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.

Multi-target immunofluorescence by separation of antibody cross-labelling via spectral-FLIM-FRET.

In biomedical research, indirect immunofluorescence labelling by use of primary and secondary antibodies is central for revealing the spatial distribution of multiple cellular antigens. However, labelling is regularly restricted to few antigens since species variation of primary and corresponding secondary antibodies is limited bearing the risk of unspecific cross-labelling. Here, we introduce a novel microscopic procedure for leveraging undesirable cross-labelling effects among secondary antibodies thereby increasing the number of fluorophore channels. Under cross-labelling conditions, commonly used fluorophores change chemical-physical properties by 'Förster resonance energy transfer' leading to defined changes in spectral emission and lifetime decay. By use of spectral fluorescence lifetime imaging and pattern-matching, we demonstrate precise separation of cross-labelled cellular antigens where conventional imaging completely fails. Consequently, this undesired effect serves for an innovative imaging procedure to separate critical antigens where antibody species variation is limited and allows for multi-target labelling by attribution of new fluorophore cross-labelling channels.

Photon arrival time tagging with many channels, sub-nanosecond deadtime, very high throughput, and fiber optic remote synchronization.

Time-Correlated Single Photon Counting (TCSPC) and time tagging of individual photon detections are powerful tools in many quantum optical experiments and other areas of applied physics. Using TCSPC, e.g., for the purpose of fluorescence lifetime measurements, is often limited in speed due to dead-time losses and pileup. We show that this limitation can be lifted by reducing the dead-time of the timing electronics to the absolute minimum imposed by the speed of the detector signals while maintaining high temporal resolution. A complementing approach to speedy data acquisition is parallelization by means of simultaneous readout of many detector channels. This puts high demands on the data throughput of the TCSPC system, especially in time tagging of individual photon arrivals. Here, we present a new design approach, supporting up to 16 input channels, an extremely short dead-time of 650 ps, very high time tagging throughput, and a timing resolution of 80 ps. In order to facilitate remote synchronization of multiple such instruments with highest precision, the new TCSPC electronics provide an interface for White Rabbit fiber optic networks. Beside fundamental research in the field of astronomy, such remote synchronization tasks arise routinely in quantum communication networks with node to node distances on the order of tens of kilometers. In addition to showing design features and benchmark results of new TCSPC electronics, we present application results from spectrally resolved and high-speed fluorescence lifetime imaging in medical research. We furthermore show how pulse-pileup occurring in the detector signals at high photon flux can be corrected for and how this data acquisition scheme performs in terms of accuracy and efficiency.