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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes a mechanism for using correlative light and electron microscopy to visualize the interaction of mitochondria and lysosomes labeled with mEosEM and APEX2, respectively.

Abstract

Cellular organelles, such as mitochondria and lysosomes, display dynamic structures. Despite the higher resolution of transmission electron microscopy for structural analysis, light microscopy is essential for the visualization of dynamic organelles by target-specific labeling. The following protocol describes a method that combines dual-color correlative light and electron microscopy (CLEM) to observe the interactions between mitochondria and lysosomes. In this study, mitochondria were labeled with mEosEM (Mito-mEosEM) and lysosomes with TMEM192-V5-APEX2. The results obtained from CLEM images enable us to observe the changes in the interactions between mitochondria and lysosomes under external stress conditions. Treatment with bafilomycin (BFA), which inhibits lysosomal function, resulted in an increase in contact between mitochondria and lysosomes, leading to the formation of fragmented mitochondria trapped inside lysosomes. Conversely, treatment with U18666A, which inhibits cholesterol export from lysosomes, caused lysosomes to be surrounded by mitochondria, indicating a distinct form of interaction. This study presents an effective method for observing the interactions between mitochondria and lysosomes in fixed cells. Furthermore, CLEM imaging with dual-color probes offers a powerful tool for future investigations of organelle dynamics and their implications for cell function and pathology.

Introduction

Mitochondria and lysosomes are the principal membrane-bound organelles that are essential for the maintenance of cellular homeostasis. Mitochondria are highly dynamic cellular organelles modulated by fission and fusion events1. In general, mitochondria are referred to as the powerhouse of the cell and are known for their important role in oxidative phosphorylation, ATP production, and metabolite storage such as calcium2. In addition to their role in energy metabolism, mitochondria are involved in a number of other cellular signaling processes, including apoptosis3,4, calcium signaling5,6, and lipid metabolism7,8. Similarly, lysosomes are highly dynamic cellular organelles that play multiple roles as cellular recycling centers to maintain cellular cleanliness through protein degradation, autophagy, endocytosis, and phagocytosis9. Apart from their role in degradation, lysosomes also play roles in cellular physiology such as nutrient sensing, homeostasis10, lipid metabolism11, and cellular dedifferentiation12.

Despite the existence of numerous studies on the functions of individual cellular organelles, our recent research has concentrated on the less-understood crosstalk between mitochondria and lysosomes, which has been demonstrated to influence both cellular physiology and pathology. Contacts between mitochondria and lysosomes have been observed in various cell types, including cancer cells, neurons, and induced pluripotent stem cell (iPSC)-derived cells13, and they are mainly observed under cellular stress or in neurodegenerative diseases such as Parkinson's disease13,14 and Charcot-Marie-Tooth disease15. The contact between mitochondria and lysosomes plays an important role in maintaining metabolic homeostasis through the exchange of ions (e.g., Ca2+)16, cholesterol17, and iron18 between the two organelles. Furthermore, the contact between mitochondria and lysosomes plays a crucial role in the quality control of cellular organelles. For example, damaged mitochondria can be targeted for degradation through the process of mitophagy, in which they are engulfed by autophagosomes and delivered to lysosomes for degradation. Consequently, the removal of dysfunctional mitochondria can be facilitated before they cause further damage to the cell or to normal mitochondria. This interaction between mitochondria and lysosomes is crucial for maintaining cellular homeostasis and preventing the accumulation of dysfunctional organelles. Thus, only damaged mitochondria are selectively removed, allowing healthy mitochondria to function optimally within the cell.

Correlative light and electron microscopy (CLEM) is an imaging technique that has recently received attention as a research method by combining the advantages of light microscopy (LM) and electron microscopy (EM) to provide detailed structural information at different scales. In CLEM, a sample is first imaged using LM to identify specific regions of interest, followed by EM sample preparation steps such as fixation, dehydration, and embedding. The same sample is then observed under an electron microscope. Although live-cell CLEM methods have been reported recently19,20,21,22, it remains challenging to accurately combine optical images of highly dynamic biological samples with electron microscopy images of fixed cells into a single plane. For highly dynamic organelles like mitochondria and lysosomes, their precise localization can change as they are observed live under the light microscope, and they continue to move even during the short delay time required for fixation.

