Theoretical And Experimental Investigations On Stimulated Emission Depletion (STED) Microscopy System Based On Continuous-wave Lasers

Stimulated emission depletion(STED)microscopy,which was proposed by Hell in 1994 and realized in 2000,has attracted much attention as a new type of microscopic imaging technology in multidiscipline field.It breaks the barriers of diffraction limit of the conventional microscopy system to achieve super-high resolution.My investigations include a series numerical,theoretical and experimental researches on STED microscopy.First,we systematically investigated the influence of wavefront phase modulation on the structures of spatial light.By applying Debye integral method,the effects of different phase plate functions and the polarizations of incident beams on the distribution of focal spot and the size of STED microscopy system were studied numerically.We found the azimuthally polarized light could generate a doughnut-like focal spot with zero intensity at the center.Compared with the conventionally applied depletion beam which was generated by right-hand circularly polarized beam modulated by vortex phase plate,the "dark spot" of the azimuthally polarized light has a smaller full width at half maximum(FWHM).Subsequently,based on the experimental results of the reported STED system,with the data from literatures(wavelengths and powers of excitation and depletion beams,the type of fluorescent dyes and spatial resolution of the STED system),the saturation intensity can be evaluated by numerical simulation.Based on the calculated saturation intensity,combined with the optimal excitation and depletion beams that numerically stimulated,we want to see how the resolution of STED system could be optimized.It was found,if using azimuthally polarized beam modulated by vortex phase plate as the excitation beam and the azimuthally polarized beam without modulation as depletion beam,the resolution of STED system can be increased by 67%,compared with that of original STED system.Later,I focused on the relevant theories of STED microscopy system that constructed by continuous-wave(CW)lasers.Although CW STED system has been investigated and developed for nearly a decade,the corresponding suppression function for CW STED has not be well established.Normally,researchers simply expanded the depletion theory developed from short pulse(picosecond,femtosecond)STED system for CW STED.This could cause discrepancies when evaluating the spatial resolution of CW STED system.In this study,we illustrate that the suppression function of CW STED is influenced by various optical and material parameters in details.For picosecond pulse STED system,the suppression function is controlled by the intensity of the depletion beam and the saturation intensity.However,for CW STED,it was found that the suppression function was dominated by the local laser intensity ratio of excitation and depletion beams.Also,the FWHM is highly sensitive to the rates of fluorescence(kfl)and quenching(kQ),but insensitive to the rate of vibrational relaxation.The spatial resolution of CW STED increases rapidly with decreasing kfl and kQ.Our model is highly consistent with published experimental works,without preacquisition of saturation intensity in the literatures.This investigation is important for guiding the development of new CW STED systems.Finally,we studied the depletion process of Coumarin 102 dyes experimentally.The effects of the power of excitation and depletion beams and dye concentration on depletion efficiency were investigated in details.In addition,the saturation intensities of Coumarin 102 dye in six different concentrations were numerically calculated.It was found that the smallest saturation intensity had been obtained when the concentration is 10 ?M.More importantly,we found the saturation intensity was not constant in the same CW STED system,but varied with the concentration of the fluorescence and the intensity of the depletion beam.This showed that the suppression model dominated by saturation intensity and developed based on the picosecond STED could not be completely applicable for the CW STED system.This further proved the importance of our previous works on developing new suppression theory for CW STED system.