Here, we present a protocol for optimized on-section correlative light-electron microscopy based on endogenous, fluorescent labeling as a tool to investigate the localization of rare proteins in relation to cellular ultrastructure. The power of this approach is demonstrated by ultrastructural localization of endogenous LC3 in starved cells without Bafilomycin treatment.
The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal details that are important for understanding the autophagic process. However, EM methods often lack molecular information, obstructing the correlation of ultrastructural information obtained by EM to fluorescence microscopy-based localization of specific autophagy proteins. Furthermore, the rarity of autophagosomes in unaltered cellular conditions hampers investigation by EM, which requires high magnification, and hence provides a limited field of view.
In answer to both challenges, an on-section correlative light-electron microscopy (CLEM) method based on fluorescent labeling was applied to correlate a common autophagosomal marker, LC3, to EM ultrastructure. The method was used to rapidly screen cells in fluorescence microscopy for LC3 labeling in combination with other relevant markers. Subsequently, the underlying ultrastructural features of selected LC3-labeled spots were identified by CLEM. The method was applied to starved cells without adding inhibitors of lysosomal acidification.
In these conditions, LC3 was found predominantly on autophagosomes and rarely in autolysosomes, in which LC3 is rapidly degraded. These data show both the feasibility and sensitivity of this approach, demonstrating that CLEM can be used to provide ultrastructural insights on LC3-mediated autophagy in native conditions-without drug treatments or genetic alterations. Overall, this method presents a valuable tool for ultrastructural localization studies of autophagy proteins and other scarce antigens by bridging light microscopy to EM data.
Autophagy is a key process for the clearance and recycling of cytoplasmic proteins and organelles. The process of macro-autophagy (hereafter called autophagy) involves the formation of double-membrane organelles, autophagosomes, which allows cells to enclose cytoplasmic molecules and organelles for lysosomal degradation. Autophagy occurs at a basal level in most cells and is upregulated in response to cellular conditions, such as starvation or cellular stress. Autophagy either occurs in a substrate-specific manner, targeting specific structures or proteins for degradation, or as a non-selective bulk process encompassing parts of the cytosol. In selective autophagy, autophagosomes are formed by the conjugation of Atg8-family proteins (microtubule associated proteins 1A/B light chain 3A/B/C [LC3] and GABARAPs) to membranes derived from recycling endosomes, the Golgi, and/or endoplasmic reticulum (ER)1. LC3 recognizes autophagic cargo in the cytosol directly or via selective autophagy adaptors such as P62/SQSTM. New autophagic membranes can then be conjugated to LC3, expand, and fuse to form a completed double membrane enclosing the cargo-called the autophagosome. The autophagosome matures and eventually fuses with an endosome or lysosome, whereafter the autophagic cargo and adaptors are degraded2.
Studies on autophagosome formation, maturation, and fusion often make use of light microscopy technologies. Fluorescence microscopy of LC3 is generally used to assess the number and cellular localization of autophagosomes under different conditions. Furthermore, by coupling LC3 to pH-sensitive GFP and pH-stable RFP in a so-called tandem probe, the overall flux of autophagy can be measured in live cells as a function of GFP fluorescence loss3. These approaches are valuable tools for researchers to understand the role and mechanism of autophagy under different conditions. Another invaluable tool is electron microscopy (EM), which reveals the ultrastructure of autophagic organelles at different stages of autophagy4,5,6,7,8. To date, EM is still the method of choice to identify the precise stages of autophagosome formation by discriminating different autophagic membranes by morphology: phagophore (double membrane not fully closed), autophagosome (closed double membrane around cytosolic cargo), and autolysosome ([partial] loss of the inner autophagic membrane). Morphology without molecular information, however, can be prone to misidentification or ambiguity. Immuno-EM is the most comprehensive method for simultaneous molecular characterization and morphological classification of autophagic organelles. For example, immunogold labeling of LC3 on thawed cryosections allows the ultrastructural localization of LC3 and precise identification of LC3-marked organelles9.
A drawback of EM is the small field of view that comes with the high magnification required to observe the fine ultrastructure of autophagic membranes and, in the case of immuno-EM, to locate the label that marks the protein of interest. Due to their scarcity and low protein levels, this generally hampers the quantitative EM analysis of autophagosomes. To increase the number of autophagosomes, cells are often starved and treated with Bafilomycin A1 (BafA1), an inhibitor of lysosomal acidification and degradation. Without BafA1 treatment, the search for autophagosomes by EM is time-intensive, due to the scarcity of these organelles. The method presented in this manuscript addresses this issue through fluorescent labeling and imaging of endogenous LC3 on thawed cryosections in a fluorescence microscope before further preparation for EM. The fluorescent images then guide the search for LC3-labeled structures in the EM. After collection, EM images are correlated with the fluorescence images to add molecular information—the presence of LC3—to the ultrastructure of the cell. This 'on-section CLEM' method greatly increases the ability to find LC3-labeled structures, especially in untreated conditions, for subsequent identification and classification by EM.
