Doubling the resolution of a confocal spinning-disk microscope using image scanning microscopy

Abstract

Fluorescence microscopy has become an indispensable tool for cell biology. Recently, super-resolution methods have been developed to overcome the diffraction limit of light and have shown living cells in unprecedented detail. Often, these methods come at a high cost and with complexity in terms of instrumentation and sample preparation, thus calling for the development of low-cost, more accessible methods. We previously developed image scanning microscopy (ISM), which uses structured illumination to double the resolution and quadruple the contrast of a confocal microscope. Implementing this technique into a confocal spinning-disk (CSD) microscope allows recording ISM images with up to ~1 frame per second, making it ideal for imaging dynamic biological processes. Here we present a step-by-step protocol describing how to convert any existing commercial CSD microscope into a CSD-ISM, with only moderate changes to the hardware and at low cost. Operation of the CSD-ISM is realized with a field programmable gate array using the software environment Micro-Manager, a popular open-source platform for microscopy. The provided software ( https://projects.gwdg.de/projects/csdism-2020 ) takes care of all algorithmic complexities and numerical workload of the CSD-ISM, including hardware synchronization and image reconstruction. The hardware modifications described here result in a theoretical maximum increase in resolution of √2 ≈ 1.41, which can be further improved by deconvolution to obtain a theoretical maximum twofold increase. An existing CSD setup can be upgraded in ~3 d by anyone with basic knowledge in optics, electronics and microscopy software.