave the cellular componentsintact” [51]. Of those methacrylic resins we have mainly usedowicryl HM20. The sample is freeze-substituted from −90 ◦C to50 ◦C in the presence of acetone and 0.1% uranyl acetate and at50 ◦C it is infiltrated with the Lowicryl resin and subsequentlyV polymerized. Since there is very little extraction of materialy the HPF–freeze substitution method, the material is very densend small gold particles are hard to be visualized even in Lowicrylmbedded material (Fig. 8A). One additional advantage of usingowicryl resin over Epon is that Lowicryl is more electron-light andllows easier visualization of small gold probes. Only fairly recentlyell biologists have also started using Scanning TEM (STEM), and inarticular High Angle Annular Dark Field (HAADF), to better visu-lize small heavy particles in/on biological samples [52]. HAADFmaging provides a better contrast and signal to noise imaging thantandard bright field TEM [53]. Indeed, visualization of internalizedmall 5 nm gold particles is much easier in STEM than in stan-ard TEM mode (Fig. 8A–D). Osmium tetroxide is generally omittedrom the freeze substitution medium and this probably is benefi-ial for the high immuno-labeling efficiency [38]. The combinationf STEM with Lowicryl immuno-labeling can provide high labelingith excellent visualization of the ultrastructure (Fig. 8E).

Besides CLEM there is one other technique that is very fashion-ble in the cell biological EM field, which is Electron TomographyET). This technique can provide a 3-dimensional view of an EMlice instead of the usual 2-D projection (for reviews on ET, see54] and [55]). ET can be done both with resin-embedded samplesFig. 9) and cryo samples. The combinations of CLEM with roomemperature and cryo ET have been described (see e.g. [35] and39], respectively). Also the combination of STEM and ET is againble to visualize small marker particles in a 3-D volume [56,52].

.1. Future

Bringing together all the described techniques is the challengeor the future. Capturing an endocytic fusion event (live cell imag-ng) using high-pressure freezing is one thing [20] but can we do

[

igh-pressure frozen and freeze substituted to Epon, and the cells were retraced forD). Shown is one slice of the tomogram and the model of an endosome (green). Twos can be observed that are modeled in other colors that were potentially connected

that with the identification of both the internalized cargo as well asthe peripherally associated regulatory proteins (Markers). Can weacquire the event in 3D with the necessary resolution and recogni-tion of the particles (visualization) and finally can we do this all in amanner 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 equipmentthat in the end will let us design the ultimate CLEM experiment.

Acknowledgements

The authors would like to thank Jana Smigova for her help withthe development of the Lowicryl freeze substitution protocol andthe sample preparation and Quintin de Robillard for his help withthe Electron Tomography and the modeling.

References

[1] Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent proteinas a marker for gene expression. Science 1994;263:802–5.

[2] Thomsen P, Roepstorff K, Stahlhut M, van Deurs B. Caveolae are highly immo-bile plasma membrane microdomains, which are not involved in constitutiveendocytic trafficking. Mol Biol Cell 2002;13:238–50.

[3] Schönle A, Glatz M, Hell SW. Four-dimensional multiphoton microscopywith time-correlated single-photon counting. Appl Opt 2000;39(December 1(34)):6306–11.

[4] Gustafsson MG. Nonlinear structured-illumination microscopy: wide-field flu-orescence imaging with theoretically unlimited resolution. Proc Natl Acad SciUSA 2005;102(September 13 (37)):13081–6.

[5] Rust MJ, Bates M, Zhuang X. Sub-diffraction-limit imaging by stochasticoptical reconstruction microscopy (STORM). Nat Methods 2006;3(October(10)):793–5.

[6] Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, etal. Imaging intracellular fluorescent proteins at nanometer resolution. Science2006;313(September 15 (5793)):1642–5.

[7] Lippincott-Schwartz J, Manley S. Putting super-resolution fluorescencemicroscopy to work. Nat Methods 2009;6(January (1)):21–3.

[8] Hayat MA. In: Hayat MA, editor. Correlative microscopy in biology, instrumen-tation and methods. Academic Press; 1987.

[9] Koster AJ, Klumperman J. Electron microscopy in cell biology: integrating struc-ture and function. Nat Rev Mol Cell Biol Sep Suppl 2003:SS6–10.

10] Polishchuk RS, Polishchuk EV, Marra P, Alberti S, Buccione R, Luini A, et al. Cor-relative light-electron microscopy reveals the tubular-saccular ultrastructureof carriers operating between Golgi apparatus and plasma membrane. J CellBiol 2000;148(January 10 (1)):45–58.

evelop

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

E. Brown et al. / Seminars in Cell & D

11] Marra P, Maffucci T, Daniele T, Tullio GD, Ikehara Y, Chan EK, et al. The GM130and GRASP65 Golgi proteins cycle through and define a subdomain of theintermediate compartment. Nat Cell Biol 2001;3(December (12)):1101–13.

