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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. 

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.