Integration of stimulated emission depletion (STED) with digital scanned light-sheet microscopy (DSLM) using a Bessel beam achieves fast, high-resolution and large field of view (FOV) imaging. Here, we report on a combination method of DSLM and STED by using a Bessel beam for excitation and a hollow Gaussian beam for depletion. Owing to the advantages of confined axial distribution around the focal plane for the hollow Gaussian depletion beam, those fluorescence signals from the periphery of the Bessel excitation pattern are depleted by stimulated emission. A super-resolved light sheet with suppressed side lobes is achieved. The numerical simulation results show that the use of the Bessel beam for the excitation and the hollow Gaussian beam for the STED results in a DSLM system with enhanced performance of the axial resolution and image contrast over a moderately large FOV.

In light-sheet fluorescence microscopy (LSFM), using Gaussian beams for light-sheet generation results in a trade-off between the thickness and the field of view (FOV). Here we present a theoretical analysis of using spherical aberration to enlarge the FOV while keeping the light-sheet thickness small. Such spherical aberration can arise when focusing beams through an interface between materials of mismatched refractive indices. The depth-of-focus extension of the Gaussian beam is achieved when using air objectives to focus light into the samples dipped in the immersion medium with a higher refractive index. By scanning this elongated beam, a thin light sheet with a wide FOV can be used for LSFM imaging. Meanwhile, the accompanied sidelobes with the spherical aberrated light sheet, which are mainly distributed in the rear part of the light sheet, are also discussed. Simulation results show that an extended FOV of 64.4 µ m is possible for an objective lens of N A = 0.3 , which is about 5 times that of the unaberrated case. For such an extended FOV, a comparatively thin thickness of 1.38 µ m as well as the first sidelobe about 11.1% of the peak intensity in the center are also demonstrated.

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