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The optical parameters of temporal-focusing multiphoton excitation microscopy (TFMPEM), which is capable of achieving varying wavelength excitation for multiple fluorophore measurement, was systematically examined to have better excitation performance. For this purpose, the approaches were adopted to quantitatively evaluate the grating groove density, focal length of the collimating lens and objective, and different excitation wavelengths. A grating with a groove density of 830 lines/mm enables the TFMPEM system to achieve a wavelength range of 700-1000 nm by adjusting the incident angle of the ultrafast laser on the grating; a diffraction efficiency of 81 ± 3 % was obtained at this wavelength range. By using the 830 lines/mm grating, a collimating lens with a 500 mm focal length, and a 60× water immersion objective, we achieved a large excitation area and a better filling effect of the spectrum band of the pulse on the back focal plane of the objective; these parameters ensure high optical sectioning and small variation in the illumination power density within this wavelength range. The variation ranges of the excitation area, optical sectioning, and illumination power density of 4272 ± 798 μm2, 2.6 ± 0.3 μm, and 71 ± 29 %, respectively, were obtained in two photon excitation fluorescence imaging at 700-1000 nm excitation wavelengths.
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