Compact Multi-Color STED Microscopy

Abstract

Stimulated emission depletion (STED) microscopy enables fluorescence imaging on the nanoscale with resolutions of 20 nm and beyond. As samples prepared for far-field fluorescence microscopy are in general also suitable for STED imaging, it has been applied advantageously in biosciences, in particular investigations of subcellular structures and assemblies.In order to expand the regime of applications to high-resolution colocalization studies we designed a setup which allows for STED imaging of up to three different labels (quasi-)simultaneously. This enables us to accurately investigate the interplay of at least three differently labeled cellular components [1]. We distinguish the labels by using different spectral properties of the dyes and, in addition, by separating dyes with similar spectral properties but different fluorescence lifetimes. The design intrinsically provides high spatial resolution for all markers simultaneously, since in STED, the diffraction barrier is overcome by reducing the effective volume in which fluorescence can occur spontaneously to a nanosized region at the very center of the STED beam. Our approach is less prone to drift artifacts than e.g. stochastic imaging concepts where the recording of even a small region of the object extends over the whole image acquisition period, and it is not subject to systematic errors due to the fluorophore orientations which have to be considered in stochastically based high-resolution techniques.[1] D. Neumann, J. Buckers, L. Kastrup, S. W. Hell, S. Jakobs: “Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms,” PMC Biophysics, 3:4 (2010).