In stimulated emission depletion (STED) microscopy, the excitation focus is overlapped by an intense doughnut-shaped spot to instantly de-excite markers from their fluorescent state to the ground state by stimulated emission. This effectively eliminates the periphery of the Point Spread Function (PSF), resulting in a narrower focal region, or super-resolution. Scanning a sharpened spot through the specimen renders images with sub-diffraction resolution. Multi-color STED imaging can present important structural and functional information for protein-protein interaction.
In this work, we presented a two-color, synchronization-free STED microscopy with a Ti:Sapphire oscillator. The excitation wavelengths were 532nm and 635nm, respectively. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. Human respiratory syncytial virus (hRSV) proteins were imaged with our two-color CW STED for co-localization analysis.