Integrated Array Tomography for High Throughput Electron Microscopy

Abstract

Volume electron microscopy (EM) is now increasingly looked to for answering complex biological questions, thanks in large part to revolutionary developments in instrumentation and sample preparation techniques. However, due to the slow nature of electron-probe-based scanning techniques, throughput has long remained the primary obstacle in pushing to larger areas at high resolution. An undesirable trade-off is therefore commonly made between imaging resolution, volume, and time. A recent approach that attempts to circumvent this trade-off involves selective imaging of the specimen in multiple iterations at increasing levels of magnification. Between iterations, the EM dataset is reconstructed and inspected to locate sites for higher magnification imaging in the next iteration [1]. While this approach can outpace indiscriminate scanning of the entire volume, overall throughput is limited by the overhead involved in reconstructing and evaluating the intermediate datasets.

Correlative Organelle Microscopy: Fluorescence Guided Volume Electron Microscopy of Intracellular Processes

“…Integrated CLEM instruments (with the LM built in the EM) greatly facilitate retracing the ROI from FM to EM ( Liv et al, 2013 ; Koning et al, 2019 ) as the coordinate systems of the FM-EM are shared. Recently, multiple integrated (confocal) FM and volume EM systems were reported ( Brama et al, 2016 ; Ando et al, 2018 ; Delpiano et al, 2018 ; Gorelick et al, 2019 ; Lane et al, 2019 ). We add to this a home-built system integrating a Confocal Laser Scanning Microscope (CLSM) into a Focused Ion Beam Scanning Electron Microscope (FIB.SEM), similar in geometry to a previously reported ( Timmermans et al, 2016 ) integrated system, as outlined in Supplementary Figure S1 .…”

Correlative Organelle Microscopy: fluorescence guided volume electron microscopy of intracellular processes

“…Integrated CLEM instruments (with the LM built in the EM) greatly facilitate retracing of the ROI from FM to EM (Koning et al, 2019; Liv et al, 2013), since the coordinate systems of the FM-EM are shared. Recently, multiple integrated (confocal) FM and volume-EM systems were reported (Ando et al, 2018; Brama et al, 2016; Delpiano et al, 2018; Gorelick et al, 2019; Lane et al, 2019). We add to this a home-built system integrating a Confocal Laser Scanning Microscope (CLSM) into a Focused Ion Beam / Scanning Electron Microscope (FIB.SEM), similar in geometry to a previously reported (Timmermans et al, 2016) integrated system and as outlined in Supplementary Figure 1.…”