Precise, Correlated Fluorescence Microscopy and Electron Tomography of Lowicryl Sections Using Fluorescent Fiducial Markers

CHAPTER 13

Precise, Correlated Fluorescence Microscopy and Electron Tomography of Lowicryl Sections Using Fluorescent Fiducial Markers Wanda Kukulski*, †, Martin Schorb*, Sonja Welsch*, Andrea Picco†, Marko Kaksonen†, *and John A.G. Briggs*, † * Structural

and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstr. 1, D-69117 Heidelberg, Germany † Cell

Biology and Biophysics Unit European Molecular Biology Laboratory, Meyerhofstr. 1, D-69117 ­Heidelberg, Germany

Abstract I. Introduction II. Rationale III. Methods A. High-Pressure Freezing B. Freeze-Substitution and Embedding C. Sectioning, Pick-Up and Application of Fluorescent Fiducial Markers D. Fluorescence Microscopy E. Electron Tomography F. Fluorescent Fiducial-Based Correlation G.  Compensating for Possible Fluorescence Image Shifts H.  Application to Thin Section 2D Electron Microscopy IV. Instrumentation and Materials A. High-Pressure Freezing of Y   east Cells B. High-Pressure Freezing of Mammalian Cells C. Freeze-Substitution/Lowicryl Embedding D. Ultramicrotomy, EM Grids, FluoSpheres E. Fluorescence Microscopy F. Electron Tomography G. Fluorescent Fiducial-Based Correlation

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V. Discussion A. Flexibility in the Sample Preparation Protocol B. The Choice of the EM Grids C. The Choice of FluoSpheres as Fluorescent Fiducial Markers D. Estimation of the Correlation Accuracy E. Photobleaching of EGFP and mCherry in Lowicryl Sections F. General Applicability of the Method References

Abstract The application of fluorescence and electron microscopy to the same specimen allows the study of dynamic and rare cellular events at ultrastructural detail. Here, we present a correlative microscopy approach, which combines high accuracy of correlation, high sensitivity for detecting faint fluorescent signals, as well as robustness and reproducibility to permit large dataset collections. We provide a step-by-step protocol that allows direct mapping of fluorescent protein signals into electron tomograms. A localization precision of <100 nm is achieved by using fluorescent fiducial markers which are visible both in fluorescence images and in electron tomograms. We explain the critical details of the procedure, give background information on the individual steps, present results from test experiments carried out during establishment of the method, as well as information about possible modifications to the protocol, such as its application to 2D electron micrographs. This simple, robust, and flexible method can be applied to a large variety of cellular systems, such as yeast cell pellets and mammalian cell monolayers, to answer a broad spectrum of structure–function related questions.

I. Introduction Many cell biological questions that aim at the functional characterization of a cellular process also involve questions on ultrastructure. Such studies often rely both on dynamic information obtained from fluorescence microscopy (FM) and on high-resolution data from electron microscopy (EM) or electron tomography (ET). FM, in particular the use of green fluorescent protein (GFP) and its variants to genetically label cellular proteins, offers unique capabilities to observe processes in living cells over time (Lippincott-Schwartz & Patterson, 2003). It provides the cellular localization and distribution pattern of the fluorescent protein (FP)-labeled proteins, and it can give dynamic information on the behavior of the proteins, such as patch lifetimes, movement, and protein turnover. The large field of view (usually over many cells) and the fact that only what is fluorescently labeled is visible, make it possible to search for rare cellular events marked by FP-labeled proteins. By labeling different proteins with different color-variants of FPs (Shaner, Steinbach, & Tsien, 2005), it is possible to distinguish multiple proteins within a single sample. Together, these capabilities allow a

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functional state of a cellular structure, an intermediate stage in a cellular process or the sequence of events during a process to be defined based upon the presence or absence of proteins labeled with different color variants, or observations of the time points at which the proteins arrive and depart. The major limitations of FM are the low attainable resolution as well as the lack of information regarding the cellular context, since most cellular components are unlabeled and therefore invisible. It is a dream of cell biologists to visualize the ultrastructure hidden under the fluorescent spot whose motion they observe and to know what its surrounding environment looks like. The repertoire of recently developed superresolution techniques can partially fill the resolution gap (Patterson, Davidson, Manley, & Lippincott-Schwartz, 2010), but remain blind to the cellular context; still only what is fluorescently labeled can be seen. In contrast, cellular EM and particularly cellular ET are unbeatable tools for providing three-dimensional structural data at nanometer-resolution within cells (Baumeister, 2002; Hoenger & McIntosh, 2009). The power of imaging by EM lies in its ability to display the full cellular landscape in the field of view, and to show the ultrastructure of interest within the crowdedness of the cell at high resolution. The drawbacks, on the other hand, are equally important: The complexity of the cellular landscape makes it sometimes very difficult to identify the ultrastructure of interest. If the structure is rare, searching for it becomes searching for a needle in a haystack, and screening large areas is tedious if not impossible. Further, the electron micrograph is a static snapshot of the system; dynamic information gets lost due to the necessity of fixing the sample. Clearly, the complementarity of FM and EM makes both equally indispensable tools for cell biology. The many efforts being made in recent years to combine both techniques on the very same specimen attest to the potential of correlative microscopy methods in cell biology. Many and varied correlative microscopy procedures are available these days, as discussed in the other chapters of this volume. The first family of methods consists of those where FM is applied to the system prior to fixation. These methods have proven to be very powerful in identifying cells expressing a certain fluorescent pattern that marks a defined stage in the cell cycle, or the developmental stage of an organism (Guizetti et  al., 2011; Kolotuev, Schwab, & Labouesse, 2010; Müller-Reichert, Srayko, Hyman, O’Toole, & McDonald, 2007; Pelletier, O’Toole, Schwager, Hyman, & Müller-Reichert, 2006; Verkade, 2008). The second family of methods identifies fluorescent cells or even organelles within the EM specimen after preparation. This can be achieved either directly by FM or indirectly by immunogold-labeling or chemical reactions which transform fluorescent signals into electron contrast (Sartori et al., 2007; Schwartz, Sarbash, Ataullakhanov, McIntosh, & Nicastro, 2007; Shu et al., 2011; van Driel, Valentijn, Valentijn, Koning, & Koster, 2009; van Rijnsoever, Oorschot, & Klumperman, 2008; Watanabe et al., 2011). To answer questions concerning suborganelle-sized ultrastructures associated with highly dynamic, complex cellular processes, two further hurdles must be overcome. Firstly, high precision of correlation must be obtained. In most methods, the positioning accuracy is sufficient to localize cells within a tissue or organelles within a cell, but

