Simulation Approach to Optimize Fluorescence Imaging Performance of Wide-Field Temporal-Focusing Microscopy with Tunable Wavelength Excitation

摘要

The optical parameters of temporal-focusing multiphoton excitation microscopy (TFMPEM), which is capable ofachieving varying wavelength excitation for multiple fluorophore measurement, was systematically examined to havebetter excitation performance. For this purpose, the approaches were adopted to quantitatively evaluate the gratinggroove density, focal length of the collimating lens and objective, and different excitation wavelengths. A grating with agroove density of 830 lines/mm enables the TFMPEM system to achieve a wavelength range of 700-1000 nm byadjusting the incident angle of the ultrafast laser on the grating; a diffraction efficiency of 81 ± 3 % was obtained at thiswavelength range. By using the 830 lines/mm grating, a collimating lens with a 500 mm focal length, and a 60× waterimmersion objective, we achieved a large excitation area and a better filling effect of the spectrum band of the pulse onthe back focal plane of the objective; these parameters ensure high optical sectioning and small variation in theillumination power density within this wavelength range. The variation ranges of the excitation area, optical sectioning,and illumination power density of 4272 ± 798 μm~2, 2.6 ± 0.3 μm, and 71 ± 29 %, respectively, were obtained in twophotonexcitation fluorescence imaging at 700-1000 nm excitation wavelengths.