SignificanceLight-sheet fluorescence microscopy (LSFM) has emerged as a powerful and versatile imaging technique renowned for its remarkable features, including high-speed 3D tomography, minimal photobleaching, and low phototoxicity. The interference light-sheet fluorescence microscope, with its larger field of view (FOV) and more uniform axial resolution, possesses significant potential for a wide range of applications in biology and medicine.AimThe aim of this study is to investigate the interference behavior among multiple light sheets (LSs) in LSFM and optimize the FOV and resolution of the light-sheet fluorescence microscope.ApproachWe conducted a detailed investigation of the interference effects among LSs through theoretical derivation and numerical simulations, aiming to find optimal parameters. Subsequently, we constructed a customized system of multi-LSFM that incorporates both interference light sheets (ILS) and noninterference light-sheet configurations. We performed beam imaging and microsphere imaging tests to evaluate the FOV and axial resolution of these systems.ResultsUsing our custom-designed light-sheet fluorescence microscope, we captured the intensity distribution profiles of both interference and noninterference light sheets (NILS). Additionally, we conducted imaging tests on microspheres to assess their imaging outcomes. The ILS not only exhibits a larger FOV compared to the NILS but also demonstrates a more uniform axial resolution.ConclusionsBy effectively modulating the interference among multiple LSs, it is possible to optimize the intensity distribution of the LSs, expand the FOV, and achieve a more uniform axial resolution.
We design a real-time laser stimulus system for laser confocal scanning microscope. By introducing the FPGA and AOM to achieve high speed modulation of a scanning laser, we can adjust the laser lighting area freely. For reducing the size of the optical path, we use MEMS-mirror instead of traditional fast and slow axis mirrors. The size of MEMS-mirror is 1.5 mm diameter and the scanning frequencies are set 16 kHz and 12 Hz at the fast and slow axis, respectively. Our system is capable of delivering stabilized large stimulus pattern (up to 500 x 500 pixels) to the biological tissues.
We demonstrate a home-made two-photon laser scanning microscopy (TPLSM) with a light stimulus system. In this system, the femtosecond pulses are produced by a picosecond fiber laser with pulse width compression. A laser diode serves as stimulus, which modulated by an AOM and coupled into the same light path of a femtosecond laser. The control signal of AOM and trigger signal of two scanning mirrors are synchronized by a FPGA board. With modulating the intensity of CW laser at precise point in one scanning frame, any pattern of light stimulus can be delivered to the sample in real time.
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