Abstract
.Stimulated emission depletion (STED) microscopy is a powerful bioimaging technique that theoretically provides molecular spatial resolution while preserving the most important assets of fluorescence microscopy. When combined with two-photon excitation (2PE) microscopy (2PE-STED), subdiffraction resolution may be achieved for thick biological samples. The most straightforward implementation of 2PE-STED microscopy entails introduction of an STED beam operating in continuous wave (CW) into a conventional Ti:sapphire-based 2PE microscope (2PE CW-STED). In this implementation, resolution enhancement is typically achieved using time-gated detection schemes, often resulting in drastic signal-to-noise/-background ratio (SNR/SBR) reductions. Herein, we employ a pixel-by-pixel phasor approach to discard fluorescence photons lacking super-resolution information to enhance image SNR/SBR in 2PE CW-STED microscopy. We compare this separation of photons by lifetime tuning approach against other postprocessing algorithms and combine it with image deconvolution to further optimize image quality.
Highlights
Stimulated emission depletion (STED) microscopy[1,2,3] is a powerful fluorescence imaging technique for nanoscale visualization of biological processes
The fluorescence lifetime is uniform along the point spread function (PSF) in 2PE microscopy while a gradient occurs with 2PE continuous wave (CW)-STED microscopy, with the shortest fluorescence lifetime occurring in the periphery of the PSF corresponding with the maximum STED intensity in the doughnut profile [Fig. 1(b)]
The separation of photons by lifetime tuning (SPLIT) technique exploits the spatiotemporal information contained in a time-resolved STED image to enhance the performance of 2PE CW laser for STED (CW-STED) microscopes
Summary
Stimulated emission depletion (STED) microscopy[1,2,3] is a powerful fluorescence imaging technique for nanoscale visualization of biological processes. The fundamental mechanism underlying STED microscopy entails spatially modulated fluorescence depletion to enable effective excitation/fluorescent volumes with size below the diffraction limit of the light. The first implementations of 2PE-STED microscopy employed CW lasers for depletion and a modelocked Ti:Sapphire laser for 2PE.[5,6,7] While the use of a CW laser for STED (CW-STED) avoids synchronization challenges and mitigates costs inherent to higher peak-power pulsed lasers, the resulting less effective depletion limits the achievable spatial resolution.[6,7] a higher resolution can be achieved using pulsed STED (pSTED) approaches,[8,9,10] which in the cases of 2PE typically require synchronization of two mode-locked ultrafast lasers at different wavelengths to provide sufficient peak power for both the excitation and the depletion beams. We refer to these implementations as 2PE-pSTED microscopy
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