Optical Sectioning Microscopy at ‘Temporal Super-Resolution’

Abstract

Within the recent years several super-resolution microscopic methods were developed, where the super-resolution refers to bringing the optical resolution beyond the diffraction limit introduced by Ernst Abbe, which was believed to be a real limit for quite some time. The popularity of the method also in cardiac related research can be followed in the chapter ‘Quantitative super-resolution microscopy of cardiac myocytes’ in this book. In parallel to this spatial super-resolution progress, within the past two decades there was a dynamic development of high speed–high resolution imaging initially towards video-rate (30 frames per second, also referred to as ‘real time’-imaging) but soon to ever increasing frame rates reaching the kHz order of magnitude these days. Many processes, especially those in excitable cells such as neurons and cardiomyocytes [1] or cells in flow like erythrocytes or leukocytes [2], require even higher temporal resolution to elucidate the kinetics of processes like the Excitation-Contraction Coupling (ECC). Such ultra high speed recordings still require a diffraction limited spatial resolution to correlate function and subcellular structures [3]. Within this chapter we review optical sectioning microscopy and their application in cellular cardiology. In this approach we focus on methods that allow to access any part of the cell, i.e. we exclude methods that are intrinsically limited to surface investigations like total internal reflection fluorescence (TIRF) microscopy [4] or scanning near field optical microscopy (SNOM) [5]. In similarity we exclude techniques that require several images to calculate an image section such as deconvolution microscopy [6] or structured illumination microscopy [7] (e.g., Apotome.2, Zeiss, Jena, Germany).