L2 - Advanced Electron Microscopy

Question 1

Why and how are biological samples stained in EM?

Answer 1

Samples are stained to provide contrast by using e- dense material like heavy metals to scatter electrons. Osmium tetroxide (heavy metal) stains bind lipid bilayer and proteins. Uranyl acetate (heavy metal) stains bind nucleic acids in EM.

Question 2

What is the main imaging difference between TEM and SEM?

Answer 2

TEM illuminates the whole sampling image at once to give a 2D projection.
SEM builds the image by scanning with an e- beam, line by line, to build up an image of the 3D surface.

Question 3

What is vEM and its main advantages over TEM? Give example vEM techniques.

Answer 3

While TEM only gives a 2D image with non depth information, vEM is a collection of techniques designed to reveal the 3D ultrastructure of a biological sample
vEM techniques - Electron tomography (ET), single section TEM (ssTEM), SBF-SEM, Array tomography, etc.

Question 4

What is electron tomography (ET)?

Answer 4

Thin EM sample section is incrementally tilted to give a set of 2D EM images which are computationally reconstructed into a 3D model of the sample.

Question 5

What is single section TEM (ssTEM)?

Answer 5

A entire sample is sectioned into thin slices, then each slice is imaged in TEM. The collection of 2D images is reconstructed into 3D.

Question 6

What is Serial Block Face SEM (SBF-SEM)?

Answer 6

An SEM technique with an inbuilt microtome (diamond knife) that slices a thin layer off the sample after imaging, exposing the next face for imaging. SBF-SEM allows a full 3D reconstruction of the ultrastructure of a biological sample.

Question 7

What is array tomography?

Answer 7

Sample is sectioned into many tiny slices, mounted in an ordered array, then imaged systematically (via LM, SEM, etc.) for high throughput.

Question 8

Compare TEM-based and SEM-based vEM techniques (resolution and volume)?

Answer 8

TEM-based vEM give higher resolution, but SEM-based vEM can deal with much greater volumes of sample more practically.

Question 9

What is CLEM and its uses? Give the CLEM workflow.

Answer 9

Correlative Light Electron Microscopy (CLEM) combines the strengths of LM (for live imaging and fluorescent tracking) with the high res of EM.

1. Use LM (often fluorescent) to locate a point-of-interest.
2. Fix/prep sample for EM.
3. Image using EM.
4. Align/overlay the LM and EM images to correlate functional/molecular data with ultrastructural info respectively.

Question 10

What is Cryo-CLEM / cryo-CLEM tomography?

Answer 10

Cryo-CLEM is correlative Light Electron Microscopy, performed under cryogenic conditions. Feature-of-interest is identified using (fluorescence) LM, then thinned using a focused ion beam to creat a thin section, then imaged using Cryo-ET. This gives a high res 3D structure of the sample.

Question 11

What is vCLEM, and how can specific feature be detected in the large 3D sample?

Answer 11

Volume CLEM is a extension of CLEM, combining LM and vEM to give correlated high res 3D structural info and molecular/locational info.

It can be very hard to locate a specific feature (identified through LM) in a large 3D EM volume. A high power laser can be used to make auto fluorescent marks encircling the feature, which can be seen in both fluorescent LM and vEM.

Question 12

what was observed in the SPARK case study, using CLEM and Cryo-CLEM?

Answer 12

SPARK protein has domains that induce LLPS, tag the protein to the membrane, and nucleate actin polymerization. The goal of the SPARK investigation is produce LLPS to drive actin polymerization and steer cell growth.

CLEM was used to observe the effects of SPARK. Fluorescence LM showed SPARK associating with (polymerizing) actin, in presumed LLPS organelles. Conversely, EM (and then Cryo-ET at higher res) showed membrane stacking and very little visible actin. Research is ongoing to resolve this issue.