12] Mironov AA, Polishchuk RS, Luini A. Visualizing membrane traffic in vivo bycombined video fluorescence and 3D electron microscopy. Trends Cell Biol2000;10(August (8)):349–53.

13] Hoebe RA, Van Oven CH, Gadella Jr TW, Dhonukshe PB, Van Noorden CJ, Man-ders EM. Controlled light-exposure microscopy reduces photobleaching andphototoxicity in fluorescence live-cell imaging. Nat Biotechnol 2007;25(Febru-ary (2)):249–53.

14] Grabenbauer M, Geerts WJ, Fernadez-Rodriguez J, Hoenger A, Koster AJ, NilssonT. Correlative microscopy and electron tomography of GFP through photooxi-dation. Nat Methods 2005;2:857–62.

15] Meiblitzer-Ruppitsch C, Vetterlein M, Stangl H, Maier S, Neumüller J, Freiss-muth M, et al. Electron microscopic visualization of fluorescent signals incellular compartments and organelles by means of DAB-photoconversion. His-tochem Cell Biol 2008;130(August (2)):407–19.

16] Gaietta G, Deerinck TJ, Adams SR, Bouwer J, Tour O, Laird DW, et al. Mul-ticolor and electron microscopic imaging of connexin trafficking. Science2002;296:503–7.

17] Nisman R, Dellaire G, Ren Y, Li R, Bazett-Jones DP. Application of quantumdots as probes for correlative fluorescence, conventional, and energy-filteredtransmission electron microscopy. J Histochem Cytochem 2004;52(January(1)):13–8.

18] Giepmans BN, Deerinck TJ, Smarr BL, Jones YZ, Ellisman MH. Correlated lightand electron microscopic imaging of multiple endogenous proteins usingQuantum dots. Nat Methods 2005;2:743–9.

19] Perinetti G, Müller T, Spaar A, Polishchuk R, Luini A, Egner A. Correlation of4Pi and electron microscopy to study transport through single Golgi stacks inliving cells with super resolution. Traffic 2009;10(April (4)):379–91.

20] Verkade P. Moving EM: the rapid transfer system as a new tool for correlativelight and electron microscopy and high throughput for high-pressure freezing.J Microsc 2008;230:317–28.

21] Lucic V, Leis A, Baumeister W. Cryo-electron tomography of cells: connectingstructure and function. Histochem Cell Biol 2008;130(August (2)):185–96.

22] Agronskaia AV, Valentijn JA, van Driel LF, Schneijdenberg CT, Humbel BM, vanBergen En Henegouwen PM, et al. Integrated fluorescence and transmissionelectron microscopy. J Struct Biol 2008;(July 1).

23] Karreman MA, Agronskaia AV, Verkleij AJ, Cremers FF, Gerritsen HC, Hum-bel BM. Discovery of a new RNA-containing nuclear structure in UVC-inducedapoptotic cells by integrated laser electron microscopy. Biol Cell 2009;101(May(5)):287–99.

24] Crang RFE, Klomparens KL. Artifacts in biological electron microscopy. NewYork: Plenum Press; 1988.

25] Kellenberger E, Johansen R, Maeder M, Bohrmann B, Stauffer E, Villiger W.Artefacts and morphological changes during chemical fixation. J Microsc1992;168:181–201.

26] Small JV. Organization of actin in the leading edge of cultured cells: influence ofosmium tetroxide and dehydration on the ultrastructure of actin meshworks.J Cell Biol 1981;91:695–705.

27] Murk JL, Posthuma G, Koster AJ, Geuze HJ, Verkleij AJ, Kleijmeer MJ, et al.Influence of aldehyde fixation on the morphology of endosomes and lyso-somes: quantitative analysis and electron tomography. J Microsc (Oxford)2003;212:81–90.

28] Dahl R, Staehelin LA. High-pressure freezing for the preservation of biologicalstructure: theory and practice. J Electron Microsc Tech 1989;13:165–74.

29] McDonald KL. High pressure freezing for preservation of high resolution finestructure and antigenicity for immunolabeling. In: Hajikbagheri MAN, editor.Electron microscopy methods and protocols. 1st edition Totowa, NJ: HumanaPress; 1999. p. 77–97.

30] McDonald KL, Morphew M, Verkade P, Müller-Reichert T. Recent advances inhigh pressure freezing: equipment and specimen loading methods. In: Kuo J,editor. Methods in molecular biology, vol. 369: electron microscopy: methods

and protocols. 2nd edition Totowa, NJ: Humana Press Inc.; 2007. p. 143–73.

31] Moor H. Theory and practice of high-pressure freezing. In: Steinbrecht RA,Zierold K, editors. Cryotechniques in biological electron microscopy. Berlin:Springer Verlag; 1987. p. 175–91.

32] Al-Amoudi A, Chang JJ, Leforestier A, McDowall A, Salamin LM, Norlen LP, et al.Cryo-electron microscopy of vitreous sections. EMBO J 2004;23:3583–8.

