Correlative two-photon and serial block face scanning electron microscopy in neuronal tissue using 3D near-infrared branding maps

Robert M Lees, Christopher Peddie, Lucy Collinson, Paul Verkade, Michael Ashby

Research output: Chapter in Book/Report/Conference proceedingChapter in a book

15 Citations (Scopus)


Linking cellular structure and function has always been a key goal of microscopy, but obtaining high resolution spatial and temporal information from the same specimen is a fundamental challenge. Two-photon (2P) microscopy allows imaging deep inside intact tissue, bringing great insight into the structural and functional dynamics of cells in their physiological environment. At the nanoscale, the complex ultrastructure of a cell's environment in tissue can be reconstructed in three dimensions (3D) using serial block face scanning electron microscopy (SBF-SEM). This provides a snapshot of high resolution structural information pertaining to the shape, organization, and localization of multiple subcellular structures at the same time. The pairing of these two imaging modalities in the same specimen provides key information to relate cellular dynamics to the ultrastructural environment. Until recently, approaches to relocate a region of interest (ROI) in tissue from 2P microscopy for SBF-SEM have been inefficient or unreliable. However, near-infrared branding (NIRB) overcomes this by using the laser from a multiphoton microscope to create fiducial markers for accurate correlation of 2P and electron microscopy (EM) imaging volumes. The process is quick and can be user defined for each sample. Here, to increase the efficiency of ROI relocation, multiple NIRB marks are used in 3D to target ultramicrotomy. A workflow is described and discussed to obtain a data set for 3D correlated light and electron microscopy, using three different preparations of brain tissue as examples.
Original languageEnglish
Title of host publicationMethods in Cell Biology
Subtitle of host publicationCorrelative Light and Electron Microscopy III
EditorsThomas Müller-Reichert, Paul Verkade
Place of PublicationAmsterdam
Number of pages32
ISBN (Print)978-0-12-809975-9
Publication statusPublished - 1 Jun 2017


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