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Cell Biology JoVE (Open Access) Citable · DOI

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

DOI: 10.3791/57059-v
What you'll learn
  • ✓ Prepare resin-embedded tissue samples using an ultramicrotome for serial sectioning
  • ✓ Use hierarchical light and electron microscopy to locate and image specific cellular targets
  • ✓ Register and align SEM image stacks for three-dimensional nanoscale reconstruction
Protocol

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

Difficulty
advanced
Total time
~3-5 days per sample (sectioning, light microscopy screening, hierarchical SEM imaging, image registration)
Model organism
Plant root tissue (Arabidopsis thaliana or equivalent)

Steps

1
Prepare ultramicrotome and resin-embedded sample

Mount resin-embedded biological material in the ultramicrotome chamber and calibrate sectioning parameters. Ensure proper block orientation and blade alignment for consistent serial section production.

▶ 01:16
2
Generate serial sections with ultramicrotome

Cut consecutive ultrathin serial sections from the resin block and collect them on slides. Monitor section thickness and quality to maintain uniformity across the series.

▶ 03:04
3
Screen and identify targets using light microscopy

Image the collected serial sections with light microscopy to rapidly survey large tissue volumes and locate the specific cellular target of interest before detailed electron microscopy imaging.

▶ 04:21
4
Acquire hierarchical SEM images of target region

Use scanning electron microscopy to image the identified target area in a hierarchical manner, progressively increasing magnification to achieve nanoscale resolution of specific cellular structures.

▶ 07:33
5
Register and align SEM image stack

Process and register the collected SEM images into a spatially aligned three-dimensional image stack to enable accurate reconstruction and analysis of the target structure.

▶ 08:59
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