EM Embraces 3D Volume (vEM)
University of Rochester - Center for Advanced Research Technologies
CART provides the equipment, personnel and analysis to move your research forward.
By Chad Galloway, PhD, CryoEM Facility Manager at the Center for Advanced Research Technologies (CART), a division of the University of Rochester Medical Center
Returning from the?Microscopy & Microanalysis meeting in Minneapolis this past July, one theme was abundantly clear in the field of electron microscopy,?that, 3 dimensional volume EM (vEM) which utilizes serial sectioning collection, is fast becoming a routine imaging modality, challenging 2D imaging where only single thin section views are used documenting structural changes to cells and organelles.?Increasingly, these studies utilize multiple modalities, the most poignant being correlative light and electron microscopy (CLEM).?These volume techniques are not novel or even newly developed, as CLEM was developed in the early 1990s.?Serial sectioning EM dates back to the early 1950’s, soon after scientists first developed methods for embedding of biologic specimens for transmission electron microscopy.?However, it was painstakingly slow to perform, as electron micrographs on film negatives?necessitated darkroom printing on photopaper prior to performing the laborious tracing of organelles for 3-dimensional representations.??
What is driving this change to vEM??Advances?in technology, instrumentation,?reagents, and methodologies have been continuously evolving while being complimented by the coordination of scientists in the vEM?community1, democratizing this technique.?Much of this is taking place in?the?software involved in the segmentation and reconstruction of data in an automated fashion.?The vEM community defines it as newly-developed imaging using transmission or scanning electron microscopy to allow 3-dimensional investigation of cells and tissue ultrastructure up to millimeters in volume with nanometer resolution.??The technique encompasses various methodologies distinguished by the nature of sectioning; Focused Ion Beam scanning electron microscopy (FIB-SEM) where the block face is shaved by a gallium or plasma beam, Serial Block Face SEM (SBF-SEM) where an ultramicrotome sections the block housed inside the SEM itself and array tomography where the individual sections are cut on an ultramicrotome and are collected serially on slides, tape and/or silicon wafers2?.?In the?EMR we have completed and published a study using the latter of these methodologies, interrogating the invasion of canaliculi in a?S. aureus?bone infection model utilizing tape collected sections on the ATUMtome and image collection in back scatter mode in an SEM3.?Improvements in detection in backscatter mode and optimization of tissue preparation,?to improve contrast, allow?for acquisition of images almost indistinguishable, ultrastructurally, from routine transmission electron microscopy.?Array tomography has the added advantage that it can be re-interrogated for multiple regions of interest (ROI).?The benefit of re-interrogation is accentuated when doing CLEM. Recent developments of fixation resistant fluorescent proteins and protected probes, normally quenched by crosslinking aldehydes and osmium tetroxide, allow for fluorescent imaging post embedment, streamlining the targeting of cells/structures of interest4.?A routine request of customers at the EMR?is to target only those rare?cells that were transfected?in a mixed-cell population of a tissue, these?vEM advancements will increasingly make the?task of finding that “needle in a haystack” easier.?As a methodology alone, vEM is becoming an essential tool in neuroscience studies whereas the routine 2D view of a 70 nm thin section, generated by ultramicrotomy, has become inadequate to describe synaptic structure and connectivity.?At an intracellular level, vEM is now the preferred tool of?mitochondrial researchers, where the restriction of 2-dimensional analysis?can result in improper interpretation of mitochondrial shape and size descriptors.?In addition to volumetric observations of mitochondria, changes in the Golgi, in a protein processing defect for example, or the ER, in the unfolded protein response, are better observed and described in 3-dimensions.?These reconstructions also better describe inter-organelle contacts, critical sites for cross-talk in response to stimuli, often underappreciated in standard 2D?electron microscopy.???
The prospect of this electron microscopy renaissance?towards 3 dimensional visualization?is exciting to us in the EMR. The journal?Nature?agrees, naming vEM one of top 7 technologies to watch in 20235.?We are familiar with the techniques and technologies and plan?to move toward acquisition of?the necessary instrumentation. If you have a project that would benefit from CLEM and/or vEM, we invite you to reach out for further discussion.???