Fluorophore photophysics and super-resolution microscopy get married in SMIS
Single-molecule localization microscopy (SMLM) is the most popular super-resolution fluorescence imaging technique. It is also the technique providing the best resolution enhancement, therefore being most suited for integrating structural biology into the cellular context. Like all super-resolution methods, SMLM fundamentally relies on specific fluorophore photophysical properties, like photoswitching. Its limitations, however, are also largely bound to –unwanted- fluorophore photophysics.
A main goal of the Pixel team of the Integrated Imaging of Stress Response Group is to understand how fluorophore photophysics and SMLM are intertwined. To this aim, since 10 years, dominique Bourgeois has been developing a Matlab-based simulation tool named SMIS (Single Molecule Imaging Simulator). SMIS simulates a SMLM microscope and generates data sets from virtual samples while taking account arbitrarily complex photophysics of the employed fluorophores. This way, all sorts of weird behaviors and artifacts can be evaluated and understood. Taking advantage of the Covid confinement, he could finalize the work in the form of a GUI (Graphical User Interface) available to the community, also showing in a number of examples how SMIS can be used to understand what’s going on. This personal project turned out to be hugely time-consuming, and kept him busy in parallel with the other projects of the I2SR group!
Single molecule imaging simulations with advanced fluorophore photophysics. Bourgeois D. Communications Biology 2023; 6, 53.