This method was applied to starved hepatoblastoma-derived HEPG210 cells to find autophagosomes in unaltered (i.e., no BafA1 was used) conditions. Relatively few fluorescent puncta (less than one per cell profile in a 90 nm section) were found, which is in agreement with the high turnover of LC311. This sparsity of LC3-puncta emphasized the value of CLEM; by selecting regions with several fluorescent puncta for imaging in the EM, LC3-positive organelles were found and characterized in a much more effective manner than through conventional immuno-EM. This revealed that the majority of LC3-positive organelles were autophagosomes, as defined by their morphology, which is in contrast to the results obtained in BafA1-treated cells, where autolysosomes are more common9. These data show that with on-section CLEM, autophagy can be studied at the ultrastructural level without the need to inhibit autophagic flow.
1. Preparation of tools and reagents
NOTE: For required reagents, buffers, and solutions, see Supplemental File 1 or 12 for more information. For details related to all materials, reagents, equipment, and software used in this protocol, see the Table of Materials.
2. Fixation and sample preparation
3. Sectioning
4. Labeling and light microscopy
5. EM
6. Correlation and analysis
An optimized immuno-EM protocol for immuno-gold labeling of LC3 on ultrathin cryosections was recently published by De Maziere et al.9. This study included starved conditions without BafA1, in which LC3 was present but relatively rare and difficult to find by EM. An on-section CLEM method was introduced in a separate study, which uses the sensitivity of fluorescence labeling to visualize relatively rare and low expressed endogenous proteins and correlate this to EM ultrastructure14. Here, these two approaches are combined by the use of the optimized LC3 labeling protocol as part of a CLEM approach.
HEPG2 cells, liver-derived cells with relatively high levels of basal autophagy22, were starved in minimal medium (Earle's balanced salt solution [EBSS]) for 2 h prior to fixation in 4% PFA. This was followed by sample preparation by the Tokuyasu method of ultrathin cryosectioning (sections 1-3; see Slot and Geuze12), which is highly compatible with on-section CLEM14,23. Thawed cryosections were fluorescently labeled (protocol section 4 and Figure 1) using mouse anti-LC3 primary antibody9. Additionally, rabbit anti-LAMP1 was used to indicate endo-lysosomes, followed by anti-mouse AlexaFluor488 and anti-rabbit AlexaFluor568 secondary antibodies. Grids were sandwiched between a coverslip and glass slide and imaged at RT on a widefield microscope (100x 1.47 NA oil objective, sCMOS camera).
An advantage of fluorescence labeling of thin sections over conventional whole-cell IF is the increased resolution in Z, since the physical thickness of the section is 60-90 nm. With this improved Z resolution, the fluorescence labeling of LC3 and LAMP1 on thin sections reveals very little colocalization (Figure 2A). In cells treated with lysosomal inhibitors, such as BafA1, high colocalization occurs, since lysosomal-enclosed LC3 remains undegraded9. In untreated cells, LC3 is rapidly degraded upon contact with enzymatically active, LAMP1-positive lysosomes, and therefore co-localization is rare in these conditions. Generally, less than one LC3 punctum per cell profile was observed. This indicates that even in starved conditions, the turnover of autophagosomes is rapid, keeping autophagosome numbers low. It also highlights the importance of using CLEM to find the rare LC3-labeled structures, using the large field of view provided by light microscopy. Moreover, the higher sensitivity of fluorescence labeling in comparison to gold labeling enables the identification of more LC3-positive organelles than in conventional immuno-EM, further aiding their characterization.
After acquiring a full tileset of the ribbon of sections, the grids were retrieved from the microscope and post-stained for EM using UA and the loop-out method (protocol steps 4.4-4.6;Â Figure 1F,G). This 'loop-out' method ensures that a thin layer of UA:methylcellulose remains on the grid, which creates the desired contrast in the EM. The thickness of the layer depends on the speed and angle with which the UA:methylcellulose is blotted off onto the filter paper. Dragging the loop too quickly can leave too much UA:methylcellulose on the grid and darken the appearance of the sections in EM. Dragging too slowly can draw too much UA:methylcellulose away, resulting in too little staining and poor morphology, and risks the grid falling out of the loop. 'Oil slick' coloring (Figure 1G) on dry grids indicates a suitable UA:methylcellulose layer thickness.