Therefore, in this protocol, two different molecular tags that remain active even within fixative solutions were employed to visualize the interaction between mitochondria and lysosomes, which play an important role in maintaining metabolic homeostasis through the exchange of ions (e.g., Ca2+)16, cholesterol17, and iron18 between these organelles. The constructs TMEM192-V5-APEX2 and Mito-mEosEM were used to observe the interactions of mitochondria and lysosomes in cultured cells at both the EM and LM levels. For mitochondria, the mEosEM tag was selected for its ability to preserve the fluorescence signal during EM sample preparation in a fixative solution and OsO423. For lysosomes, the APEX2 tag, a protein of approximately 28 kDa, was used to study the localization of a specific protein at both EM and LM levels24. In the presence of hydrogen peroxide, the APEX2's substrate, Amplex-Red, forms an insoluble polymer that can be observed by confocal microscopy at the tag-targeted location. In this protocol, dual-color CLEM was used to visualize the structure of the interactions between lysosomes and mitochondria in fixed cells.

Protocol

The step-by-step workflow for sample preparation is shown in Figure 1.

1. Cell culture and transfection

  1. Culture HeLa cells at a density of 1 × 105 cells in 35 mm glass grid-bottomed culture dishes containing DMEM with 10% fetal bovine serum and 100 U/mL of penicillin and streptomycin at 37 °C.
  2. The day after seeding the cells, co-transfect the cells at 30%-40% confluency with TMEM192-V5-APEX2 and Mito-mEosEM plasmids using the transfection reagent according to the manufacturer's instructions(1 µg each of TMEM192-V5-APEX2 and Mito-mEosEM plasmid DNA with 3 µL of transfection reagent). After transfection, treat the cells with DMSO or U18666A (2 µg/mL), and Bafilomycin A1 (100 nM) for 18 h.

2. Confocal microscopic imaging

  1. After treating the cells with Bafilomycin and U18666A for 18 h, remove the culture media and immediately add 250 µL of warm (30 °C-37 °C) fixative solution (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate solution [pH 7.0]) by gentle pipetting. Immediately remove the fixative, replace it with 1.5 mL of fresh fixative, and incubate on ice for 30 min. Then, wash the cells for 3 x 10 min in 1 mL of ice-cold 0.1 M sodium cacodylate buffer.
    NOTE: Aldehyde fumes are toxic. All experiments should be conducted in a ventilated fume hood.
  2. Add 1 mL of cold 20 mM glycine solution to quench the aldehyde and incubate on ice for 10 min. Wash 3 x 10 min in cold 0.1 M sodium cacodylate buffer.
  3. Prepare a fresh solution containing 300 µM Amplex-Red by adding 10 mM H2O2 to a 0.1 M cacodylate solution. Add 500 µL of Amplex-Red solution to the sample dish and incubate for 5 min on ice.
  4. Remove the Amplex-Red solution and rinse 3 x 10 min with 0.1 M sodium cacodylate buffer.
  5. After Amplex-Red staining, use a confocal microscope to image the area around the cells of interest in 3 x 3 tile scan mode. Capture both differential interference contrast (DIC) and fluorescence signal to identify and record the grid and letters on the grid dish using a 40x (or 60x) objective.
    NOTE: These overview images serve as a map for identifying specific target cells in the plastic-embedded specimens.
  6. Image the target cells at high magnification using a 40x (or 60x) objective and acquire a Z-stack image.
    NOTE: This high-magnification image is superimposed on an electron microscope image to create a correlative image.

