RT Journal Article
SR Electronic
T1 Enabling spectrally resolved single-molecule localization microscopy at high emitter densities
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.06.29.498127
DO 10.1101/2022.06.29.498127
A1 Koen J.A. Martens
A1 Martijn Gobes
A1 Emmanouil Archontakis
A1 Roger R. Brillas
A1 Niels Zijlstra
A1 Lorenzo Albertazzi
A1 Johannes Hohlbein
YR 2022
UL http://biorxiv.org/content/early/2022/07/02/2022.06.29.498127.abstract
AB Single-molecule localization microscopy (SMLM) is a powerful technique for elucidating structure and dynamics in the life- and material sciences with sub-50 nm spatial resolution. The simultaneous acquisition of spectral information (spectrally resolved SMLM, sSMLM) enables multiplexing using spectrally distinct fluorophores or enable the probing of local chemical environments by using solvachromatic fluorophores such as Nile Red. Until now, the widespread utilisation of sSMLM was hampered by several challenges: an increased complexity of the optical detection pathway, limited software solutions for data analysis, lower accessible emitter densities or smaller field-of-views, and overall compromised spatio-spectral resolution. Here, we present a low-cost implementation of sSMLM that addresses these challenges. Using a blazed, low-dispersion transmission grating positioned close to the image plane here represented by the camera sensor, the +1st diffraction order is minimally elongated compared to the point spread function of the 0th order and can therefore be analysed using common subpixel single-molecule localization algorithms. The distance between both PSFs provides accurate information on the spectral properties of the emitter. The minimal excess width of 1st order PSFs enables a fivefold higher emitter density compared to other sSMLM approaches whilst achieving a spatio-spectral localization accuracy sufficient to discriminate between fluorophores whose peak emission are less than 15 nm apart as demonstrated using dSTORM, DNA-PAINT and smFRET. We provide an ImageJ/Fiji plugin (sSMLMAnalyzer) and suitable Matlab scripts for data analysis. We envision that our approach will find widespread use in super-resolution applications that rely on distinguishing spectrally different fluorophores under low photon conditions.Competing Interest StatementWageningen University filed a patent application describing this method (WO2022053649).