After loop-out and drying, the grids were imaged in a TEM at ROIs selected by fluorescence. The IF and EM datasets were correlated by overlaying the DAPI signal to the outlines of nuclei visible in the EM, generating an integrated image containing information of both modalities.
Finding the same area in EM as selected in IF can be challenging. It is therefore recommended to keep an overview image of the IF tileset at hand while searching in the EM. Users should look for recognizable features in both modalities, such as folds or tears in the sections, grid bars, or arrangement of nuclei. It is also important to keep in mind that the sample can appear rotated and mirrored in EM. 'Finder grids' with specific features to identify areas can be used to ease correlation (see Table of Materials).
Correlation of the LC3-positive organelles to EM ultrastructure revealed that the different puncta represented distinct stages of autophagy (Figure 2B). Although the preservation of autophagosomal ultrastructure is challenging in cryosections, organelles with cytoplasmic content and double membranes were frequently observed (Figure 2C, arrows in organelles 1-5; Supplemental Figure S1), which are defining morphological features of autophagosomes. Interestingly, rather weak fluorescent spots were identified by EM as LC3-positive autolysosomes (Figure 2C, organelle 6; autophagic content is marked *), characterized by dense content and intraluminal vesicles. This showed that very small amounts of LC3 are visible in IF of ultrathin cryosections, and indicated that despite the degradative milieu, some LC3 is detectable in steady-state autolysosomes. However, the majority of LC3-positive puncta represented autophagosomes, whereas autolysosomes were very rare. This is in contrast with BafA1-treated cells, which primarily accumulate autolysosomes and not autophagosomes9.
In summary, this protocol describes an on-section CLEM method for linking molecular information obtained by fluorescence microscopy to the ultrastructure of EM. This method increases the sensitivity of immuno-EM, since only fluorophores are used for labeling and these generally yield more signal than EM probes. The method is especially suited for using ultrathin cryosections, in which high levels of specific fluorescence over negligible background staining can be obtained. By using fluorescence to screen for rare structures or events and correlating selected ROIs to EM, the EM operation time and associated costs can be greatly decreased. The sensitivity and feasibility of the method is demonstrated by the visualization of LC3 in untreated, starved cells, showing that LC3 predominantly associates to autophagosomes in these conditions, with very low levels visible in autolysosomes.
Figure 1: Schematic overview of on-section CLEM. (A) Cryosections from gelatin-embedded cells are collected on a formvar-coated copper grid. (B) Grids are processed section-down on droplets of the appropriate solutions. (C) Grids are labeled with primary and fluorescent secondary antibodies. (D) Grids are sandwiched between a coverslip and glass slide in 50% glycerol. (E) Fluorescence images are collected in a widefield microscope. (F) Grids are retrieved from the glass slide and further processed by uranyl staining for EM. (G) After drying, the grids can be imaged by TEM. (H) High magnification TEM image tileset is acquired from an area selected from fluorescence data. (I) Images from fluorescence microscopy and EM are correlated and overlaid. Please click here to view a larger version of this figure.
Figure 2: CLEM of LC3 and LAMP1 in starved HEPG2 cells. HEPG2 cells were starved for 2 h in EBSS prior to fixation with 4% PFA for 2 h. (A) IF imaging of LC3 (green) and LAMP1 (red) on sections reveals relatively few LC3 puncta and little colocalization with LAMP1. (B) Linking molecular information from IF (left panel) to the ultrastructural information obtained in EM (middle panel) by overlaying the two imaging modalities based on DAPI and nuclear outlines (dashed lines, right panel). The ultrastructure of the individual LC3-labeled compartments, as exemplified by box 1 (right panel), is shown in C. (C) Ultrastructure of LC3-positive compartments. CLEM images are shown on the left and pseudocolored (beige) EM images on the right (uncolored EM images are shown in Supplemental Figure S1). Inner and outer autophagosomal membranes are indicated by white and black arrowheads, respectively. The autophagic content inside the autolysosome in example 6 is indicated by *. Scale bars = 10 µm (A), 1 µm (B), 200 nm (C). Please click here to view a larger version of this figure.
Supplemental Figure S1: Uncolored EM images of LC3-positive organelles. (A-F) Uncolored EM images of pseudocolored examples 1-6 shown in Figure 2C. The organelles were selected by LC3 fluorescence, as described for Figure 2. Inner and outer autophagosomal membranes are indicated by white and black arrowheads, respectively. The autophagic content inside the autolysosome in example 6 is indicated by *. Scale bars = 200 nm. Abbreviations: AL = autolysosome; AP = autophagosome; M = mitochondrion. Please click here to download this File.
