Sted Based Super-Resolution Imaging of Transverse Tubules in Ventricular Myocytes

Abstract

Transverse tubules (TTs) are specialized membrane invaginations in mammalian cardiomyocytes (CMs) that facilitate excitation-contraction coupling. TTs are reported to be about 300 nm in diameter (or less) by electron microscopy precluding structural characterization by conventional light microscopy (widefield or confocal). However, investigation of TTs and the associated signaling domains like ryanodine receptor containing junctions or caveoli in live cells is important towards understanding of dynamic subcellular signaling processes. To overcome the limitations in conventional cell imaging, we have examined TTs using Stimulated Emission Depletion (STED) super-resolution microscopy. The sarcolemmal and TT membranes were stained with lipophilic fluorescent dyes (di-8-ANNEPS). We used isolated CMs under quiescent conditions and confocal images were acquired and compared to STED images using the same optical path, microscope and sample. The apparent diameter of the TTs was determined both by confocal and STED imaging in live heart cells: fitting a Gaussian function to the fluorescence signal distribution yielded the following full width at half maximum (mean ± SEM): 271 ± 4 nm (confocal) versus 224 ± 5 nm (STED). Accordingly, STED-determined TT diameters were significantly smaller by 47 ± 6 nm (n=111; P<0.05) than those measured by confocal imaging. While TT sections from confocal images typically showed a 1-peak intensity distribution devoid of structural information, the STED intensity distribution showed a 2-peak pattern consistent with morphometric identification of tubular cross-sections. Thus, STED provides real-time data of tubule structures in living cells. How TTs are related to other subcellular structures will be presented. These findings suggest that STED microscopy may improve our quantitative understanding of complex subcellular structures in heart cells and enable study of their dynamic reorganization in normal and diseased states.