Dark-based Optical Sectioning assists Background Removal in Fluorescence Microscopy

Cao, R.; Li, Y.; Wang, W.; Zhang, G.; Wang, G.; Sun, Y.; Renc, W.; Sun, J.; Hou, Y.; Xu, X.; Hu, J.; Lu, Y.; Li, C.; Wu, J.; Li, M.; Qu, J.; Xi, P.

doi:10.1101/2024.03.02.578598

bioRxiv · 10.1101/2024.03.02.578598

Dark-based Optical Sectioning assists Background Removal in Fluorescence Microscopy

Cao, R.·Li, Y.·Wang, W.·Zhang, G.·Wang, G.·Sun, Y.·Renc, W.·Sun, J.·Hou, Y.·Xu, X.·Hu, J.·Lu, Y.·Li, C.·Wu, J.·Li, M.·Qu, J.·Xi, P.

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Abstract

A fundamental challenge in fluorescence microscopy is the defocused background caused by scattering light, optical aberration, or limited axial resolution. Severe defocus backgrounds will submerge the in-focus information and cause artifacts in the following processing. Here, we leverage a priori knowledge about dark channels of biological structures and dual frequency separation to develop a single-frame defocus removal algorithm. It stably improves the signal-to-background ratio and structural similarity index measure of images by approximately 10-fold, and recovers in-focus signal with 85% accuracy, even when the defocus background is 50 times larger than in-focus information. Our Dark-based optical sectioning approach (Dark sectioning) is fully compatible with various microscopy techniques, such as wide-filed microscopy, polarized microscopy, laser-scanning / spinning-disk confocal microscopy, stimulated emission depletion microscopy, lightsheet microscopy, and light-field microscopy. It also complements reconstruction or processing algorithms such as deconvolution, structure illumination microscopy, and super-resolution optical fluctuation imaging.

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Cao, R., Li, Y., Wang, W., Zhang, G., Wang, G., Sun, Y., Renc, W., Sun, J., Hou, Y., Xu, X., Hu, J., Lu, Y., Li, C., Wu, J., Li, M., Qu, J., Xi, P.. 2024-03-03. Dark-based Optical Sectioning assists Background Removal in Fluorescence Microscopy. https://doi.org/10.1101/2024.03.02.578598

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