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does not permit to unambiguously identify and localize features with a precision below 100 nm. Secondly, high sensitivity is required to detect faint fluorescent signals. Most approaches abrogate the fluorescent signal during sample preparation, or do not allow the use of high numerical aperture and oil-immersion objectives, which require short working distances. The achieved sensitivity is therefore far behind the state-of-the-art light microscopy setups during live cell imaging. Approaches which perform live cell imaging before fixation can potentially produce high-quality FM images, but the temporal delay between the FM and the EM image is at least a few seconds, which does not permit the study of fast processes or mobile features. These limitations mean that a number of cell biological problems are challenging to address. Such questions include the following: What is the conformational state of a flexible cellular component when it binds to a specific auxiliary protein? Is there a defined 3D ultrastructure underlying the diffraction-limited fluorescent spot which represents the cellular function of my interest? Can we unambiguously identify and assign an unknown structure to our fluorescently labeled protein of interest? How do ultrastructural intermediates of a very dynamic cellular process look like in 3D at a precisely defined time point? These types of questions require a correlative microscopy approach by which the specimen is imaged by FM and EM at the same time point (i.e., after preparation) in a manner compatible with high-sensitivity FM as well as with state-ofthe-art ET. The method must be sufficiently robust and reliable to permit large datasets to be recorded and must allow correlation of FM and EM data with high precision. We have recently presented a correlative procedure that fulfills these conditions (Kukulski et al., 2011). This approach is based on the observation that the fluorescent signal of GFP, expressed in cells which have been high-pressure frozen, can be retained in Lowicryl resin sections (Nixon et al., 2009). The freeze-substitution and embedding protocols are optimized such that the ultrastructural preservation is as good as in current state-of-the-art ET studies. At the same time, faint fluorescence signals are sufficiently well preserved that a detection sensitivity similar to live cell imaging can be achieved. RFPs are preserved as well as GFPs. FM is performed after the sections have been placed on EM grids, and the imaging setup is designed to permit the use of high numerical aperture oil-immersion lenses. To achieve the required accuracy of correlation, a fluorescent fiducial marker system is introduced. As fluorescent fiducial markers, we use fluorescent microspheres, which are added onto the section surface and are visible by FM in a fluorescent channel distinct from GFP or RFP. By ET, they become visible on the section surface after tomogram reconstruction. The set of coordinates provided by the positions of the beads in FM and in ET allows the precise position of the FP spot of interest within the electron tomogram to be calculated. In addition, the fluorescent fiducial system provides means for estimating the accuracy of the correlation. The potential of this method has been illustrated by application to different types of cell biological questions. We have demonstrated its power to find rare events such as virus–cell interactions during virus entry, by pinpointing fluorescent HIV particles on the surface of MDCK cells. We could determine the tip conformation of growing microtubules in fission yeast, marked by an RFP-labeled TIP-binding protein. Finally, we have used the method to describe intermediate membrane states within a

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10-seconds-time window during endocytosis in budding yeast (Kukulski et al., 2011). The diversity of these examples demonstrates that the spectrum of possible applications of the method is large. The method is simple, robust, and does not require any specially designed equipment. It can therefore be established and used in any cell biology lab where there is access to both FM and EM including high-pressure freezing technology.

II. Rationale This chapter is intended as a description of our recently developed correlated FM and ET method (Kukulski et al., 2011) providing comprehensive and detailed information on the protocol, focusing on critical points. An overview of the complete workflow and time line of the procedure is shown in Fig. 1. Within the chapter we will also present technical findings and conclusions drawn while establishing the method. We describe variations on the method, including application of the correlative procedure to thin sections for 2D EM.

III. Methods A. High-Pressure Freezing The primary specimens are cells that express the proteins of interest tagged with fluorescent proteins. The first stage of the method is cryo-immobilization of the sample by high-pressure freezing. For high-pressure freezing of yeast cells, we follow the protocols which were described in detail earlier in this book series (Höög & Antony, 2007; McDonald, 2007). In brief, yeast cells are pelleted using a vacuum filtration device onto a nitrocellulose filter, which is then placed onto an agar plate. The yeast paste is loaded into membrane carriers with a toothpick, and high-pressure frozen using the Rapid Transfer System of the EMPACT2. For high-pressure freezing of MDCK cells, we also use an established protocol described in detail in an earlier volume of this series (Walther, Wang, Liessem, & Frascaroli, 2010). To give a brief description, cells are grown on precleaned, carbon-coated sapphire discs. For high-pressure freezing with the BAL-TEC HPM-010, sapphire discs are clamped between aluminum planchettes (Sapphire disc placed on the flat side of a Wohlwend platelet type 242, covered with the 50 µm deep space of a Wohlwend platelet type 390), and the residual space in the planchette cup is filled with 1-hexadecene. The planchette sandwich is disassembled under liquid nitrogen prior to freeze-substitution. B. Freeze-Substitution and Embedding The embedding procedure we employ is based on the protocol published by Nixon et al. (2009), which reports GFP signal retention in Lowicryl resin. For this, high-pressure frozen samples are processed by freeze-substitution and embedding in Lowicryl HM20.

