Precise targeting for 3D cryo-correlative light and electron microscopy volume imaging of tissues using a FinderTOP

Abstract

Cryo-correlative light and electron microscopy (cryoCLEM) is a powerful strategy to high resolution imaging in the unperturbed hydrated state. In this approach fluorescence microscopy aids localizing the area of interest, and cryogenic focused ion beam/scanning electron microscopy (cryoFIB/SEM) allows preparation of thin cryo-lamellae for cryoET. However, the current method cannot be accurately applied on bulky (3D) samples such as tissues and organoids. 3D cryo-correlative imaging of large volumes is needed to close the resolution gap between cryo-light microscopy and cryoET, placing sub-nanometer observations in a larger biological context. Currently technological hurdles render 3D cryoCLEM an unexplored approach. Here we demonstrate a cryoCLEM workflow for tissues, correlating cryo-Airyscan confocal microscopy with 3D cryoFIB/SEM volume imaging. Accurate correlation is achieved by imprinting a FinderTOP pattern in the sample surface during high pressure freezing, and allows precise targeting for cryoFIB/SEM volume imaging.

Fig. 1. FinderTOP for aligning cryo-light and electron imaging modalities.

a Schematic representation of (i) a HPF carrier representing the featureless surface of tissue sample after vitrification using a conventional flat top carrier compared to (ii) vitrification using the FinderTOP, showing the imprinted pattern on the ice surface. b Reflection image of the vitrified sample using cryo-light showing the FinderTOP pattern. c Overlay of the reflection image in the confocal fluorescence microscope (green) with the SEM image in the FIB/SEM (gray). Letters and numbers can be seen in both modalities, like the highlighted “8”, “9”, “F” and “G”.

Fig. 2. Cryo fluorescence microscopy of the scale.

a Overview images of the scale taken with the 5× objective to generate a complete overview of the carrier. a-i reflection mode shows the FinderTOP imprint. White box indicates the medium resolution area in b. a-ii Composite fluorescence image taken with the 5× objective, showing the scale for the different probes in the three channels. a-iii–v Images of the three different fluorescence channels. b Medium resolution imaging around the region of interest (ROI) using the 10× objective. b-i Reflection image; the white box denotes the ROI in c. b-ii Composite image showing the three fluorescent probes and the FinderTop imprint. b-iii–v Images of the individual fluorescence channels. c-i–iv High resolution images of the ROI taken with the 100× objective. Z- projection images (11 slices) of the reflection mode and each fluorescent channel. d Orthogonal views of the ROI indicated in c. The image shows the active osteoblast layer with the mitochondrial network. The ortho slices show the cross-sectional planes (yz and xz), where also the ice surface (reflection channel - gray) and the mitochondria in the posterior epithelial cell layer are observed. The ortho slices images are used to calculate the absolute depth of the ROI.

Fig. 3. Localization of the region of interest (ROI) in cryoFIB/SEM.

a (i) Low magnification CACM image of the sample showing the FinderTOP imprint using reflection microscopy. The scale is highlighted as white outline. (ii) Overlay of medium resolution reflection microscopy with the ROI (orange box). This shows the ROI location in square “7H”. b (i) Low magnification SEM image of the sample, showing the FinderTOP imprint. The scale is highlighted as white outline (ii) Alignment of the SEM image with the reflection image and the ROI in high resolution CACM (orange box) using the FinderTOP pattern. c FIB image overlayed with the ROI, taken in the coincident point and after tilting the sample to 54° (FIB beam perpendicular to the surface). d Overlay high resolution CACM image with the high magnification FIB image after generating the trench. The start position of the volume stack is highlighted (red line). e Overlay of high magnification FIB image with high resolution CACM image of the ROI. The end position of the volume stack is highlighted (yellow line). f Overlay of the resliced volume stack (white box) with the high magnification FIB image and CACM image of the ROI.

Fig. 4. CryoFIB/SEM volume imaging.

a Single secondary electron (SE) image (inLens detector) showing the layered structure of the zebrafish scale. The cellular elasmoblast layer forms the top layer of the tissue and borders with the vitrified dextran solution. Below the cellular layer, there is a distinct collagen layer before reaching the bone-like mineral layer. b-i–iv Ultrastructural preservation in the elasmoblast showed by the selection of organelles (i; multivesicular body (MVB), ii; lysosome, iii; mitochondrion, iv; endoplasmic reticulum (ER)).

Fig. 5. Correlation of cryoFIB/SEM and CACM images.

a Overlay images of single x-y slices from CACM and the resliced cryoFIB/SEM volume. The white dashed box shows the region in figures b–f. b Zoom-in of the fluorescent image showing five distinct regions labeled for mitochondria. Z-values indicate the total thickness of the single slice. c–f Image pairs at different depths of different resliced FIB/SEM images corresponding with the CACM image in b with and without the fluorescence overlay.