Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

Journal of Microscopy
Katharina HöhnC Dietrich

Abstract

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells.

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Citations

Apr 28, 2016·FEBS Letters·Janina HanneHans-Georg Kräusslich
Mar 9, 2017·The Biochemical Journal·Petr Chlanda, Jacomine Krijnse Locker
Jan 3, 2019·Trends in Microbiology·Allon Weiner, Jost Enninga
Jun 24, 2021·The Journal of Cell Biology·Paolo RonchiYannick Schwab

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Methods Mentioned

BETA
electron microscopy
imaging
scanning electron microscopy
correlative
confocal microscopy
transfection
correlative microscopy
fluorescence microscopy
light microscopy
transgenic

Software Mentioned

ImageJ
Amira
LSM

Related Concepts