  Quality control in molecular optical sectioning microscopy is indispensable
for transforming 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, we
develop a simple tool based on an absolute measurement of bulk fluorophore
solutions with related Poisson photon statistics, to overcome this obstacle.
Demonstrated in a prototypical multiphoton microscope, our 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.