Supplemental File 1: Buffers and solutions used in this study. This supplemental file contains the recipes and protocols needed to make the buffers and solutions used in this study. Please click here to download this File.
The method presented here takes advantage of recent advances in cryosection-based on-section CLEM - the high sensitivity of IF labeling and accurate (<100 nm error) correlation between FM and EM14,24. This results in a method with the sensitivity to fluorescently label scarce, endogenous proteins and the capability to overlay this with high precision to the EM ultrastructure. Thus, this method avoids the need for (over)expression of exogenously tagged proteins and the use of less sensitive EM labels. The feasibility of the method is shown by examples of CLEM on endogenous LC3 in starved cells, without the use of lysosomal inhibitors.
Thawed cryosections obtained with the Tokuyasu method are ideal samples for immuno-EM, as unlike resin sections, they are permeable for antibodies. Combined with mild fixation and contrasting procedures, this generally yields superb labeling efficiency over other methods without compromising the detailed ultrastructure, and excellently visualizes cellular membranes12,25,26. Moreover, cryosections are highly compatible with fluorescence microscopy, which make them valuable substrates for CLEM. Both classic immunogold labeling and CLEM on cryosections have provided seminal insights in understanding subcellular organization14,27,28,29,30.
Currently, applications of CLEM on thawed cryosections are becoming more prevalent, as a result of continuous developments and optimizations14,20,24,31,32,33,34 that have improved the quality, applicability, and accuracy of the approach. Now, by accurate correlation of large IF and EM image tilesets, the technique facilitates screening for the ultrastructure of fluorescently-labeled endogenous cellular components14,32,33. This is an advantage over classic immuno-EM, where the search for gold-labeled structures typically requires high magnification and is, therefore, more laborious and time-intensive. It is for this reason that localization of LC3 to the ultrastructure greatly benefits from CLEM. LC3-positive organelles are common when autophagic clearance is blocked (i.e., when cells are treated with BafA1 or pH-raising agents), whereas autophagic organelles are rapidly cleared in unaltered or starved cells, resulting in very low steady-state levels. In such conditions, finding LC3-labeled organelles using classical immuno-EM can be challenging, and CLEM offers a clear advantage.
Previously, CLEM on resin sections was applied in studies using ectopic expression of LC3-GFP or an LC3-GFP-RFP tandem probe35,36,37,38,39. In these studies, fluorescence imaging was performed prior to embedding or directly in acrylic resin sections40, and samples were subsequently screened by EM. There are several advantages of resin embedding; the autophagosomal ultrastructure is generally well-preserved, especially if the material is high-pressure frozen40. Moreover, the contrast of heavy metal-stained resin-embedded material is generally more pronounced than that of uranyl-stained cryosections. Resin-embedded sections are compatible with volumetric EM methods, such as array tomography, FIB-SEM, or serial blockface SEM, while cryosections are not. In approaches that perform imaging before embedding, live-cell imaging is an option41 that is not available in CLEM on cryosections. The key advantage of CLEM on cryosections over these alternatives is the high IF signal, allowing for immuno-localization of rare proteins without the need for membrane permeabilization or overexpression. This avoids potential membrane extraction, overexpression artefacts42 and genetic modification of the subject, which, combined with the possibility to correlate large areas in IF and EM, makes it an excellent tool to study LC3 and autophagy.
Here, the application of on-section CLEM to starved HEPG2 cells revealed that LC3 predominantly localized to structures identified as autophagosomes. Additionally, a few weakly fluorescent spots were found in autolysosomes. This is in direct contrast to cells treated with BafA19 and reflects the rapid degradation of autophagosomal proteins once the autophagosome fuses with lysosomes. Overall, the data demonstrated that CLEM of thawed cryosections can provide insights on LC3-mediated autophagy in native conditions. The data also highlight the sensitivity of the technology, since LC3 was detected even in autolysosomes that contain only low levels of intact LC3 epitopes. Further application of this technique by imaging LC3 in different models and conditions will improve our understanding of autophagy and other LC3-mediated biological processes, such as LC3-associated phagocytosis or conjugation of ATG8 to single membranes.