3. Sample preparation for EM block

  1. After LM imaging, add 1 mL of 1% reduced osmium tetroxide (prepared by containing 3% potassium ferrocyanide in 0.3 M cacodylate buffer with an equal volume of 2% aqueous osmium tetroxide) for postfixation at 4 °C for 1 h, and then rinse for 3 x 10 min with distilled water at 4 °C.
    NOTE: Osmium tetroxide fumes are toxic. All experiments should be performed in a ventilated fume hood.
  2. Rinse 3 x 10 min with distilled water at room temperature, then dehydrate in a graded ethanol series for 15 min each time (50%, 60%, 70%, 80%, 90%, 95%, 100%, and 100%) at room temperature.
  3. Mix ethanol with epoxy resin in volume ratios of 3:1, 1:1, and 1:3, preparing a total of 300 µL of each mixture. Pour each epoxy resin into the dish, allowing it to incubate at room temperature for 1 h. Then, use the 100% epoxy resin for overnight impregnation.
  4. Prepare the embedding capsule or PCR tube by filling it with fresh epoxy resin, place the tube or capsule upside down on the region of interest, and place it in an oven at 60 °C for 48 h to polymerize.

4. Sectioning and TEM imaging

  1. After the epoxy resin has completely polymerized, separate the bottom glass from the polymer block by immersing it in liquid nitrogen.
  2. Place the specimen block in the sample holder of the ultramicrotome and set it in the trimming block. Use a razor blade to trim the resin into a trapezoidal or rectangular shape around the object of interest.
  3. Place the trimmed specimen block in the sample holder and adjust the diamond blade. Ensure that the leading edge of the specimen block is perfectly parallel to the edge of the diamond blade.
  4. Using an ultramicrotome, cut ultrathin sections approximately 60-70 nm from the flat-embedded cells. Collect the serial sections on a formvar-coated slot grid and dry them on a hot plate.
  5. Stain the section with the contrast stain for 2 min and lead citrate for 1 min.
  6. Place the grid in the TEM sample holder for observation. Identify the target cell of interest based on the DIC image from the light microscope. Image the entire cell area using tiled, overlapping images at 1,700x magnification.

5. Image stitching using imageJ Fiji.

  1. To begin image stitching, start imageJ Fiji25 and click on File | New | TrakEM2 (blank) | Select storage folder to create a new TrakEM226 project (Figure 2A1).
  2. Open the tile image dataset by dragging and dropping the raw tile image data into the workspace (Figure 2A2).
  3. Click the right mouse button and select Align | Montage all images in this layer | elastic (non-linear block correspondences), and then click OK to accept the default values remaining parameters (Figure 2A3).
  4. Click the right mouse button and select Export | Make flat image to create a stitched image, then click File | Save As to save the image.

6. Resizing the fluorescence image

  1. Open the photo enlargement software (see Table of Materials). Click on File | Open and select the fluorescence image.
  2. Enter the width and height to be the same size as the electron microscope image, and select the algorithm to be applied in the resize method (Figure 2B1).
  3. Click on File | Save AS to save the resized image.

7. Landmark-based image registration with the Big Warp plugin

  1. Open ImageJ Fiji and drag and drop the resized fluorescence micrograph (FM image) and the stitched electron micrograph (EM image).
  2. Click on Plugins | BigData Viewer | Big Wrap to start the registration , then click on the dropdown menu to select the FM image as the moving image and the EM image as the target image (Figure 2C1).
    NOTE: Each FM and EM image will have three new popup windows (BigWrap moving Image, BigWrap fixed image, and Landmarks) to display landmarks.
  3. Compare the FM and EM images using functions such as zoom (mouse wheel), image rotation (moving the mouse while holding down the left button), and move (moving the mouse while holding down the right button), and then press the space bar on the keyboard to switch Landmark mode on, and press the left mouse button to mark at least three landmarks identified in the FM and EM images (Figure 2C2, pink dots and red arrow).
  4. After marking all the identified landmarks, go to File | Export Moving Image | OK and a new popup window will appear with the transformed FM image (Figure 2C3).
    NOTE: The newly created popup window is an FM image transformed based on the basis of the landmark. Review the alignment and make adjustments, if necessary, by adding or modifying landmarks and recalculating the transformation.
  5. Click on File | Save As to save the transformed FM image.