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TIME LINE flexible time fixed time possible break

WORKFLOW cell culture cryo fixation (HPF) freeze substitution

3 - 7 days Lowicryl embedding 2 days

0 - 2 weeks

choice of freeze substitution solution choice of embedding resin

UV box hardening of blocks in dark trimming and sectioning of blocks

within 1- 2 days

section pickup onto grids

choice of grids

addition of fluospheres

distribution control by EM

sandwich assembly

washing fluospheres

fluorescence microscopy

choice of fluospheres color

addition of gold fiducials 1 hour post-staining 1 day (5-10 fluorescent spots)

electron tomography

2D electron microscopy

tomogram reconstruction 1 -2 days

extraction of digital slices for correlation correlation procedure

Fig. 1  Workflow and time schedule of the complete correlative microscopy protocol. In the central column,

the individual work steps are listed. In the left side column, the estimated time required for different parts of the process is given. For steps marked by a continuous line, time is critical and should be kept at a minimum, whereas for steps marked by a dashed line the duration is flexible. The right side column shows optional steps including preparation or controls to be done. Grey boxes indicate steps which are ideally combined into one wet-lab session.

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We use a temperature-controlling AFS2 (Leica) with an FPS robot, and apply the following protocol to both yeast and MDCK cells. Freeze-substitution occurs at −90°C for 48–58 h with 0.1% (w/v) uranyl acetate in glass distilled acetone. The temperature is then raised to −45°C (5°C/h), samples are washed 3 times with acetone and infiltrated with increasing concentrations (10%, 25%, 50%, 75%, 4 h each) of Lowicryl HM20 in acetone while the temperature raises further to −25°C. 100% Lowicryl is exchanged 3 times in 10 h steps, and UV-polymerized at −25°C for 48 h after which the temperature is raised to 20°C (5°C/h) and UV polymerization continued for 48 h. After polymerization, the samples should be stored protected from light, and should be processed further within a few weeks. We noticed that the fluorescent signals in samples stored for several months tend to be weak or even completely bleached. C. Sectioning, Pick-Up and Application of Fluorescent Fiducial Markers Sections of 300 nm are cut with a diamond knife in a microtome and picked up on carbon-coated 200 mesh copper grids. As fluorescent fiducial markers, FluoSpheres (Molecular Probes) are adhered to the section (see Discussion for information on choice of fluorescent fiducials). We use 0.02 µm Blue FluoSpheres (excitation 365 nm/ emission 415 nm), because they do not fluoresce strongly in the GFP and RFP channel, but a small amount of signal bleeds through into these channels. This effect can be used for drift-correction during the correlation procedure (see below). To reduce their intensity and improve their adhesion properties, FluoSpheres are diluted 20× in a 0.1% Tween-20 in PBS solution, incubated for 10 min at room temperature, washed twice by 30 min ultracentrifugation at 100,000 g, 4°C, resuspended in PBS to a concentration approximately 50× diluted compared to the initial solution, and sonicated for 5 min. Before applying on sections, it is advisable to check the monodispersity and concentration of the FluoSpheres preparation by EM. Large or fuzzy bead aggregates instead of monodisperse spheres indicate a too harsh treatment with Tween-20. A pretreated FluoSpheres preparation can be stored at 4°C for several weeks. To adsorb FluoSpheres to sections, pretreated FluoSpheres are sonicated for 5 min, then grids are placed sectionface down onto a 15 µl drop of FluoSpheres for 10 min, blotted with filter paper and washed with 3 drops of water with blotting between the washing steps. D. Fluorescence Microscopy To achieve fluorescence image quality and signal sensitivity equivalent to that obtained in high-end live cell imaging, it is desirable to use high numerical aperture oil-immersion lenses. This requires that there is no air layer between the sections and the objective. To realize this, sections can be dried onto coverslips, which gives excellent fluorescence imaging conditions (Watanabe et al., 2011), but makes recovery for transmission EM impossible. We therefore decided to image sections adhered to an EM grid, which is immersed in a thin water layer on a coverslip. To minimize the working distance, the section-side of the EM grid has to face toward the objective during imaging. The water layer between coverslip and sections ensures good imaging

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conditions and allows the use of oil immersion objectives. To prevent drying, the grid in water can be sandwiched between the coverslip and a glass-slide, and sealed with vacuum grease. In practice, we found it more convenient to sandwich the grid between two circular coverslips held together by a ring holder (custom-made or purchased “Attofluor cell chamber” from Invitrogen) (Fig. 2). For this, drops of 15 µl of water are placed onto two round coverslips, of which one has a layer of vacuum grease at the rim (Fig. 2(A and B)). The grid is placed on one of the drops, and the sandwich is very carefully closed, avoiding the formation of an air layer or bubbles between sections and coverslip, as well as preventing the grid from sliding into the rim of vacuum grease (Fig. 2(C and D)). The assembled sandwich can then be imaged in a fluorescence microscope of choice. The choice of fluorescence microscope depends upon the sample of interest, but in all cases it should have sufficient sensitivity to detect the signals of interest, and appropriate filters for detecting all FPs as well as the fluorescent fiducials. In general, the microscopy setup used for characterization of the signals by live cell or conventional

A

B

C

D

Fig. 2  Assembly of the sandwich setup for FM of sections on EM grids. (A) The ring holder consists of two

parts which can be screwed together to hold the coverslips in between. An O-ring ensures tight fixation of the coverslips while avoiding breakage. The sandwich is assembled from two coverslips each carrying a drop of water. The arrow indicates a rim of vacuum grease on one of the coverslips. (B) The grid is placed section-face down onto one drop of water, and the sandwich is carefully closed with the second coverslip, avoiding that the grid moves toward the rim of the coverslips. (C) The closed sandwich is placed in the lower part of the ring holder. (D) The two parts are screwed together, just as tight as necessary to keep the sandwich fixed. Too tight screwing will press the water out of the coverslip sandwich and cause the sections to stick to the coverslip.

