INTRODUCTION
OVERVIEW OF THIS PROTOCOL
1 Overview of the protocol. A–E Sample preparation. Fluorescence-labelled cells (A) are centrifuged, resuspended in dextran solution (B) and vitrified with a high-pressure freezer (C). D 0.2 μm dark red fluorescent microspheres (red spheres in the inset), 50 nm gold nanoparticles (larger golden dots in the inset) and 10 nm gold nanoparticles (smaller golden dots in the inset) are applied over the EM grid as fiducial markers for image registration and tomographic tilt series alignment. E The vitrified sample is sectioned with a cryo-ultramicrotome, and the cryosections are collected with the EM grid prepared in Panel D. F, G Cryogenic SMLM. F Channel B (red channel) of the cryogenic fluorescence microscope is calibrated by image transformation to match Channel A (green channel) with multicolour fluorescent microspheres. G When the sample is illuminated by laser beams, photon emission events of single molecules are captured as a video (upper), from which a superresolution fluorescence micrograph can be reconstructed (green, lower left and lower right). Dark red fluorescent microspheres are also captured in diffraction-limited wide-field mode (red, lower right). H Cryo-ET. Tomographic tilt series are acquired at fluorescence-positive locations. Electron tomograms are generated from the tilt series. I Image registration. A fluorescence micrograph (dark red fluorescent microspheres and green fluorescence signals on a dark background), a low-magnification electron micrograph (darker grey) and a projection of a tomogram (brighter grey) are aligned to each other. See Fig. 2 for details about the image registration. Scale bars: 2 μm for Panels F and G |
2 Workflow for image registration. A, B Establishment of T1 transformation that aligns the fluorescence micrograph to the low-magnification electron micrograph. Fluorescent microspheres are localised on the fluorescence micrograph (A, white points) and the low-magnification electron micrograph (B, red points, mostly covered by green points). A point pair corresponding to the same microsphere is labelled with the same number. Based on the coordinates of these point pairs, an image transformation T1 is established. Green points on Panel B mark the positions of the white points on Panel A after the T1 transformation. C, D Establishment of T2 transformation that aligns the low-magnification electron micrograph to the tomogram. The boxed area of Panel B is enlarged to be shown as Panel C. The 50 nm gold nanoparticles are localised on the low-magnification electron micrograph (C, red points) and the minimum intensity projection of the tomogram (D, red points, mostly covered by green points). A point pair corresponding to the same gold nanoparticle is labelled with the same number. An image transformation T2 is similarly established. Green points on Panel D mark the positions of the red points on Panel C after the T2 transformation. E–F Alignment of the fluorescence micrograph to the low-magnification electron micrograph. T1 is applied to the superresolution fluorescence micrograph (E, to better demonstrate the process, the diffraction-limited red channel is included) to obtain a transformed image that aligns with the low-magnification electron micrograph (F, to better demonstrate the process, the low-magnification electron micrograph is also shown as a reference). F–G Alignment of the fluorescence micrograph to a tomogram slice. T2 is applied to the transformed image (F), and the twice-transformed superresolution fluorescence image is superimposed on a tomogram slice to obtain Panel G. The tomogram corresponds to the boxed area in Panel F. Scale bars: 5 µm for Panels A, B and E; 500 nm for Panels C and D; 2 µm for Panel F; 200 nm for Panel G |
3 Fluorescence-annotated tomographic visualisation of an ER–mitochondrial membrane contact site. A When a diffraction-limited wide-field fluorescence micrograph is aligned to and superimposed on a tomogram slice, the blurred fluorescence pattern hardly presents any information. B When a superresolution fluorescence micrograph obtained with SMLM is aligned to and superimposed on the tomogram slice, the fluorescence marks an ER–mitochondrial membrane contact site. C In the position where fluorescence exists, the electron density (pink arrowheads) can be observed spanning the cleft of the membranes of the two organelles. D 3D segmentation of the ER membrane (cyan), the mitochondrial membranes (orange) and the density (yellow) between the ER membrane and the outer mitochondrial membrane. The insets in Panels C and D correspond to the same region. Watch Video S1 for an animated 3D presentation of Panels C and D. Scale bars: 100 nm |
Sample preparation
Cryogenic SMLM
Cryo-ET
Image registration
MATERIALS, INSTRUMENTATION AND SOFTWARE
Cell line
Reagents
Equipment
Software
PROCEDURE
Stage 1: Sample preparation [ TIMING ~ 1 week]
Step 1.1: Application of fiducial markers over the EM finder grids [ TIMING 1–2 h]
Step 1.2: Culture of fluorescence-labelled cells [ TIMING 3–5 d]
Step 1.3: High-pressure freezing [ TIMING 2-4 h]
Step 1.4: Cryo-ultramicrotomy [ TIMING 1–2 d]
Stage 2: Cryogenic SMLM [ TIMING ~ 1 week: ~ 4 d for Steps 2.1–2.4, ~ 3 d for Steps 2.5 and 2.6]
Step 2.1: Initialisation of the cryogenic fluorescence imaging system
Step 2.2: Calibration of fluorescence channels
Step 2.3: Wide-field cryogenic fluorescence imaging
Step 2.4: Acquisition of SMLM data
Step 2.5: File preparation for reconstruction of superresolution fluorescence micrographs
Step 2.6: Reconstruction of superresolution fluorescence micrographs
Stage 3: Cryo-ET [ TIMING variable: 24 h for Steps 3.1 and 3.2, variable time for Step 3.3]
Step 3.1: Acquisition of low-magnification EM maps
Step 3.2: Acquisition of tomographic tilt series
Step 3.3: Reconstruction and visualisation of electron tomograms
Stage 4: Image registration [ TIMING 2–4 d]
Step 4.1: Registration between FM images and low-magnification electron micrographs [ TIMING 1–2 d]
Step 4.2: Registration between low-magnification and high-magnification electron micrographs [ TIMING 1–2 d]
Step 4.3: Generation of superresolution fluorescence-annotated tomographic slices [ TIMING ~ 2 h]
1 Troubleshooting |
Step | Problem | Possible cause | Solution |
1.1 | Fibrous contaminants on grid films | If the edge of the filter paper is not smooth and clean, it may spread fibrous contaminants to the suspension when touching it | Take a new piece of filter paper, gently fold it and use the folding edge to blot the grid |
2.1 | The temperature of the sample stage cannot be stabilised at 93 K (–180 °C) | The liquid nitrogen inflow is not stable or not sufficiently strong and the temperature of the cryo chamber is consequently not stabilised | Adjust the pressure of the liquid nitrogen pump to a moderate level |
2.4 | The sample falls out of focus during video acquisition | The temperature of the sample stage is not stabilised so a noticeable mechanical drift occurs | Stabilise the temperature (see above) |
Unspecified environmental disturbances destabilise the stage | Terminate the acquisition, refocus by adjusting stage Z and restart the acquisition | ||
2.6 | Error report | The entire folder of the program package is not added to the path | Add the entire folder, including all of the subfolders, to the path before running the main2.m script |
CUDA is not properly installed | Reinstall the CUDA Toolkit |
ANTICIPATED RESULTS
Abbreviations
CLEM | Correlative light and electron microscopy |
EM | Electron microscopy |
FM | Fluorescence microscopy |
SMLM | Single-molecule localisation microscopy |
ET | Electron tomography |
csCLEM | Cryogenic superresolution correlative light and electron microscopy |
ER | Endoplasmic reticulum |
ROI | Region of interest |