Zebrafish fluorescence imaging platform based on Bessel light sheet illumination

We developed a three-dimensional (3D) zebrafish fluorescence imaging platform based on Bessel light sheet fluorescence microscopy (LSFM). During the 3D imaging process, the excitation light sheet remains static and the axial scanning is realized by moving the sample with one motorized positioning stage. To solve the defocusing problem caused by the optical path length change in 3D imaging, an electrically tunable lens (ETL) is adopted in the detection optical path. An auto-refocusing method that considers the sample structural anisotropy and has no limitation on the mathematical form of signals added to the ETL is designed. The results show that ETL can provide a satisfactory refocusing effect using detection objectives with a low numerical aperture (NA). In addition, the effects of the ETL on the system magnification and resolution are explored. A magnification calibration method is devised to refine the precision of the volume synthesis. The system design also facilitates the recording of ambient noise, which can help improve image quality with simple background image subtraction. This hardware-based background elimination method is compared with several state-of-the-art fluorescence image denoising algorithms, and the comparison results verified the high performance of this method. The imaging results of live zebrafish lymphatic and vascular structures, as well as blood flow, prove the reliability of this platform without necessitating further image deconvolution.