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FM will also be appropriate for imaging sections. We found that a wide-field imaging set-up with a 100× oil objective was ideal for our needs (see Materials). During FM, the positions on the grid of the imaged areas of interest should be recorded so that they can be found back easily by EM (Fig. 3). We recommend the use of grids with landmarks in the grid center (see Materials), which aid in locating the region of interest to a defined gridsquare. In addition, 200 mesh grids have a gridsquare size of about 90 µm, which roughly matches the entire field of view by FM in our setup, making orientation relative to grid bars easy. It is thus best to mark the gridsquare imaged by FM on a grid scheme (Fig. 3(A)). The section is usually not perfectly flat; therefore, several focal planes will be needed to obtain in-focus images of the full area of interest. This means that for each of the three channels (GFP, RFP, and blue fluorescent fiducials), a number of 2–5 images need to be taken in different focal planes. It is important to plan the recording scheme to avoid any unnecessary exposure that will cause bleaching of the FP signal. Since photobleaching of the bright FluoSpheres is insignificant, they should be imaged after all the more critical GFP and/or RFP images have been collected on the given area. It is recommended to start imaging in the bright field mode by finding the lowest (or highest) focal plane, record both GFP and RFP images, change the focus, and then record both GFP and RFP again. This can be repeated until the highest (or lowest) focus is reached. The number of focal planes that can be taken may be limited to about 3 or 4 planes due to the bleaching of the GFP signal, depending on its brightness (see “Photobleaching of EGFP and mCherry in Lowicryl sections”). After collecting all GFP and RPF images, the focal stack of blue fluorescent fiducial images is recorded by working the way back from the highest to the lowest focal plane. Collecting a sufficiently large series of blue fluorescent fiducial images ensures coverage of the whole area of

A

B

C

Fig. 3  Visual orientation on the EM grid from the fluorescence to the electron microscope. (A) The gridsquare

imaged by FM is marked on a grid scheme (white dashed box). This helps to find this gridsquare back in the electron microscope. (B) An overlay of the whole GFP and RFP image frames, which correspond to approximately the size of one gridsquare. The gridbars are indicated by grey shading (top right and bottom left corner). Their positions are identified in bright field mode (not shown). The approximate area recorded in the low magnification tomogram is marked by a white dashed square. This mark greatly helps to re-find the area during the fluorescent fiducial-based correlation procedure. (C) An enlarged view of the area marked in (B). Here the dashed square represents the dimensions of the high magnification tomogram. Scale bars are 10 µm (B) and 2 µm (C). (See color plate.)

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interest by in-focus images of FluoSpheres. Because the flatness of the sections varies from area to area, the spacing between focal planes may need to be adjusted to obtain a maximum area of coverage with a minimum of exposure. For this reason, we did not find it helpful to automatically collect focal stacks for all areas. Fluorescence imaging of EM grids should be done within approximately 1 day after sectioning, as we noticed loss of fluorescence signal in sections on EM grids after longer time periods. Fluorescent signals in sectioned material appear to deteriorate significantly faster than in the resin blocks, which can be stored in the dark for at least a few weeks. We recommend to make composite images from the three channels and to have these at hand when sitting at the electron microscope during the subsequent ET session (Fig. 3(B)). They can be used to quickly find the cell of interest in the electron microscope based on the pattern of cells, and to note the approximate area where tomograms are taken. This mark will be helpful in the later correlation procedure to restrict the area in which the fluorescent signals of fluorescent fiducial beads are found (see Fig. 4). E. Electron Tomography To enhance the contrast, grids with yeast sections are poststained after FM imaging with Reynolds lead citrate for 12 min, whereas we found the contrast in MDCK cell sections sufficient without further staining. As tomographic fiducial markers, 15 nm protein A–coupled gold beads are adsorbed on both sides of the grids. Grids are placed in a high-tilt holder, and digital images are collected as dual-axis tilt series over a −60° to 60° tilt range (1° increment). Typical pixel sizes we use are 1.18 nm, 1.52 nm, or 1.97 nm at the specimen level. With a tomogram frame size of 2048 pixels (we acquire binned images on a 4k × 4k CCD camera), a pixel size of 1.18 nm results in a tomogram field of view of 2.4 µm. This corresponds to a frame size of 37 pixels in FM (considering a pixel size of 64.5 nm as we have on our camera). The fluorescent signal from a point source such as one FluoSphere (or any diffraction-limited spot of interest) typically spreads over 5–10 pixels, depending on its brightness. It is thus likely that in such a small area, not enough FluoSpheres will be resolved by fluorescence imaging (see also “Fluorescent fiducialbased correlation”) to perform the correlation procedure. Therefore, if the structures of interest require the collection of high magnification tomograms, it is recommended to additionally record lower magnification tilt series where the field of view will contain more FluoSpheres for correlation. Low magnification tomograms are typically collected as single-axis tilt series at 2.53 nm or 5.07 nm pixel size (corresponding to 5.2 or 10.4 µm frame size) and at 3° or 2° increment, respectively. For all tomogram reconstructions, we use the IMOD software package (Kremer, Mastronarde, & McIntosh, 1996). Lower magnification tomograms, which are only used for fluorescent fiducial-based correlation and not for showing the ultrastructure of interest at high resolution, can be reconstructed using the patch-tracking algorithm available in IMOD version 4.1.4, which is much faster than the gold fiducial alignment procedure.

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input for fiducial-based correlation procedure fluorescence microscopy

electron microsocopy

1. assigning fiducial markers in FM and EM

A fiducial fluorescence

B

C

2. assigning FP spot of interest

D RFP fluorescence

calculate optimal transformation between fiducial and EM image based on fiducial positions

E centroid of FP spot of interest

apply transformation to fluorescent spot coordinates

G 3. shift between fluorescent images

F identify fiducial fluorescent

signal bleedthrough in RFP image calculate shift and apply to fluorescent spot coordinates

Result: transformed FP spot coordinates: x=1106 y=1210

Fig. 4  Fluorescent fiducial-based correlation procedure. The example area shown here corresponds to the one

shown in Fig. 3. Yellow frames indicate input images (fiducial fluorescence image (A), average EM image (C), and fluorescence image containing the RFP spot of interest (D)). The central column contains enlargements of the FM area of interest (B and E). In the first panel, the manual assignment of fluorescent fiducials in FM and EM is presented (yellow circles). The second panel illustrates the input of the RFP spot of interest (yellow circle), and the Gauss-fitting-based centroid determination is indicated as a red cross (E). In the third row, an overlay of the fluorescent fiducial and RFP fluorescence images is shown (F), and the fluorescent fiducials which bleed-through to the RFP channel are marked by yellow circles. These are used to calculate the average shift between the fiducial fluorescence image and the RFP image. This shift is then applied to the RFP spot coordinates shown in (E). The new coordinates of the RFP spot of interest are then converted into EM coordinates (red mark in G) by applying the calculated optimal transformation between fluorescence and EM fiducial coordinates. Scale bars are 10 µm (A, D, F) and 1 µm (C, G). (See color plate.)