[

[

mental Biology 20 (2009) 910–919 919

33] Biel S, Kawaschinski K, Wittern KP, Hintze U, Wepf R. From tissue to cellularultrastructure: closing the gap between micro- and nanostructural imaging. JMicrosc 2003;212:91–9.

34] Pfeiffer S, Beese M, Boettcher M, Kawaschinski K, Krupinska K. Combined useof confocal laser scanning microscopy and transmission electron microscopyfor visualisation of identical cells processed by cryotechniques. Protoplasma2003;222:129–37.

35] Muller-Reichert T, Srayko M, Hyman A, O’Toole ET, McDonald K. Correlativelight and electron microscopy of early Caenorhabditis elegans embryos in mito-sis. Methods Cell Biol 2007;79:101–19.

36] Jones MK, Bong SH, Green KM, Holmes P, Duke M, Loukas A, et al. Correla-tive and dynamic imaging of the hatching biology of Schistosoma japonicumfrom eggs prepared by high pressure freezing. PLoS Negl Trop Dis 2008;2(11):e334.

37] Sims PA, Hardin JD. Fluorescence-integrated transmission electron microscopyimages: integrating fluorescence microscopy with transmission electronmicroscopy. Methods Mol Biol 2007;369:291–308.

38] Nixon SJ, Webb RI, Floetenmeyer M, Schieber N, Lo HP, Parton RG. A singlemethod for cryofixation and correlative light, electron microscopy and tomog-raphy of zebrafish embryos. Traffic 2009;10(February (2)):131–6.

39] Plitzko JM, Rigort A, Leis A. Correlative cryo-light microscopy and cryo-electrontomography: from cellular territories to molecular landscapes. Curr OpinBiotechnol 2009;20(February (1)):83–9.

40] Sartori A, Gatz R, Beck F, Rigort A, Baumeister W, Plitzko JM. Correlativemicroscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography. J Struct Biol 2007;160(November (2)):135–45.

41] Schwartz CL, Sarbash VI, Ataullakhanov FI, McIntosh JR, Nicastro D.Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching. J Microsc2007;227(August (Pt 2)):98–109.

42] Giepmans BN, Adams SR, Ellisman MH, Tsien RY. The fluorescent toolbox forassessing protein location and function. Science 2006;312:217–24.

43] Giepmans BN. Bridging fluorescence microscopy and electron microscopy. His-tochem Cell Biol 2008;130(August (2)):211–7.

44] van Rijnsoever C, Oorschot V, Klumperman J. Correlative light-electronmicroscopy (CLEM) combining live-cell imaging and immunolabeling of ultra-thin cryosections. Nat Methods 2008;5(November (11)):973–80.

45] Takizawa T, Robinson JM. Ultrathin cryosections: an important tool forimmunofluorescence and correlative microscopy. J Histochem Cytochem2003;51(June (6)):707–14.

46] Robinson JM, Takizawa T, Pombo A, Cook PR. Correlative fluorescence andelectron microscopy on ultrathin cryosections: bridging the resolution gap. JHistochem Cytochem 2001;49:803–8.

47] van Donselaar E, Posthuma G, Zeuschner D, Humbel BM, Slot JW. Immuno-gold labeling of cryosections from high-pressure frozen cells. Traffic 2007;8:471–85.

48] Kandela IK, Bleher R, Albrecht RM. Multiple correlative immunolabeling forlight and electron microscopy using fluorophores and colloidal metal particles.J Histochem Cytochem 2007;55(October (10)):983–90.

49] Kandela IK, Albrecht RM. Fluorescence quenching by colloidal heavy met-als nanoparticles: implications for correlative fluorescence and electronmicroscopy studies. Scanning 2007;29(July–August (4)):152–61.

50] Anger P, Bharadwaj P, Novotny L. Enhancement and quenching of single-molecule fluorescence. Phys Rev Lett 2006;96(March 24 (11)):113002.

51] Schwarz H, Humbel BM. Correlative light and electron microscopy usingimmunolabeled resin sections. Methods Mol Biol 2007;369:229–56.

52] Sousa AA, Aronova MA, Kim YC, Dorward LM, Zhang G, Leapman RD. On thefeasibility of visualizing ultrasmall gold labels in biological specimens by STEMtomography. J Struct Biol 2007;159(September (3)):507–22.

53] Ziese U, Kübel C, Verkleij A, Koster AJ. Three-dimensional localization ofultrasmall immuno-gold labels by HAADF-STEM tomography. J Struct Biol2002;138(April–May (1–2)):58–62.

54] Lucic V, Förster F, Baumeister W. Structural studies by electron tomography:

from cells to molecules. Annu Rev Biochem 2005;74:833–65.

55] Hoenger A, McIntosh JR. Probing the macromolecular organization of cells byelectron tomography. Curr Opin Cell Biol 2009;21(February (1)):89–96.

56] Yakushevska AE, Lebbink MN, Geerts WJ, Spek L, van Donselaar EG, JansenKA, et al. STEM tomography in cell biology. J Struct Biol 2007;159(September(3)):381–91.