8. Overlay of CLEM images

  1. In ImageJ, drag and drop the transformed FM image and the stitched EM image.
  2. Click on Image | Type | 8-Bit Color to set the same image type for both the LM and EM images (Figure 2D1).
    NOTE: The EM image must be converted twice in the following order: 8-bit -> RGB Color -> 8-bit Color.
  3. Click on Image | Color >| Merge Channels to combine the images into a composite image by selecting the FM and EM images from the dropdown menu, respectively (Figure 2D2).
  4. Click on File | Save As to save the CLEM image.

Results

To visualize the interactions between mitochondria and lysosomes using this dual-color CLEM protocol, we employed Mito-mEosEM and TMEM192-V5-APEX2. Fixed cells were first imaged using a light microscope, followed by EM sampling and imaging. The results of the correlated images are shown in Figure 3A,B and Figure 4A. Following the protocol described above, we checked whether we could observe the changes in the interaction between mitochondria and...

Discussion

This protocol describes dual-color CLEM to observe the interaction of mitochondria and lysosomes in fixed cells using genetically encoded mEosEM and APEX2 tags. Over the past few decades, advances in microscopy have greatly improved the ability to observe changes in organelle networks32,33, and the study of organelle interactions has become increasingly interesting. Super-resolution microscopy has been used to observe changes in highly dynamic contacts between mi...

Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

This research was supported by the KBRI basic research program through Korea Brain Research Institute funded by Ministry of Science and ICT (24-BR-01-03). TMEM192-V5-APEX2 plasmids were kindly provided by Hyun-Woo Rhee (Seoul National University). TEM data were acquired at Brain Research Core Facilities in KBRI.

Materials

NameCompanyCatalog NumberComments
Chemicals and reagents
25% GlutaraldehydeElectron Microscopy Sciences16200Aldehyde fumes are extremely toxic. Use only in fume hood.
30% Hydrogen peroxide solutionMerck107210
4% Paraformaldehyde solutionBiosesangPC2031-100-00ldehyde fumes are extremely toxic. Use only in fume hood.
Amplex RedThermoFisherA12222
Epon 812Electron Microscopy Sciences14120
EthanolMerck100983
Fugene HDPromegaE2311Plasmid transfection
GlycineSIGMAG8898
Lead Citrate 3%Electron Microscopy Sciences22410
Osmium tetroxide 4 % aqueous solutionElectron Microscopy Sciences16320Very toxic. Use only in fume hood.
Potassium hexacyanoferrate(II) trihydrate SIGMAP3289
Sodium cacodylate trihydrateSigma-AldrichC0250-500G
Uranyl acetateElectron Microscopy Sciences22400
UranyLess EM stainElectron Microscopy Sciences22409
Plasmid construction
Mito-mEosEMaddgene132706DOI: 10.1016/j.chembiol.2024.02.007
TMEM192-V5-APEX2Sharma N.  et al.N/ADOI: 10.1016/j.chembiol.2024.02.007
Tools
0.22 um syringe filterSartorius16534
35 mm Gridded coverslip dishMattekP35G-1.5-14-CGRD
BEEM Embedding CapsulesTed Pella130
Formvar Supported Single slot copper gridTed Pella01705
Diamond knifeDiATOMEDU4530
Ultra-microtomeLeicaARTOS 3D
Microscopes
Inverted confocal microscopyNikonA1 Rsi/Ti-E
Transmission electron microscopyFEITecnai G2
Software and Algorithms
Fiji/Image JNIH / open sourcehttps://imagej.nih.gov/ij/
ImageJ BigWarp software packageNIH / open sourcehttps://imagej.net/plugins/bigwarp
Photozoom Pro 8 BenVistahttps://www.benvista.com/photozoompro

Alternative software
Image Resizer : https://imageresizer.com/
Gigapixel AI : https://www.topazlabs.com/gigapixel
ON1 resize : https://www.on1.com/products/resize-ai/

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Dual color Correlative Light And Electron MicroscopyCLEMMitochondriaLysosomesMEosEMTMEM192 V5 APEX2External Stress ConditionsBafilomycinU18666AOrganelle DynamicsCell FunctionPathology

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