Pixelation with Concentration‐Encoded Effective Photons for Quantitative Molecular Optical Sectioning Microscopy

Irreproducibility in molecular optical sectioning microscopy has hindered the transformation of acquired digital images from qualitative descriptions to quantitative data. Although numerous tools, metrics, and phantoms have been developed, accurate quantitative comparisons of data from different microscopy systems with diverse acquisition conditions remains a challenge. Here, they develop a simple tool based on an absolute measurement of bulk fluorophore solutions with related Poisson photon statistics, to overcome this obstacle is developed. Demonstrated in a prototypical multiphoton microscope, this tool unifies the unit of pixelated measurement to enable objective comparison of imaging performance across different modalities, microscopes, components/settings, and molecular targets. The application of this tool in live specimens identifies an attractive methodology for quantitative imaging, which rapidly acquires low signal‐to‐noise frames with either gentle illumination or low‐concentration fluorescence labeling. Irreproducibility in molecular optical sectioning microscopy hinders converting digital images into quantitative data. Existing tools and metrics struggle with data comparisons across diverse systems. A simple tool (PCEP) based on absolute measurements of bulk fluorophore solutions and Poisson photon statistics is presented. Demonstrated in a multiphoton microscope, it standardizes pixel measurementsand enables objective comparisons across various imaging conditions and systems.