Light-sheet microscopy has become a valuable tool for the three-dimensional imaging of biological samples, offering high resolution, fast imaging speeds, and minimal photodamage. Two-photon excitation further enhances its capabilities by providing deeper tissue penetration and reduced phototoxicity. However, conventional designs with orthogonal illumination and detection geometry often restrict the flexibility required for imaging large or diverse samples that require varying immersion media. To address the limitations of conventional systems for seamless imaging of both living organisms and cleared tissues, we developed an upright two-photon Bessel beam light-sheet microscope. Equipped with a middlerange magnification multi-immersion objective lens (16x, NA 0.6), the microscope accommodates refractive indices ranging from 1.33 to 1.51. Built on a previously established two-photon Bessel beam illumination system, this microscope maintains a large field of view while achieving a cellular resolution. We demonstrated its versatility by applying it to live specimen imaging and cleared tissue observation. To image optically cleared tissues, we implemented a method in which samples were immersed in a clearing agent and observed using coverslips. This enabled the high-speed, large-scale imaging of human breast cancer tissues and mouse brain slices. The adaptability of the microscope to different immersion media and its compatibility with coverslips offers significant flexibility for sample mounting, and this supports the imaging of large samples. By combining a laterally unconstrained configuration with two-photon excitation, this system advances the applicability of light-sheet microscopy in a wide range of biological research fields.

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