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

Abstract

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.

Synergizing exchangeable fluorophore labels for multi-target STED microscopy

“…Attempts to reduce fluorophore photobleaching include adaptive illumination schemes 2 , the development of photostable fluorophores 3 or the application of fluorescent proteins with multiple off-states 4 . In addition, the number of cellular structures and proteins that can be imaged within the same cell is limited due to spectral overlap of fluorophore absorption and emission spectra 5 . Both challenges can be addressed with weak-affinity, non-covalent fluorophore labels that transiently bind to a target structure and are continuously replaced by intact fluorophores from a large buffer reservoir.…”

Synergizing Exchangeable Fluorophore Labels for Multitarget STED Microscopy

“…Attempts to reduce fluorophore photobleaching include adaptive illumination schemes, the development of photostable fluorophores, or the application of fluorescent proteins with multiple off-states . In addition, the number of cellular structures and proteins that can be imaged within the same cell is limited due to spectral overlap of fluorophore absorption and emission spectra . Both challenges can be addressed with weak-affinity, noncovalent fluorophore labels that transiently bind to a target structure and are continuously replaced by intact fluorophores from a large buffer reservoir.…”

“… 4 In addition, the number of cellular structures and proteins that can be imaged within the same cell is limited due to spectral overlap of fluorophore absorption and emission spectra. 5 Both challenges can be addressed with weak-affinity, noncovalent fluorophore labels that transiently bind to a target structure and are continuously replaced by intact fluorophores from a large buffer reservoir. These “exchangeable” labels provide an elegant way to bypass photobleaching and facilitate multiplexing and 3D imaging.…”

Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation

“…Complementing fluorescence lifetime and emission spectra, utilization of the absorption spectra was suggested for multi-color STED 18 , but so far the number of fluorochromes was limited to three.…”

“…Another study made use of cross labelling antibodies to create new lifetime species via Förster resonance energy transfer (FRET), later unmixed by the same spectral FLIM separation approach 17 . However, for high precision color separation a commercially not available spectral detector was used in both studies and the applied pattern matching for color separation requires relatively high photon numbers, at least if structures are overlapping 15,18 . The method was also applied to STED, but here the separation of only two dyes in a single color channel was demonstrated on images with 5,000 - 10,000 photons in the brightest pixels 15 .…”