Obtaining super-resolved images at the mesoscale through Super-Resolution Radial Fluctuations

Widefield fluorescence microscopy is commonly used for diffraction limited imaging of fluorescently labelled cellular structures. Super-resolution methods such as scanning electron microscopy have been able to overcome this diffraction limit to achieve significantly higher spatial resolutions and this has led to a greater understanding of cellular structures, but current super-resolution methods often have a restricted field of view, minimising the available information and limiting the understanding of behaviours and interactions on a larger scale. Here, we have applied Super-Resolution Radial Fluctuations (SRRF)[1,2] in conjunction with the Mesolens[3] to obtain super-resolution images over a field of view of 4.4 mm x 3.0 mm. We demonstrate that SRRF processed images display a notable improvement in resolution over Mesolens images obtained with diffraction limited widefield fluorescence illumination. We also observe enhanced contrast in the SRRF data at the mesoscale. We assessed the accuracy of SRRF images through error maps calculated using a secondary analysis method, super-resolution quantitative image rating and reporting of error locations (SQUIRREL)[4]. Our data shows consistent structural agreement between the original raw image data and the SRRF processed images. This work demonstrates a cost-efficient and accurate method of obtaining super-resolved images over a uniquely large field of view.

[1] Gustafsson, N. et al. Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations. Nat. Commun. 7, 12471 (2016).
[2] Culley, S., Tosheva, K. L., Matos Pereira, P. & Henriques, R. SRRF: Universal live-cell super-resolution microscopy. Int. J. Biochem. Cell Biol. 101, 74–79 (2018).
[3] McConnell, G. et al. A novel optical microscope for imaging large embryos and tissue volumes with sub-cellular resolution throughout. eLife 5, e18659 (2016).
[4] Culley, S. et al. Quantitative mapping and minimization of super-resolution optical imaging artifacts. Nat. Methods 15, 263–266 (2018).