Non‐Uniform Illumination Correction for Quantitative Fluorescence Imaging

Illumination uniformity is essential for quantitative analysis in fluorescence microscopy. However, fewer images or sparse intensity information can compromise correction quality of retrospective methods, whereas traditional prospective methods necessitate recalibration whenever imaging conditions change. In this paper, we theoretically for the first time analyze the impact of non-uniform illumination on quantitative fluorescence resonance energy transfer (FRET), which is also demonstrated experimentally. We proposed an illumination correction method based on fluorescent microscope slides, termed Systematic Uniform Fluorescence Image Correction (SUFIC). And we evaluated SUFIC using the images of fluorescent microspheres, Argo-HM slide, and live cells expressing FRET construct. After SUFIC correction, the field uniformity of the "field of rings" image in Argo-HM slide increased from 27.45% to 65.30%. The signal-to-background ratios of the FRET three-channel images increased by 12.3%, 7.9%, and 20.9%. Compared to the CIDRE and BaSiC methods, which require 100 input images for prospective correction, SUFIC requires only three reference images at minimum. In summary, SUFIC is a highly efficient prospective illumination correction method for quantitative microscopic imaging.