研究目的
Investigating the effects of specimen-induced aberrations on the performance of two-photon light-sheet microscopy (TP-LSM) systems and evaluating the effectiveness of adaptive optics (AO) in mitigating these effects.
研究成果
The study concludes that high-order aberrations have little effect on the axial resolution and excitation efficiency of the TP-LSM system, and only low-order components require correction. A general formula is provided for estimating the number of Zernike modes that should be corrected under different aberration strengths and numerical apertures of the objective, offering guidance for the design and evaluation of AO units in TP-LSM systems.
研究不足
The study is based on simulations and assumes uniform amplitude distributions of aberrations at different depths. The actual biological specimens may introduce more complex aberration patterns not fully captured in the simulations.
1：Experimental Design and Method Selection:
The study employs a numerical simulation method to analyze the effects of aberrations on the TP-LSM system. The simulation is based on the point spread function (PSF) of the TP-LSM system, and new evaluation indexes based on integral intensity are introduced for assessing the aberrated light-sheet.
2：Sample Selection and Data Sources:
The study simulates the formation of a two-photon light sheet and investigates the influences of each Zernike mode and random aberrations on the illumination path of the TP-LSM system.
3：List of Experimental Equipment and Materials:
The simulation assumes an equivalent focal length of the objective (f) set to 5 mm, with the effective aperture (d) varied to achieve different illumination numerical apertures (NAill). The wavelength of the femtosecond pulse laser is set to 1000 nm.
4：Experimental Procedures and Operational Workflow:
The study simulates the distribution of illumination laser intensity (PSFill) along the beam propagation direction and integrates the intensity along the y-axis to consider the effect of the detection camera. The scanning of the x-axis is simulated to obtain a virtual aberration-free light sheet at the x-z plane.
5：Data Analysis Methods:
The effects of aberrations on the thickness and intensity of the two-photon light sheet are evaluated using new indexes: the ratio between aberrated thickness and aberration-free thickness (TR) and the ratio of aberrated intensity to aberration-free intensity (IR).
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