Optical Sectioning In Bright-Field Microscopy

The bright-field (BF) optical microscope is a traditional bioimaging tool that has been recently tested for depth discrimination during the evaluation of specimen morphology. However, existing approaches require dedicated instrumentation or extensive computer modeling. Now, researchers have developed a direct method for three-dimensional (3D) imaging in BF microscopy, applicable to label-free samples, where they use Köhler illumination in the coherent regime and conventional digital image processing filters to achieve optical sectioning.

The classic bright-field (BF) microscope is an indispensable tool in any biological laboratory, routinely used to evaluate cellular or tissular morphology in stained two-dimensional (2D) samples. However, BF images of phase objects display little contrast in the absence of staining.

The reconstruction of an input object from the corresponding output image constitutes a well-known inverse problem in microscopy. Deconvolution, a standard method that addresses this problem, typically analyzes images in the Fourier domain and has been successfully applied to BF in 2D. Here, the researchers introduce optical sectioning in bright-field microscopy (OSBM), a 3D image reconstruction method that analyzes high-contrast BF images of phase objects in real space.

By visualizing fungal, animal tissue, and plant samples and comparing them with light-sheet fluorescence microscopy imaging, the researchers demonstrate the accuracy and applicability of the method, showing how the standard microscope is an effective 3D imaging device.

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