How to label a protein with one fluorophore

Super-resolution microscopy is a rapidly evolving class of fluorescence-based visualisation methods allowing non-destructive biological imaging below the diffraction limit. One approach, direct stochastic optical reconstruction microscopy (dSTORM), is a single-molecule imaging technique which commonly uses fluorescently labelled antibodies to detect the cellular protein of interest. In this method, only a subset of fluorophores is fluorescent at the same time, with the majority of fluorophores residing in a long-lived dark state. This allows the localisation of individual fluorophores with an accuracy of approximately 20 nm. Unfortunately, this method cannot currently be used for the quantitative analysis of nanoscale biochemical reactions. One of the problems lies with the labelling of target proteins with multiple antibodies each of which is conjugated to a number of fluorophores. To count the number of target proteins, a ratio of one fluorophore to each target protein should be assured. The aim of this seminar is to present the strategies and challenges involved in labelling proteins with a single fluorophore and describe our group’s progress in this field to enable quantitative analysis of molecules in dSTORM.