Automated STED nanoscopy for high-throughput imaging of cellular structures

Abstract

STimulated Emission Depletion (STED) nanoscopy uniquely combines a high spatial resolution (20-50nm in cells) with relatively fast imaging (frame rate of [~]1-30Hz), straightforward sample preparation and direct image output (no postprocessing required). Although these characteristics in principle make STED very suitable for high-throughput imaging, only few steps towards automation have been made. Here, we have developed fully automated STED imaging, eliminating all manual steps including the selection and characterisation of the relevant (cellular) regions, sample focusing and positioning, and microscope adjustments. This automatic STED image acquisition increases the data output by roughly two orders of magnitude, resulting in a more efficient use of the high-end microscope, and the ability to detect and characterise objects that are only present in a small subset of the sample.