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F. Fluorescent Fiducial-Based Correlation The concentration of fluorescent fiducial markers needs to be adjusted to achieve the appropriate distribution density. The density must be low enough to clearly resolve the beads from each other in the FM, but high enough that about 6–10 fluorescent fiducials fall within the region reconstructed in the electron tomogram. We found that approximately 50-fold dilution after pretreatment of FluoSpheres usually gives the right distribution. To aid in visualizing all fluorescent fiducials included in the EM field of view, those tomographic slices which contain the fluorescent fiducials adhered to the section surface were averaged. To do so, we use the “Stack” functions of the ImageJ software. An overview of the correlation procedure is presented in Fig. 4. The correlation process is performed with a simple in-house-written MATLAB procedure. As inputs it requires the averaged EM image as well as those fluorescence images containing fluorescent fiducials, GFP and RFP in-focus signals from the area of interest. After entering the input data, the fluorescence micrographs are shown on screen next to the average EM image (it is possible to adjust the brightness and contrast). Next, using the MATLAB Control Point Selection Tool, the signals of FluoSpheres in the fluorescent image are identified and assigned to the corresponding FluoSpheres visible in the averaged EM images by clicking with the mouse on 6–10 FluoSpheres in each image. The precise coordinates of the fluorescent signals of the fluorescent fiducials are determined using a centroid fit with subpixel accuracy, which is carried out on a fluorescence image which is high-pass-filtered with a smooth cut-off of 70 pixels. The filter excludes gradient effects from cellular background, which would otherwise affect the centroid fitting. The centroid coordinate of the GFP or mCherry labeled signal of interest is determined in the same manner, after manually marking its position in the fluorescence image. The coordinates of the fluorescent fiducial pairs are used as landmarks for generating linear conformal transformations which relate the fluorescence image with the EM image. Transformations are calculated using all possible combinations of subsets of fluorescent fiducials containing a minimum of three fluorescent fiducial pairs. The transformations are then scored by comparing the sum of squared residuals of the predicted fluorescent fiducial positions from the coordinates of the centroids of fluorescent fiducials in the EM image, and the best-scoring transform is selected. This transformation is then applied to the fluorescence coordinates of the spot of interest to calculate its coordinates in the electron tomogram. To verify the success of the correlation, the predicted positions of the transformed fluorescent fiducials are presented to the user to visually assess their deviation from the beads’ actual positions in the EM image. In this way, any incorrectly picked fluorescent fiducials can be identified. Although the above procedure excludes most outliers, in rare cases they are included in the calculation. Thus, if the correlation is inaccurate due to wrongly assigned fluorescent fiducials or outliers which are positioned on locally deteriorated section areas, the fluorescent fiducial assignment can be repeated at this stage after they are discarded. Once the bead positions are accepted by the user, the final transformed coordinates are saved in a log file. In case a further visual check of the correlation is desired,

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transformed fluorescent images as well as images showing the positions of fluorescent fiducials and the transformed spot in the EM image are created and can be overlaid. If a higher magnification tomogram was recorded, a further transformation of the coordinates of the fluorescent spot from the low magnification to the high magnification tomogram is necessary (Fig. 5). This is done in a second correlation step using a MATLAB Control Point Selection Tool-based procedure, which is equivalent to the first, but which uses the tomographic gold fiducials to calculate the transform. ­Averages of high and low magnification tomogram slices showing the tomographic gold fiducials are calculated in ImageJ, and serve as input images displayed in MATLAB to assign pairs of tomographic gold fiducials. Since the EM image showing the FluoSpheres, which was used in the first round of correlation, also shows the section surface, it may contain enough tomographic gold fiducials to be readily used in this step as well. The tomographic gold fiducials are generally distributed more densely than the FluoSpheres and are therefore better suited for correlating small fields of view. G. Compensating for Possible Fluorescence Image Shifts The desired accuracy of the correlation is below 100 nm, which is in the range of the FM pixel size. This means that chromatic aberration or small sub-pixel to pixelsized shifts between the fluorescence image containing the fluorescent fiducials and the image containing the FP signal of interest will cause localization errors of the FP coordinates in the electron tomograms in the 50–100 nm range. It is therefore absolutely critical to either avoid any shift between the fluorescence images or to correct for any shift or aberration which has occurred during imaging.

input for 2nd correlation step if higher magnification tomogram required assigning gold fiducials in low mag and high mag

A

B

C

Result: transformed FP spot coordinates in high mag tomogram: x=1034 y=964

Fig. 5  Correlation of FP signal with high magnification tomograms. If a higher magnification tomogram has

been recorded, the FP spot coordinates are transformed onto this high magnification tomogram based on tomographic gold fiducials. Average images of the low magnification (A) and high magnification tomogram (B) are used to assign gold fiducials. A detail from a tomographic slice of the high magnification tomogram is shown in (C); the transformed FP spot coordinates reveal the structure of interest. The inner and outer circles mark the 50% and 80% accuracy, respectively. Scale bars are 1 µm (A), 200 nm (B), and 100 nm (C).

