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.