Multi-color, quantitative single-molecule localization microscopy (qSMLM) based on kinetic analysis of fluorophore blinking cycles

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We will establish the hybrid technology of multi-color single-molecule super-resolution imaging with quantitative read-out at the nanoscale, termed qSMLM. This effort will exploit the unique type of raw data generated in an SMLM experiment, i.e. the coordinates of single emitters and the time of the detection event. In most cases, this data is used to reconstruct an image with subdiffraction spatial resolution. However, it also contains valuable photokinetic information: photoactivatable/photoswitchable fluorescent proteins and fluorophores exhibit the photophysical phenomenon of blinking (a fluorescence intermittency), which follows well-defined kinetic laws and allows to determine how many fluorophores are present in a multi-molecular complex (Fricke et al., 2015). The time course of blinking of a single fluorophore is directly available from the raw data of an SMLM experiment. We aim to establish multi-color quantitative SMLM (qSMLM) by simultaneously generating super-resolution images and determining protein stoichiometries by analyzing the kinetics of blinking. We will investigate fluorescent proteins and organic fluorophores in combination with a variety of tagging technologies for protein labeling. For calibration and validation purposes, we will exploit well-defined standards of membrane proteins and homo-oligomers (Finan et al., 2015) as well as DNA origami (Schmied et al., 2012). Our approach will allow extracting quantitative information at the molecular level, representing an extremely useful extension for anyone applying single-molecule super-resolution microscopy. It will in particular be useful to study homo- and heterooligomeric signaling protein complexes in the plasma membrane of an intact cell with molecular resolution.