Beyond autophagy, on-section CLEM can be applied to other rare events or structures, such as cell division, infection, rare cell types in tissues, kinetochores, primary cilia, or cell type-specific organelles. Effective screening for the subject of interest by IF can greatly facilitate the ultrastructural study of these rarities. Furthermore, it was shown14 that the technique can be used to localize proteins in a more sensitive manner than classical immuno-EM. Adjusting the fixation length can further extend this sensitivity, allowing for the ultrastructural localization of very low-abundant or poorly antigenic proteins. Finally, the on-section CLEM method eases rapid selection of a quantitative number of organelles, facilitating a more robust analysis of the ultrastructural distribution of a given protein.
CLEM on cryosections requires the equipment and expertise for cryosectioning. In groups with access to these tools (e.g., cryomicrotomes), the implementation of on-section CLEM is straightforward and only requires the availability of an automated widefield microscope, a setup most labs have access to. Furthermore, the method is available in EM facilities worldwide. Since on-section CLEM combines the application of established IF and EM methods, the method is easily adapted and can be combined with, for example, tomography20,33,43, serial section volume EM of a limited number of sections44, or super-resolution microscopy45. This versatility of the method supports applications to a wide range of biological questions.
We thank our colleagues at the Center for Molecular Medicine of the University Medical Center Utrecht for fruitful discussions and feedback. We thank past and present colleagues of the Klumperman lab for making continuous improvements in our microscopy technologies. The EM infrastructure used for this work is part of the research program National Roadmap for Large-Scale Research Infrastructure (NEMI) financed by the Dutch Research Council (NWO), project number 184.034.014 to JK.
Name | Company | Catalog Number | Comments |
Chemicals and reagents | |||
Antibody donkey anti-mouse Alexa Fluor 488 | Life Technologies | #A21202 | use 1:250 |
Antibody donkey anti-rabbit Alexa Fluor 568 | Life Technologies | A#10042 | use 1:250 |
Antibody mouse anti-LC3 | Cosmo Bio | CTB-LC3-2-IC | use 1:100 |
Antibody rabbit anti-LAMP1 | Cell Signaling | 9091 | use 1:250 |
Bovine serum Albumin, fraction V | Sigma-Aldrich | A-9647 | |
BSA-c | Aurion | 900.099 | |
BSA-conjugated gold | Cell Microscopy Core, UMC Utrecht | BSAG 5 nm | |
Water-free Chloroform | Merck | 1.02447.0500 | |
DAPI | Invitrogen | 10184322 | Use at end concentration of 10 µg/ml |
EGTA | Sigma-Aldrich | E4378 | |
Fish-skin Gelatin | Sigma-Aldrich | G7765 | |
Food-grade gelatin | Merck | G1890 | |
Formvar, Vinylec E | SPI | 02492-RA | |
Gluteraldehyde | Serva | 23115.01 | See CAUTION note |
Glycerol | Boom | MBAK 7044.1000 | |
Glycine | Merck | 1042010250 | |
HEPES | Sigma-Aldrich | H3375 | |
Methylcellulose, 25 centipoises | Sigma-Aldrich | M-6385 | |
MgSO4 | Riedel-de Haen | 12142 | |
Na2HPO4 (PB component A) | Merck | 106580-0500 | |
NaBH4 | Merck | 806373 | |
NaH2PO4 (PB component B) | Merck | 106346 | |
NH4OH | Sigma-Aldrich | 221228-0025 | |
Oxalic acid | Merck | 100495 | |
Paraformaldehyde prills | Sigma-Aldrich | 441244 | See CAUTION note |
PIPES | Merck | 110220 | |
Protein-A conjugated gold | Cell Microscopy Core, UMC Utrecht | PAG 5, 10, 15 or 20 nm | |
Sucrose D(+) | VWR | 27483294 | |
Uranyl acetate | SPI | 020624-AB | See CAUTION note |
Tools and consumables | |||
Pick-up loop | Electron Microscopy Sciences | 70944 | |
Filter paper, qualitative, medium-fast | LLG | 6.242 668 | |
Finder grids | Ted Pella | G100F1 | |
Grids | Cell Microscopy Core, UMC Utrecht | CU 100 mesh | |
Microscopes | |||
Leica Thunder widefield microscope | Leica | Components: 100x, 1.47 NA TIRF objective; Photometrics prime 95B sCMOS camera; LAS X software; | |
Leica UC7 ultracryomicrotome | Leica | ||
Tecnai T12 | FEI | Components: Veleta VEL-FEI-TEC12-TEM camera; SerialEM software | |
Software | |||
ec-CLEM in icy | open source | Paul-Gilloteaux et al., 2017 | |
Fiji | open source | Schindelin et al., 2012 | |
IMOD | open source | Mastronarde et al., 2017 | |
Photoshop | Adobe | ||
SerialEM | open source | Mastronarde et al., 2018 |
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