Many biological processes occur in 3D across large volumes. In order to get an accurate representation of the underlying structure with single molecule localisation microscopy (SMLM) stringent labelling requirements must be met. This critical limit is defined by the Nyquist criterion where, for a 200 × 200 × 600 nm volume ∼40,000 molecules are required. This constitutes a lower threshold, and stochastic sampling typical of SMLM techniques means this is often insufficient. Even if an appropriate density were to be achieved, it is still necessary to be able to contrast the localisation from the background. PALM/STORM techniques are capable of achieving good contrast, but can suffer from photobleaching out of plane which reduces the attainable molecular density. Meanwhile points for accumulation in nanoscale topography (PAINT) utilises the transient binding of fluorescent probes. Although this circumvents photobleaching and facilitates collection of high localisation density data, PAINT is inherently limited by unwanted background exhibited at high probe concentrations. Here we present ‘reservoir'-PAINT (resPAINT), in which we combine PAINT with active control of probe photophysics to achieve up to a 50-fold increase in localisation rate for a constant background. We apply resPAINT to extended depth-of-field imaging and then the concept is generalised by implementing multiple activation strategies, including photoactivation and spontaneous photoswitching. We show the use of different imaging modalities such as tetrapod PSF and light-field microscopy. Notably, we demonstrate the extended operating regime of resPAINT by directly imaging membrane proteins using a Fab, which could open up the possibility of imaging low affinity antigen-protein interactions.
|Number of pages||1|
|Issue number||3 Supplement 1|
|Publication status||Published - 11 Feb 2022|