Wavelength-tunable depletion expands fluorophore compatibility in two-photon STED nanoscopy

We report the development of a novel two-photon excitation stimulated emission depletion microscopy system, namely 2PE-scSTED, featuring a custom light source setup that integrates a supercontinuum (SC) laser light source with our proprietary 1064 nm pulsed semiconductor excitation laser. This SC source provides the capability of emitting a broadband spectral range, enabling highly reliable selection of depletion wavelength. Utilizing 2PE-scSTED, we investigated the depletion characteristics of several fluorophores. By employing a tunable filter to precisely select depletion wavelength from 640 to 800 nm, we systematically measured the depletion efficiency and identified the optimal wavelengths that maximize fluorescence depletion efficiency for four distinct fluorescent probes. This approach yielded super-resolution images with a lateral spatial resolution as fine as 45 nm (approximately λ _2PE /24) with a relatively lower depletion power than our previous reports, and can be principally applicable to all fluorescent probes. Our results demonstrate that the integration of a broadband SC source removes the spectral constraint of fluorescence depletion, enabling tailored 2PE-STED microscopy for individual fluorophores. This enhances the versatility and applicability of 2PE-STED microscopy for super-resolution imaging of biological specimens.