203 / 2024-07-11 21:56:54

Correction of uneven illumination in microscopic image based on fluorescent microscope slides

Optical Field Inhomogeneity; Vignetting Phenomenon; Imaging Quality; FRET Analysis; Quantitative Fluorescence Measurements

Quantitative fluorescence resonance energy transfer microscopy (FRET) is a widely used imaging method to study biomolecular activity in living cells. However, the effect of optical field inhomogeneity on FRET is unknown, and theoretical analysis and experimental results show that optical field inhomogeneity affects the accuracy of quantitative FRET. In order to improve the accuracy and reliability of E-FRET, we developed a flat-field correction algorithm based on the fluorescent microscope slides. This method not only effectively improves the imaging quality by correcting the vignetting phenomenon in fluorescence imaging, but also improves the accuracy of FRET analysis. This flat-field correction algorithm can be used in any fluorescence microscope to obtain high-throughput high-quality images and quantitative fluorescence measurements.