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Such shifts might typically be caused by slight drift of the microscope stage between collection of the FP image and collection of the fluorescent fiducial image. We decided to correct for the shift after imaging, which could be comfortably done during the correlation procedure. In order to correct for any shift between the fiducial fluorescence image and the fluorescence image containing the FP spot of interest, it is necessary to measure the amount and direction of the shift. To do this, we look at an overlay of these two images and click the positions of bright FluoSpheres signals which bleed-through from the fiducial fluorescence channel into the fluorescence image containing the FP spot of interest. We then measure the differences between their positions in the two images. From these measurements, the average shift in x and y directions is calculated, and used to correct the coordinates of the fluorescent spot of interest. H. Application to Thin Section 2D Electron Microscopy For some ultrastructural questions, 3D information is not required, and it is sufficient to collect conventional 2D EM images instead of tomograms. We therefore tested the applicability of our correlative procedure to 2D images. We found that FluoSpheres were visible on poststained thin sections and that it is therefore very straightforward to apply the method for 50 nm sections (Fig. 6). The protocol is generally the same as for 300 nm sections. Instead of tomograms, we collected EM images of the spot of interest at various magnifications and applied the fluorescent fiducial-based procedure to correlate these images to the corresponding GFP and RFP images. If higher magnifications are desired, gold fiducials can be adsorbed to the sections in addition to FluoSpheres and used to correlate low and high magnification images, analogously to the procedure described above using low and high magnification tomograms.

IV. Instrumentation and Materials A. High-Pressure Freezing of Yeast Cells Instrumentation: Vacuum pump, filtration apparatus (McDonald, 2007). As highpressure freezer, we use the Leica EMPACT2 equipped with the Rapid Transfer ­System. Materials: 0.45 µm pore size nitrocellulose filters, toothpicks, YPD agar plate, 0.2 mm membrane carriers (Leica Microsystems) B. High-Pressure Freezing of Mammalian Cells Instrumentation: For mammalian cells grown on sapphire discs, we use the ­BAL-TEC HPM-010. Materials: Carbon-coated sapphire discs and aluminum planchettes (M. Wohlwend, Sennwald-CH) (Walther et al., 2010) Reagents: 1-hexadecene

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Fig. 6  The correlation procedure applied to 2D images of 50 nm Lowicryl sections. (A) Overlay of EGFP and mCherry images of a 50 nm section of yeast cells expressing proteins labeled with both FPs. A spot where EGFP and mCherry colocalize has been targeted, and the dashed rectangle marks the area that corresponds to the electron micrograph shown in (B). In (C) a part of the fiducial fluorescence image is shown, and the selected fluorescent fiducial signals assigned to beads seen in (D) are illustrated by yellow circles. The green cross represents the transformed EGFP spot coordinates. (E) A higher magnification micrograph of the region of interest. Scale bars are 10 µm (A), 2 µm (C), and 250 nm (E). (See color plate.)

C. Freeze-Substitution/Lowicryl Embedding Instrumentation: Leica AFS2 with FPS robot for automated reagent handling Materials: AFS2 consumables (reagent bottles, flow-through rings, reagent baths, dispenser syringes) Reagents: Glass-distilled acetone, 20% uranyl acetate in dried methanol, Lowicryl HM20 (Polysciences, Inc.)

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D. Ultramicrotomy, EM Grids, FluoSpheres Instrumentation: Microtome (Leica Ultracut UCT), diamond knife (Drukker), ultracentrifuge (Tabletop Beckmann Instruments Inc., with rotor TLA 120.1) Materials: Centrifuge tubes (Beckman Instruments, Inc.), 200 mesh copper grids with carbon support film (Plano, product number S160) Reagents: Blue FluoSpheres (365/415), 0.02 µm in size (Invitrogen, catalog number F8781, a large selection of colors and sizes is available at Invitrogen), Tween 20 (Sigma-Aldrich), phosphate buffered saline (PBS) E. Fluorescence Microscopy Instrumentation: We use an Olympus IX81 microscope equipped with a 100× NA1.45 objective, Orca-ER CCD camera (Hamamatsu), electronic shutters, and filter wheels (Sutter Instruments). An X-Cite 120 PC lamp (EXFO) is used for fluorescence excitation with the following filters: 470/22 nm for GFP, 556/20 nm for mCherry and 377/50 nm for Blue FluoSpheres. For GFP and Blue FluoSpheres we use 520/35 nm, and for mCherry 624/40 nm emission filters. We use Metamorph software (Universal Imaging) to control the CCD camera, filter wheels, and shutters. Materials: Round coverslips (Menzel-Gläser, diameter 25 mm, number 1), custommade ring-holder or Attofluor cell chamber (Invitrogen) Reagents: Beckman vacuum grease silicone (Beckman Instruments Inc.) F. Electron Tomography Instrumentation: High-tilt tomography holder (Model 2020; Fischione Instruments) or a DualAxis tomography holder (Model 2040, Fischione Instruments). The electron microscope we use for data collection is a FEI Tecnai TF30 operated at 300 kV. We record digital images on a FEI 4k Eagle camera as dual-axis tilt series. Software: SerialEM for automated tilt series acquisition (Mastronarde, 2005), IMOD software package for tomogram reconstruction (Kremer et al., 1996) Reagents: 15 nm Protein-A covered gold beads, Reynolds lead citrate for poststaining of yeast sections. G. Fluorescent Fiducial-Based Correlation Software: Matlab 7.4 (The MathWorks, Inc.) with the Image Processing Toolbox installed. ImageJ 1.44 (National Institutes of Health, USA)

V. Discussion In this chapter, we have described a correlative microscopy method for the study of unknown, rare, transient, and dynamic cellular ultrastructures on the 100 nm scale, which are identified by tagging proteins of interest with FPs. We aimed to provide the

