isoSTED microscopy with water-immersion lenses and background reduction

Authors

Siegmund R, Werner F, Jakobs S, Geisler C, Egner A

Journal

Biophysical Journal

Citation

Biophys J. 2021 Jul 8:S0006-3495(21)00556-7.

Abstract

Fluorescence microscopy is an excellent tool to gain knowledge on cellular structures and biochemical processes. Stimulated emission depletion (STED) microscopy provides a resolution in the range of a few ten nanometers at relatively fast data acquisition. As cellular structures can be oriented in any direction, it is of great benefit if the microscope exhibits an isotropic resolution. Here, we present an isoSTED microscope that utilizes water-immersion objective lenses and enables imaging of cellular structures with an isotropic resolution of better than 60 nm in living samples at room temperature and without CO2 supply or another pH control. This corresponds to a reduction of the focal volume by far more than two orders of magnitude as compared to confocal microscopy. The imaging speed is in the range of 0.8 s/μm3. Since fluorescence signal can only be detected from a diffraction-limited volume, a background signal is inevitably observed at resolutions well beyond the diffraction limit. Therefore, we additionally present a method which allows to identify this unspecific background signal and to remove it from the image.