A 3D super-resolution method based on sparse-tagging and point spread function
(PSF) analysis was developed for use with optical microscopes, suitable for imaging
transcytosis or similar phenomena in dynamic live-cell imaging. The method was
based on utilising previously acquired images of the full 3D PSF to estimate the axial
position of newly acquired 2D data, removing the need for sequential axial acquisition
and thus rendering the process much faster than comparable super-resolution
techniques. The method made use of careful parameterisation of the PSF to allow
for the robust analysis of the previously acquired data. This, combined with a genetic
algorithm, allows for greater accuracy and the flexibility to use the developed
technique on many optical platforms with a wide range of fluorophores. Using microbeads
and tagged low-density-lipoprotein on both an Elyra PS.1 widefield microscope
and a Leica SP8 confocal microscope it was demonstrated that the method can
achieve single particle localisation with a precision of 80 nm and accuracy of 15-30
nm in prepared gelatin samples. Further we detail some initial steps to translate the
use of the technique to live cell in vitro.