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Optical microscopy has vastly expanded the frontiers of structural and functional biology, due to the non-invasive probing of dynamic volumes in vivo. However, traditional widefield microscopy illuminating the entire field of view (FOV) is adversely affected by out-of-focus light scatter. Consequently, standard upright or inverted microscopes are inept in sampling diffraction-limited volumes smaller than the optical system’s point spread function (PSF). Over the last few decades, several planar and structured (sinusoidal) illumination modalities have offered unprecedented access to sub-cellular organelles and 4D (3D + time) image acquisition. Furthermore, these optical sectioning systems remain unaffected by the size of biological samples, providing high signal-to-noise (SNR) ratios for objective lenses (OLs) with long working distances (WDs). This review aims to guide biologists regarding planar illumination strategies, capable of harnessing sub-micron spatial resolution with a millimeter depth of penetration. 

Background: In the study of early cardiac development, it is essential to acquire accurate volume changes of the heart chambers. Although advanced imaging techniques, such as light-sheet fluorescent microscopy (LSFM), provide an accurate procedure for analyzing the heart structure, rapid, and robust segmentation is required to reduce laborious time and accurately quantify developmental cardiac mechanics. Methods: The traditional biomedical analysis involving segmentation of the intracardiac volume occurs manually, presenting bottlenecks due to enormous data volume at high axial resolution. Our advanced deep-learning techniques provide a robust method to segment the volume within a few minutes. Our U-net-based segmentation adopted manually segmented intracardiac volume changes as training data and automatically produced the other LSFM zebrafish cardiac motion images. Results: Three cardiac cycles from 2 to 5 days postfertilization (dpf) were successfully segmented by our U-net-based network providing volume changes over time. In addition to understanding each of the two chambers' cardiac function, the ventricle and atrium were separated by 3D erode morphology methods. Therefore, cardiac mechanical properties were measured rapidly and demonstrated incremental volume changes of both chambers separately. Interestingly, stroke volume (SV) remains similar in the atrium while that of the ventricle increases SV gradually. Conclusion: Our U-net-based segmentation provides a delicate method to segment the intricate inner volume of the zebrafish heart during development, thus providing an accurate, robust, and efficient algorithm to accelerate cardiac research by bypassing the labor-intensive task as well as improving the consistency in the results.