Triple-Color STED Nanoscopy: Sampling Absorption Spectra Differences for Efficient Linear Species Unmixing

Stimulated emission depletion (STED) in confocal fluorescence microscopy enables a visualization of biological structures within cells far below the optical diffraction limit. To meet the demand in the field for simultaneous investigations of multiple species within a cell, a couple of different STED techniques have been proposed, each with their own challenges. By systemically exploiting spectral differences in the absorption of fluorescent labels, we present a novel, beneficial approach to multispecies STED nanoscopy. By using three excitation wavelengths in nanosecond pulsed interleaved excitation (PIE) mode, we probe quasi simultaneously multiple species with fluorescent labels having absorption maxima as close as 13 nm. The acquired image is decomposed into its single species contributions by application of a linear unmixing algorithm based on present reference patterns. For multispecies images containing single species regions, we introduce the image correlation map (ICM). Here, the single species regions easily can be identified in order to generate the necessary single species reference patterns. This avoids the otherwise cumbersome and artifact prone preparation and recording of additional reference samples. The power of the proposed imaging scheme persists in species separation quality at high speed shown for up to three species with established reference samples and dyes commonly used for cellular STED imaging.

Automated summary

STED technique allows visualization of biological structures within cells below diffraction limit by exploiting spectral differences in absorption of fluorescent labels for simultaneous detection of multiple species with close absorption maxima. This novel approach using multiple excitation wavelengths in nanosecond pulsed interleaved excitation mode decomposes acquired images into single species contributions through a linear unmixing algorithm. The introduced image correlation map (ICM) facilitates identification of single species regions for generating necessary single species reference patterns, showing high speed species separation quality for up to three species in cellular STED imaging.