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Quality control in molecular optical sectioning microscopy is indispensablefor transforming acquired digital images from qualitative descriptions toquantitative data. Although numerous tools, metrics, and phantoms have beendeveloped, accurate quantitative comparisons of data from different microscopysystems with diverse acquisition conditions remains a challenge. Here, wedevelop a simple tool based on an absolute measurement of bulk fluorophoresolutions with related Poisson photon statistics, to overcome this obstacle.Demonstrated in a prototypical multiphoton microscope, our tool unifies theunit of pixelated measurement to enable objective comparison of imagingperformance across different modalities, microscopes, components/settings, andmolecular targets. The application of this tool in live specimens identifies anattractive methodology for quantitative imaging, which rapidly acquires lowsignal-to-noise frames with either gentle illumination or low-concentrationfluorescence labeling.

Pixelation with concentration-encoded effective photons for molecular optical sectioning microscopy