Scattering Reduction and Axial Resolution Enhancement in Light‐Sheet Fluorescence Microscopy

Light-sheet fluorescence microscopy (LSFM) provides an ideal tool for long-term observation of live specimens due to its low photodamage and fast volumetric imaging speed. The wavefront distortions in the illumination path of LSFM will reduce the intensity and broaden the light-sheet thickness, thereby degrading the image quality. We propose to use the wavefront shaping technique to reduce the scattering effect and shrink the light-sheet thickness. Scanning the refocused laser beam to generate LS improves both the fluorescence intensity and the axial resolution. The axial resolution can be further enhanced by subtracting the two images captured via double scanning the samples with the refocused beam and the uncorrected scattered beam for each slice. The axial resolution is improved from 2.2 ± 0.3 to 1.5 ± 0.2 μm across the field of view of 270 μm × 270 μm. The effectiveness of the wavefront shaping subtraction method is demonstrated by imaging fluorescent beads and Aspergillus conidiophores behind a scattering medium.