Achromatic light patterning and improved image reconstruction for parallelized RESOLFT nanoscopy

TL;DR

This paper introduces a novel achromatic light patterning technique for RESOLFT nanoscopy, enabling highly parallelized, multicolor super-resolution imaging of living cells with improved image reconstruction methods.

Contribution

It presents a wavelength-independent patterning method using light splitting and recombination, enhancing multicolor RESOLFT nanoscopy capabilities.

Findings

Achieved 60-80 nm resolution in live cell imaging.

Enabled multicolor imaging with up to three patterns.

Demonstrated large field of view imaging (100x100 μm).

Abstract

Fluorescence microscopy is rapidly turning into nanoscopy. Among the various nanoscopy methods, the STED/RESOLFT super-resolution family has recently been expanded to image even large fields of view within a few seconds. This advance relies on using light patterns featuring substantial arrays of intensity minima for discerning features by switching their fluorophores between on and off states of fluorescence. Here we show that splitting the light with a grating and recombining it in the focal plane of the objective lens renders arrays of minima with wavelength-independent periodicity. This colour-independent creation of periodic patterns facilitates coaligned on- and off-switching and readout with combinations chosen from a range of wavelengths. Applying up to three such periodic patterns on the switchable fluorescent proteins Dreiklang and rsCherryRev1.4, we demonstrate highly parallelized, multicolour RESOLFT nanoscopy in living cells at 60-80 nm resolution for 100 times 100 square micrometers fields of view. We discuss the impact of novel image reconstruction algorithms featuring background elimination by spatial bandpass filtering, as well as strategies that incorporate complete image formation models.