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MIRAVA POLYSCOPE – All in one and on for all: the perfect image
Science beyond Barriers

abberior instruments

Cell Biology

2023
bioRxiv

Analysis of RyR2 distribution in HEK293 cells and mouse cardiac myocytes using 3D MINFLUX microscopy

Authors:

Alexander H Clowsley, Anna Meletiou, Evelina Lučinskaitė, Isabelle Jansen, Peter P. Jones, William E. Louch, Christian Soeller

Keywords:

MINFLUX, RyR2, calcium, calcium release, Ca2+, cardiac myocytes

Abstract:

The cardiac type 2 ryanodine receptor (RyR2) is a large homotetramer of a ∼560 kD subunit and is the molecular pathway through which the majority of Ca2+ enters the cytosol during cardiac activation. It constitutes the molecular basis of the process of calcium induced calcium release where activation of RyR2s can be locally regenerative giving rise to local release events termed Ca2+ sparks. Accordingly, the molecular distribution of RyR2 in cardiac myocytes has been of great interest. Here we present the first purely optical data of RyR2 distribution with sub-molecular resolution by applying 3D MINFLUX fluorescence super-resolution microscopy. We demonstrate that by using single-domain antibodies (sdABs) against fluorescent protein domains in engineered RyR2 fluorescent protein fusions we can determine the location of individual RyR2 subunits with high precision (∼3 nm) in all directions. This allows determining not only the location but also the 3D orientation of individual RyR2 channels in intact cells. In practice, this capability is currently limited by a relatively modest effective labeling efficiency (∼10 % subunit detection efficiency translating into ∼35% RyR2 labeling efficiency) which we measure in-situ using a novel procedure enabled by the true molecular resolution of MINFLUX microscopy. The new data suggests a resolution to apparent discrepancies between previous data from electron microscopy and super-resolution data that may be at least partially explained by effects of labeling efficiency. The methodology developed here will be critical to reveal the full complexity of RyR2 and related Ca2+ handling proteins in 3D as well as their relationship to contractile function. Our new approaches may be applicable to other multi-subunit complexes in cardiac muscle and other cell types.

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Superresolution & Confocal Systems

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Superresolution & Confocal Modules

  • Overview
  • MINFLUX Module
  • MATRIX Detector
  • TIMEBOW Imaging
  • FLEXPOSURE Illumination
  • RAYSHAPE Mirror
  • TRUESHARP Deconvolution
  • EASY3D
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