Enhancing STED Microscopy via Fluorescence Lifetime Unmixing and Filtering in Two-Species SPLIT-STED

Abstract Simultaneous super-resolution imaging of multiple fluorophores remains a major challenge in STimulated Emission Depletion (STED) microscopy due to spectral overlap of STED-compatible fluorophores. The combination of STED microscopy and Fluorescence Lifetime Imaging Microscopy (FLIM) offers a powerful alternative for super-resolved, multiplexed imaging of biological samples but is hindered by lifetime convergence at high depletion powers. Here, we present an analysis method, two-species Separation of Photons by LIfetime Tuning (SPLIT)-STED, that uses a linear system of equations in phasor-based STED-FLIM to enhance both fluorophore unmixing and spatial resolution. It defines the fluorescence signal as a mixture of three lifetime components: the two target fluorophores and a short-lifetime contribution from undepleted peripheral fluorescence photons. Two-species SPLIT-STED disentangles overlapping lifetimes and selectively filters low-resolution signal. The method enables accurate unmixing of spectrally overlapping fluorophores and, by enhancing resolution through lifetime-based filtering, allows the use of lower depletion powers, thereby improving fluorescence lifetime separation. Competing Interest Statement The authors have declared no competing interest. List of Acronyms - STED - STimulated Emission Depletion - FLIM - Fluorescence Lifetime Imaging Microscopy - SPLIT - Separation of Photons by LIfetime Tuning - PSF - Point Spread Function - TCSPC - Time Correlated Single Photon Counting - DIV - days in vitro - PAB - primary antibodies - SAB - secondary antibodies - PFA - paraformaldehyde - PBS - Phosphate-Buffered Saline - NGS - normal goat serum - RT - room temperature - APD - Avalanche Photodiode Detectors - IRF - Instrument Response function - FWHM - Full Width at Half Maximum - MLE - Maximum Likelihood Estimation - ROI - regions of interest - SLSQP - Sequential Least Squares Programming - CWF - Complex Wavelet Filter - SQUIRREL - Super-resolution QUantitative Image Rating and Reporting of Error Locations - SE - Squared Error - SEM - Standard Error of the Mean - SNR - signal to noise ratio - STD - standard Deviation - BFP - Back Focal Plane - CW - Continuous Wave - FCS - Fluorescence Correlation Spectroscopy