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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. 

Abstract BackgroundThe developing zebrafish ventricle generates higher intraventricular pressure (IVP) with increasing stroke volume and cardiac output at different developmental stages to meet the metabolic demands of the rapidly growing embryo (Salehin et al. Ann Biomed Eng, 2021;49(9): 2080‐2093). To understand the changing role of the developing embryonic heart, we studied its biomechanical characteristics during in vivo cardiac cycles. By combining changes in wall strains and IVP measurements, we assessed ventricular wall stiffness during diastolic filling and the ensuing systolic IVP‐generation capacity during 3‐, 4‐, and 5‐day post fertilization (dpf). We further examined the anisotropy of wall deformation, in different regions within the ventricle, throughout a complete cardiac cycle. ResultsWe found the ventricular walls grow increasingly stiff during diastolic filling with a corresponding increase in IVP‐generation capacity from 3‐ to 4‐ and 5‐dpf groups. In addition, we found the corresponding increasing level of anisotropic wall deformation through cardiac cycles that favor the latitudinal direction, with the most pronounced found in the equatorial region of the ventricle. ConclusionsFrom 3‐ to 4‐ and 5‐dpf groups, the ventricular wall myocardium undergoes increasing level of anisotropic deformation. This, in combination with the increasing wall stiffness and IVP‐generation capacity, allows the developing heart to effectively pump blood to meet the rapidly growing embryo's needs. 

Light-sheet fluorescence microscopy (LSFM) provides access to multi-dimensional and multi-scale in vivo imaging of animal models with highly coherent volumetric reconstruction of the tissue morphology, via a focused laser light sheet. The orthogonal illumination and detection LSFM pathways account for minimal photobleaching and deep tissue optical sectioning through different perspective views. Although rotation of the sample and deep tissue scanning constitutes major advantages of LSFM, images may suffer from intrinsic problems within the modality, such as light mismatch of refractive indices between the sample and mounting media and varying quantum efficiency across different depths. To overcome these challenges, we hereby introduce an illumination correction technique integrated with depth detail amelioration to achieve symmetric contrast in large field-of-view images acquired using a low power objective lens. Due to an increase in angular dispersion of emitted light flux with the depth, we combined the dehazing algorithm with morphological operations to enhance poorly separated overlapping structures with subdued intensity. The proposed method was tested on different LSFM modalities to illustrate its applicability on correcting anisotropic illumination affecting the volumetric reconstruction of the fluorescently tagged region of interest.