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reader with detailed information on how to perform correlative FM and ET based on preserved fluorescent protein signals in resin sections of embedded cells, and how to achieve an accuracy of correlation of better than 100 nm. The method is robust and allows acquisition of large correlative datasets, and thus facilitates quantitative analysis of the system under study. A. Flexibility in the Sample Preparation Protocol Several points in the protocol must be stringently followed, others can be adapted to the needs of the actual project. Stringent requirements of the method include cryo-immobilization of the specimen, the limited use of chemicals in the freeze-­substitution solution, and fast processing of the samples after UV-polymerization of Lowicryl through fluorescence imaging, in particular after sectioning. Aspects of the protocol which can be tuned for the cellular system or biological question of interest include, for example, the temperature program of the freeze-substitution and embedding. The freeze-substitution solution also tolerates some changes and small amounts of fixative, if necessary. We tested freeze-substitution solutions with 0, 0.1, and 0.2% uranyl acetate and found that with 0.2% uranyl acetate, the fluorescence preservation was decreased, and that with 0% uranyl acetate the ultrastructural preservation was impaired. We also tested the addition of 1–3% water, which is in some cases desired to improve membrane contrast (Walther & Ziegler, 2002), and found it had no effect on fluorescence retention, and that it did not significantly improve membrane contrast in our samples. The addition of 0.1% glutaraldehyde in the freeze-substitution solution was tested as well: we observed no significant improvement in ultrastructure preservation, but found that weak fluorescent signals were no longer preserved. Signals originating from a large number of FPs, such as MA-EGFP-labeled HIV particles, could, however, still be visualized. Since the ultrastructural preservation was equally satisfying without chemical fixatives, we did not further investigate the effects of higher glutaraldehyde concentrations. Although we did not explore this ourselves, various resins have been reported to retain the fluorescent signal from FPs. These are LR White for YFP fluorescence, particularly when the sample is not fully dehydrated (Micheva & Smith, 2007), or GMA resin to retain Citrine, tdEOS and Dendra fluorescence (Watanabe et al., 2011). These reports suggest that the embedding medium does not necessarily need to be Lowicryl HM20. In addition, while our proof of principle experiments focused on GFP and RFP variants (Kukulski et al., 2011), the above-mentioned studies showed the retention of other FPs, indicating that it is likely to be possible to retain the fluorescence of a wide range of FPs. B. The Choice of the EM Grids While exploring procedures to image resin sections by FM, we tested various grid types as support for the fluorescent Lowicryl sections. We observed that Formvar and Pioloform, both used as support for carbon film on EM grids or as exclusive support film on slot grids, show significant autofluorescence in both GFP and RFP channels,

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creating a speckle-type background which obscures faint fluorescent spots during FM. We therefore recommend the use of grids coated only with carbon (see Materials). Alternatively, for example, if the use of slot grids is important, using a confocal microscope for fluorescence imaging can permit the resin section plane to be focally separated from the plastic support film. C. The Choice of FluoSpheres as Fluorescent Fiducial Markers When selecting the type of beads suitable to form the fluorescent fiducial system for correlating fluorescent images to electron tomograms, the requirements were defined as follows: (i) a bright fluorescent signal with a spectrum distinct from the FPs of choice; (ii) a good visibility by EM on cellular sections; (iii) monodispersity as well as good adsorptive properties on cellular sections; and (iv) commercial availability. We tested different variants of FluoSpheres, as well as fluorescent gold beads and quantum dots as fluorescent fiducial markers. We tested Rhodamine-labeled 8 nm gold beads by adsorbing them to resin sections on EM grids and imaged them both by FM and by EM. We were able to assign fluorescent signals to the corresponding gold beads, which in most cases were in fact clusters of 3–10 gold beads with the fluorescence intensity correlating with the number of gold beads in the cluster. Single gold beads were rarely detected by FM. Although their high contrast in electron micrographs, even on strongly stained cellular background, was an advantage, we found several disadvantages in using fluorescently labeled gold. Firstly, it is necessary to discriminate them from tomographic gold fiducials used for tomogram reconstruction. The same fiducials cannot be used for both correlation and tomogram reconstruction, because the latter must be present on the section at a much higher density than can be used for correlation. Secondly, single beads give a signal that is too weak to be detected, and most reliable signals originate from bead clusters, which hinder precise localization of the signal. Thirdly, the beads are not commercially available, and need to be prepared in several centrifugation cycles with sequential coating steps. We also tested imaging of quantum dots Q655 adsorbed to carbon-coated EM grids by FM and EM. The main disadvantage of quantum dots is their “blinking” behavior in FM. This requires imaging over a long time period in order to collect signals from all fiducials in the imaged areas. In addition, the small size, low contrast, and nonspherical shape of the quantum dots make it difficult to detect them on stained sections of cells. In contrast to this, commercially available FluoSpheres give a bright, constant fluorescent signal, can be easily distinguished from tomographic gold fiducials, and their concentration and monodispersity on the section surface can be well controlled. Their color can be chosen according to the color of the FP signals. Although FluoSpheres appear less electron dense than gold beads or quantum dots, which makes them difficult to see in projection images of 300 nm sections of stained resin sections, they can easily be detected after tomographic reconstructions and on thin 50 nm sections. The diameters we observe for FluoSpheres with a nominal size of 20 nm vary between approximately 20 and 200 nm, and correlate rather well with their fluorescence intensity, which can be helpful when assigning fluorescent signals to the fluorescent fiducials seen in the EM image.

