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

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: 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.