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Three-dimensional super-resolution imaging: from cellular surface to interior Tafteh, Reza

Abstract

Single-molecule localization microscopy has greatly improved our understanding of biology by providing super-resolution images of biological processes and structures. However, it is still very challenging to apply this technique to thick tissues. A 3D imaging system based on single-molecule localization microscopy is presented to allow high-accuracy drift-free (< 0.7 nm lateral; 2.5 nm axial) imaging many microns deep into a cell. When imaging deep within a cell, distortions of the point-spread function result in an inaccurate and very compressed Z distribution. For the system to accurately represent the position of each molecule, a series of depth-dependent calibrations are required. The system and its allied methodology were developed to image the type-2 Ryanodine receptor (RYR2) in the cardiac myocyte at a depth of several microns. It enabled us to resolve the structure of the individual (30 nm square) receptors giving a result similar to that obtained with electron tomography. We also present an optical setup using an electrically tunable lens to actively stabilize a single-molecule localization microscope in three dimensions (RMS ~ 0.7 nm lateral; ~ 2.7 nm axial). The effectiveness of the ETL was demonstrated by imaging endosomal transferrin receptors near the apical surface of B-lymphocytes at a depth of 8 µm. This stabilization system enables a more accurate topological cluster analysis. We have used these super-resolution imaging approaches to examine overlap between the RYR2 and the L-type Calcium channel (Cav1.2) on the cellular surface and within the rat ventricular myocyte. We accurately imaged receptors down to a depth of 6 µm below the surface, and for the first time, using light microscopy, we were able to image individual receptors. The distribution of RyR2 and Cav1.2 parallel each other and vary greatly between the surface, just below the surface, and deep in the interior. We have also used two-color super-resolution microscopy to quantify receptor organization on the plasma membrane of follicular (FO) and marginal zone (MZ) B cells. We have found that B cell receptors (BCR) on the surface of MZ B cells were more dispersed and exhibited less clustering than those on FO B cells.

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