Aberration correction and complementary beam subtraction in light-sheet fluorescence microscopy

Abstract

Abstract. Significance Scanning nondiffracting Bessel beams in light-sheet fluorescence microscopy (LSFM) can expand the field of view (FOV), but side lobes of the Bessel beams produce the out-of-focus background and limit the axial resolution. Furthermore, optical aberrations degrade the image quality and affect the imaging depth. Aim To compensate wavefront distortions and enhance the imaging performance in LSFM, we propose a method that combines the adaptive optics (AO) with the complementary beam subtraction (CBS) method in LSFM, referred as AO-CBS. Approach In the AO-CBS method, the sensorless AO is employed to correct optical aberrations in LSFM, resulting in a notable enhancement of image contrast and signal intensity by 48%. Higher axial resolution is achieved by effectively removing the out-of-focus background through subtracting the two image stacks acquired by scanning the Bessel beam and its corresponding complementary beam. Results The final axial resolution is improved from 2.13±0.18  μm to 1.19±0.02  μm with AO-CBS methods. The effectiveness of AO-CBS in correcting aberrations is demonstrated through imaging of fluorescent beads, Aspergillus conidia, cleared mouse liver section, and other biological tissues. Conclusions The AO-CBS method effectively addresses key issues existing in traditional Bessel beam-based LSFM, including the sidelobe-induced out-of-focus background, the limited axial resolution, and the degraded image quality caused by optical aberrations. The successful application of this idea in LSFM can serve as a design strategy to enhance the light-sheet-based imaging systems, thereby promoting the development and the application of LSFM and providing more powerful tools for biomedical imaging.