Harald Hess: Larger Volume Imaging with Electrons and Cryo-Super-Resolution Microscopy
Friday, November 8, 2024 11am to 12pm
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29 Oxford Street, Cambridge, MA 02138
Volume or 3D electron microscopy EM continues to expand its potential for imaging ever larger biological entities. Images from serial diamond knife cut sections launched the field of volume EM. An alternative of imaging the sequentially cut block face offered easier use and registration. FIB-SEM or Focused Ion Beam Scanning Electron Microscopy liberated the z resolution from the limits of minimal diamond section thickness and achieves a step edge resolution of 5-10 nm in the xy and most importantly the z directions. While this resolution is not up to the standards of Transmission Electron Microscopes, such resolution is of unique value when it encompasses whole cells and complete tissues. We will review the capabilities of FIB-SEM, which has imaged a complete fly brain and can routinely image 0.1 mm^3 sized volumes. Bigger volumes can be accessed by using thick (~20 microns thick) with a hot diamond knife followed by the finer FIBSEM imaging and milling. A cryogenic sample preparation protocol enables cryo-super-resolution microscopy to be paired with the FIB-SEM microscopy for correlative insight into biology. Examples are presented and prospects of future challenges are discussed. Finally, a MultiBeam imaging system which we call IBEAM-MSEM, Ion Beam Etching And Milling Multi Scanning Electron Microscope can image much larger volumes approaching 1.0 mm^3. It uses a hybrid cutting approach too, with samples cut into 100-1000 nm thickness and milled with an oblique ion or ion cluster beam followed by etching and wider area imaging with a 91 beam multiSEM. This opens a microscopy regime where entirely new questions can be asked.
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