Multicolor super-resolution imaging made easy

© 2020 EPFL - Scientists at EPFL have developed robust and easy-to-implement multicolor super-resolution imaging. The approach is based on the simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing. They exploit fluorophore blinking and spectral crosstalk for the generation of additional color channels with super-resolved images. Multicolor fluorescence microscopy is an important tool for the life sciences to study the relative arrangements of cellular structures or the interactions of different proteins. However, conventional microscopes, the workhorses for many biological studies, can only resolve details on the order of the wavelength of light. In the past two decades, several super-resolution microscopy concepts helped researchers to overcome this diffraction limit and to make new discoveries. These new methods are only slowly finding their way into routine biological applications. For some of the new techniques, this is due to complex microscope hardware, but also increased demands on sample preparation and fluorescent labels pose significant hurdles. The requirements for successful super-resolution imaging are even more challenging to meet for multicolor applications. EPFL's Laboratory of Biomedical Optics headed by Theo Lasser has been extensively working on Super-resolution Optical Fluctuation Imaging (SOFI) to increase the spatial resolution and sampling in 2D and 3D. SOFI is an alternative to Single-molecule Localization Microscopy techniques such as STORM and PALM. It analyzes higher order spatio-temporal statistics of a time-series of blinking fluorophores and does not require the isolation of individual fluorophores?