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D. Estimation of the Correlation Accuracy For interpretation of the data, it is important to determine the accuracy and confidence with which a specific FP signal is localized within the electron tomogram. This accuracy will be sample-dependent: the FP signal itself, the cellular system under study, the density of the fluorescent fiducials and the degree of distortion of the sample during sectioning may all have an impact on how precisely the correlation will work. If a reasonably large dataset has been collected, it is possible to use the data to estimate the accuracy with which the position of the fluorescent signal can be determined in the tomogram. In the ­correlation procedure described above, the coordinates of fluorescent fiducials within fluorescence and EM images serve as a basis to calculate the transform that maps the FP signal of interest onto EM images. To estimate the accuracy of the correlation, one fluorescent fiducial bead is excluded from calculating the transformation, but is instead treated like the FP spot of interest: this means that its position in the EM image is predicted based on the transformation calculated using the coordinate pairs of the other fluorescent fiducials. The predicted fluorescent fiducial position can then be compared to its actual position. This procedure is repeated automatically for all the fluorescent fiducials in the dataset, resulting in a set of deviations of the predicted positions from the true positions. From this set, the fraction of predicted positions which fall within a certain radius from the actual position can be derived. For example, in the case of the RFP-labeled microtubuletip binding protein mal3p localized in fission yeast cells, we used this method to estimate that the accuracy of RFP-mal3p localization was 52 nm for 50% of the correlations, and 121 nm for 80% of the correlations (see dashed circles in Fig. 5(C)). This calculation does not take into account any error in the determination of the shift between the fiducial fluorescent image and the fluorescent image of the FP of interest (see Section III.G). This error is typically on the order of 30 nm and can be removed completely if the correlation is carried out using only fluorescent fiducials that bleed through into the fluorescent channel of the object of interest, and if their coordinates are measured in that channel. E. Photobleaching of EGFP and mCherry in Lowicryl Sections When collecting several fluorescence images to cover the entire focal depth of a section (see Section III.D), we noticed that GFP signals bleach rather fast, while RFP signals remain almost constant. We compared the fluorescence photobleaching in sections of budding yeast co-expressing Lsp1-mCherry and Pil1-EGFP, which are the major components of eisosomes, and are present at eisosomes in roughly equal, high copy numbers of 2000 to 5000 molecules (Brach, Specht, & Kaksonen, 2011; Walther et al., 2006). The used excitation intensities gave similar signal-to-noise ratios with 1 s exposure times for both channels, but after 60 frames of 1 s exposure time each, Pil1-EGFP is almost completely bleached, whereas Lsp1-mCherry still shows significant fluorescent signals (Fig. 7). These observations indicate that the photostability of mCherry is greater than that of EGFP when embedded in resin sections. We have not quantified this effect, but have observed it repeatedly in various samples. It should therefore be considered when selecting the FP tag for a set of experiments. This choice will of course

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shown in (A) and of Pil1-EGFP in (B). The gallery shows a selection of frames from a series of 60 frames, 1 s exposure each. The numbering of frames corresponds to the cumulative exposure when the given frame was recorded. Below, the fluorescence intensity along a line (positioned as shown in the inset frame) is plotted for the frames corresponding to a total exposure of 1 s, 30 s, and 60 s. The plot shows that spot and background intensities (peaks and baseline) of mCherry and EGFP are similar after 1 s of exposure. After a total exposure of 60 s, the mCherry signal over background remains clearly visible, whereas EGFP spots are almost undetectable after the same amount of exposure.

also depend on the same factors considered for live cell imaging experiments such as the cytosolic background, fluorescence intensity, protein maturation rate, etc. F. General Applicability of the Method It is important to emphasize that this correlative microscopy method can be applied to a large variety of cell biological questions in various cell types or organisms, provided that the following requirements of the specimen are fulfilled: First, cryo-immobilization (i.e., high-pressure freezing) protocols for the given organisms must be available and result in good ultrastructural preservation. For many model systems, high-pressure freezing protocols can be found in various chapters of Volume 96 of Methods in Cell Biology (Electron Microscopy of Model Systems). Second, signals from the FP-tagged proteins of interest must be unambiguously identifiable and well characterized by conventional FM or live cell imaging. Information should be available or obtainable on the cellular distribution of

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the signal, its brightness, and its shape (i.e., whether the signal originates from structures which are seen as resolution-limited spots in FM). The lower detection limit of the signal will depend on a combination of copy number, brightness of the selected FP, and the cytosolic background of the tagged protein: these are the same considerations as for live cell imaging. We have found that in general, even at low copy number, if a signal is clearly detectable by conventional FM or live cell imaging, it will also be detectable in resin sections (Kukulski et al., 2011). If a transient or dynamic structure is to be described, dynamic information such as signal lifetimes and movement are also essential for a comprehensive interpretation of the “static” correlative microscopy data derived from plastic sections. A simple variant of the method described here is its application on 2D electron micrographs, instead of electron tomograms. This approach is faster because tomographic reconstruction is avoided, and it does not require the availability of a high-end electron microscope equipped for automated tomography. In some cases, visualization or identification of the ultrastructure of interest may not require 3D information. However, acquiring electron tomographic data instead of 2D micrographs in order to correlate cellular ultrastructures to fluorescence data has great advantages: for example, when studying endocytic invaginations in yeast, only in 3D does their tubular shape become obvious and can be unambiguously distinguished from sections through furrow-like invaginations of the plasma membrane (Kukulski et al., 2011; Stradalova et al., 2009). Subtle conformational variants such as the different structures of microtubule tips can only be reliably determined in electron tomograms (Höög et  al., 2007, 2010). A 2D electron micrograph is a projection through the section volume (with a minimal thickness of approximately 50 nm), which means that subtle fine structural features such as microtubule protofilaments will be largely obscured by the dense cytoplasm above and below it. In contrast to that, virtual tomographic slices represent volumes of few nanometers thickness, giving crisp and clear images. On the other hand, many problems do not require 3D information, and these can be addressed by the 2D correlative approach. For instance, projection images are adequate for questions on the subcellular localization of the protein of interest, or the compartment with which the labeled protein is associated. The 2D approach can in some cases serve as an alternative to immuno-EM if, for example, immunogold labeling is impractical due to the lack of suitable antibodies. Correlative light and electron microscopy methods have enormous potential to answer fundamental cell biological questions. The correlated FM and ET method described in this chapter allows 3D spatial resolution to be combined with dynamic information from FM on the ultrastructural scale. The procedure is simple, robust, and provides the flexibility to be applied to many different cell biological questions in ­various cellular systems. References Baumeister, W. (2002). Electron tomography: towards visualizing the molecular organization of the cytoplasm. Current Opinion in Structural Biology, 12, 679–684. Brach, T., Specht, T., & Kaksonen, M. (2011). Reassessment of the role of plasma membrane domains in the regulation of vesicular traffic in yeast. Journal of Cell Science, 124, 328–337. Guizetti, J., Schermelleh, L., Mäntler, J., Maar, S., Poser, I., Leonhardt, H., et al. (2011). Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments. Science, 331, 1616–1620.

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