Development of quantitative dSTORM

Abstract

Direct stochastic optical reconstruction microscopy (dSTORM) is a singlemolecule imaging technique which involves tagging molecular targets with fluorescently labelled antibodies. In this method, only a subset of fluorophores emit photons at the same time, while the majority of fluorescent tags are pushed into an optically inactive state. This powerful technique, where resolution of 20 nm can be achieved, suffers from two major drawbacks which prevent quantitative analysis. The first problem lies with labelling of proteins of interest, where a single protein is typically labelled by multiple secondary antibodies tagged with a variable number of fluorophores. To count the number of proteins only one fluorophore per protein of interest must be assured. To solve this problem, I aimed to develop a novel linker molecule which, together with Fab’, an antigen-binding fragment, would produce a detection agent for 1:1 fluorophore to protein labelling. An alternative approach was also employed, in which an anti-EGFP nanobody was homogeneously mono-labelled with Alexa Fluor 647. Binding to EGFP was analysed both qualitatively and quantitatively and an excellent nanomolar affinity was demonstrated. The degree of labelling investigation revealed 1:1 nanobody to fluorophore ratio. The analysis of the nanobody was also performed using dSTORM, both on glass and in cells. The monolabelled nanobody produced significantly less localisations per single target as compared to the commercially available F(ab’)2 fragment and showed excellent colocalisation with EGFP in EGFP-SNAP-25 and EGFP-Lifeact transfected cells. The second problem in dSTORM is connected with the photophysical process itself. This is because the same fluorophore in dSTORM can enter light and dark cycles multiple times, so it is impossible to establish if closely neighbouring signals originate from one or multiple sources. A polarisation-based method was developed allowing measurement of polarisation of each fluorophore’s dipole. My strategy involved a change in the microscope pathway employing a polarisation splitter to separate light coming from each fluorophore into two components with orthogonal polarisations. Finally, the single labelling was combined with the polarisation experiments to achieve quantitative dSTORM, where the neighbouring signals could be assigned to the same or different targets, based on the polarisation value of each signal.