A workflow for streamlined acquisition and correlation of serial regions of interest in array tomography

Abstract

Background Array tomography (AT) is a high-resolution imaging method to resolve fine details at the organelle level and has the advantage that it can provide 3D volumes to show the tissue context. AT can be carried out in a correlative way, combing light and electron microscopy (LM, EM) techniques. However, the correlation between modalities can be a challenge and delineating specific regions of interest in consecutive sections can be time-consuming. Integrated light and electron microscopes (iLEMs) offer the possibility to provide well-correlated images and may pose an ideal solution for correlative AT. Here, we report a workflow to automate navigation between regions of interest. Results We use a targeted approach that allows imaging specific tissue features, like organelles, cell processes, and nuclei at different scales to enable fast, directly correlated in situ AT using an integrated light and electron microscope (iLEM-AT). Our workflow is based on the detection of section boundaries on an initial transmitted light acquisition that serves as a reference space to compensate for changes in shape between sections, and we apply a stepwise refinement of localizations as the magnification increases from LM to EM. With minimal user interaction, this enables autonomous and speedy acquisition of regions containing cells and cellular organelles of interest correlated across different magnifications for LM and EM modalities, providing a more efficient way to obtain 3D images. We provide a proof of concept of our approach and the developed software tools using both Golgi neuronal impregnation staining and fluorescently labeled protein condensates in cells. Conclusions Our method facilitates tracing and reconstructing cellular structures over multiple sections, is targeted at high resolution ILEMs, and can be integrated into existing devices, both commercial and custom-built systems.

Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

“…In a first round, this will be achieved by combining preserved fluorescence images with corresponding lower contrast unstained EM-images. Then, after the image contrast has been improved by post-staining, the navigation parameters determined and stored by our navigation software ( Gabarre et al, 2021 ) during the first round will be re-used to automatically re-acquire the same but contrast-enhanced set of correlated EM-images. Thus, this two-step ILEM-SEM procedure allows defining cellular identity using antibody labeling and FM, followed by high resolution, fine ultrastructural imaging of subcellular contents and relationships with surrounding cell types.…”

Integrated Array Tomography for 3D Correlative Light and Electron Microscopy

“…The workflow demonstrated here extends the work of Liv et al ( Liv et al, 2013 ), which introduced the integrated microscope, and Haring et al ( Haring et al, 2017 ), which presented the fiducial-free CL registration procedure, to targeted correlative imaging of serial sections. Gabarre et al ( Gabarre et al, 2021 ) presented an alternative method for integrated array tomography in which light microscopy and EM are combined to localize structures through a series of feedback loops. Our approach differs in several ways.…”

“…By detecting fluorescence expression in-situ , it can further be decided in an automated fashion which areas to scan at high magnification and which areas to omit for the sake of higher throughput ( Delpiano et al, 2018 ). For array tomography applications, ROIs can be targeted with increasing magnification through a sequence of feedback loops ( Gabarre et al, 2021 ). Similarly, strategies for rapidly screening sections have been developed for sequential CLEM to limit volume acquisitions to select ROI ( Burel et al, 2018 ; Ronchi et al, 2021 ).…”

Field-Emission Scanning Electron Microscope as a Tool for Large-Area and Large-Volume Ultrastructural Studies

“…The term array tomography, depending on application, comprises three different techniques: (i) fluorescence microscopy array tomography, which delivers volumetric, high-resolution data on the distribution of molecules and enables the detection of several antigens in the same section [ 59 , 64 ], (ii) electron microscopy array tomography, which enables the capturing of ultrathin sections for 3D ultrastructural studies [ 65 ], and (iii) correlative array tomography [ 66 , 67 , 68 ], which combines fluorescence imaging and electron microscopy imaging to obtain voxel-level associations between structure and chemistry. The new concept is the use of array tomography to locate targets for z -axis high-resolution imaging through FIB-SEM [ 69 ].…”

“…However, note that the best results are obtained after the intensive infiltration of samples with heavy metals because of the lack of charging and section contrasting artifacts. Array tomography enables correlative light and electron microscopy studies, and their combination with 3D analyses [ 66 , 68 , 74 ]. The disadvantages of this method are the time-consuming preparation of sections for imaging and the alignment of sections into z-stacks.…”