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Among many LSFM designs, we found that the Axially Swept Light-sheet Microscope ASLM and its descendants demonstrated outstanding potential for high-performance, large-FOV imaging. Therefore, we aim to develop a high-performance LSFM with a simple design.
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Still, the high design complexity and low availability of these advanced systems limit their use in a broader range of biomedical research applications.Ī simpler LSFM design with good imaging performance would greatly benefit the biomedical research community. For instance, scanned non-Gaussian beams such as Bessel beams and structured light demonstrated promising capability for generating a thinner sheet over a wider FOV in modern LSFM designs. Optical engineers have developed various methods to improve both axial resolution and FOV at the same time. Therefore, a simple Gaussian beam-based light-sheet microscope often suffers from either limited axial resolution or reduced FOV. While the beam waist determines the light-sheet thickness and the optical sectioning resolution, the Rayleigh length determines the effective FOV in the direction of the illumination propagation. A physical tradeoff exists between the beam waist width and the Rayleigh length (or confocal parameter) with a focused Gaussian beam. The optical sectioning resolution and FOV of the system are intrinsically limited.Ī fundamental challenge for designing a simple Gaussian-based LSFM is simultaneously improving the axial resolution and the FOV. Besides, simple customized LSFM often uses Gaussian-beam-based illumination using cylindrical lens focusing. It is still challenging for biomedical researchers to start a customized light-sheet project with limited expertise in optical engineering. However, compared to commercial systems, customized optical systems often require extra resources to build and maintain. Several research groups have also developed open-hardware LSFM projects such as OpenSPIM, allowing researchers to build their LSFM without extensive knowledge of optical engineering. While some LSFM systems are commercially available, it is common for research groups to build customized light-sheet microscopes due to lower costs and more design flexibility. Since then, LSFM has rapidly evolved with growing needs for cutting-edge imaging performance (e.g., high speed, large FOV with high resolution, etc.) in biomedical research. further demonstrated the efficacy of LSFM on zebrafish and drosophila melanogaster, achieving up to 6-µm lateral resolution and 6-20-µm optical sectioning in a 2.5-mm FOV.
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The light sheet was generated by focusing a collimated laser beam using a cylindrical lens, which enabled 3D imaging using a digital microscope with a 10-µm lateral resolution, a 26-µm axial resolution, and a 1.5-mm field of view (FOV). in 1992 as orthogonal-plane fluorescence optical sectioning (OPFOS) to create a 3D image of an optically cleared guinea pig cochlea. The modern LSFM was first introduced by Voie et al. Light scattered from colloidal gold was observed using a conventional wide-field microscope. The light sheet was generated by projecting sunlight through a rectangular slit aperture using a finite conjugate microscope objective. invented the first light-sheet microscope. This configuration provides the optical sectioning advantage of confocal microscopes and the speed advantage of camera-based systems. Instead of using transmissive or epi-illumination, a sheet of light is used to illuminate a thin plane inside the sample.
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The light-sheet microscope is an upgrade of the fluorescence wide-field microscope. With the emerging need for 3D imaging of whole-mount tissue samples, light-sheet fluorescence microscopy (LSFM, also called selective/single plane illumination microscopy (SPIM)) has become a popular technique in biomedical research.