Correlative Microscopy Workflows to Link Functional Spine Imaging and Volume Electron Microscopy

13 December 2021 · 55 min watch
  • Correlative Microscopy
  • Life Sciences
  • Confocal Laser Scanning Microscopy
  • Scanning Electron Microscopy
Author Dr. Naomi Kamasawa Head of the Imaging Center Company:
Max Planck Florida Institute for Neuroscience (USA)

Correlative Microscopy Workflows to Link Functional Spine Imaging and Volume Electron Microscopy

Correlative light and electron microscopy (CLEM) is a powerful method to investigate the structure-function relationship in biology. We have established two different CLEM workflows to visualize the ultrastructural basis of neuronal functions with volume EM. First, we captured functional properties of the dendritic spines of neurons with live 2-photon calcium imaging, and targeting the same cell, dendrites, and spines in serial block-face scanning EM (SEM) to analyze the structural characteristics of synapses and their surroundings. Second, we induced morphological changes in spines using glutamate uncaging, applied an immuno-EM protocol after fixing the tissues, and performed serial-section array tomography SEM to identify the exact same spine among other unstimulated spines.

Key Learnings:

  • Several practical tips for finding “a needle in a haystack,” including the optimization of sample preparation protocols, selection of detectors for SEM imaging, and application of the focal charge compensation device.
  • Ideas will be provided on how to choose the best workflow depending on your research goals.
  • An advanced cryo-CLEM workflow for capturing biological phenomena that require a better temporal resolution is introduced using the ZEISS cryo-Airyscan technique for imaging cryo-fixed cultured brain slices followed by array tomography SEM.

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