Fluorescence-Activating and absorption-Shifting Tag (FAST) is a new class of genetically encoded optical highlighter probes based on stochastic labelling. FAST is a protein that is half the size (14 kDa) of conventional fluorescent proteins (e.g. GFP). This genetically encoded protein forms fluorescent assemblies by association with cell permeable and nontoxic fluorogens, displaying various spectral properties. As the FAST fluorogen interaction is dynamic, constant replenishment of the fluorogens in the media leads to a pool of naïve fluorogens for binding, thereby resulting in negligible photobleaching. Nanoorganization laboratory at the Centre for Neuroscience combined FAST-Fluorogen binding with a new analytical method based on intensity fluctuation analysis named Super-Resolution Radial Fluctuations (SRRF) to achieve spatial resolution better than the diffraction limit of a regular epifluorescent microscope. Combining FAST and SRRF, we obtained sub – 100 nm resolution images of live cells with high temporal resolution. The flexibility and diversity of the fluorogen probes allowed multiplexing with other fluorescent molecules to study subcellular dynamics. This protocol can be adopted for any epifluorescent microscopes using widefield detection for resolution doubling in both live and fixed samples.
Stochastic labelling and super resolution microscopy using genetically encoded fluorogen binding tags: FAST can be reversibly labelled using ligands which will render it fluorescent. When the fluorogen-tag complex cannot emit fluorescence anymore, the fluorogen will be dissociated making way for a new fluorogen to take its place and emit fluorescence again. This is advantageous over conventional labelling techniques as there is no bleaching of fluorescence signal. The fluorescence of proteins of interest can acquired using a regular widefield microscope. Due to stochastic labelling, intensity variations can be analysed using a radial-fluctuation analysis (SRRF) from a stream of widefield microscopy images to obtain a super-resolution image.
Venkatachalapathy M, Belapurkar V, Jose M, Gautier A, Nair D (2019) Live cell super resolution imaging by radial fluctuations using fluorogen binding tags. Nanoscale 11: 3626-3632
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