Ciprocity

Confocal fluorescence microscopy is the gold standard for imaging 3D samples such as organoids, spheroids or large tissue samples but the thickness of these samples leads to light absorption and scattering. This makes it hard to inspect images visually as we cannot differentiate between a lack of marker (e.g. a necrotic core) or light loss. Our physics-based innovation measures the amount of absorption and scattering of both excitation and emission light. Using these measurements we can calculate the transmittance and correct for absorption and scattering in the sample to obtain a measure of the fluorophore concentration that is unaffected by the sample thickness, or which plane is being imaged. Our solution allows, for the first time using confocal microscopy, measuring spatial differences in fluorophore concentration within and between organoids. Using this, we understand whether a lack of signal in the center of an organoid is due to a lack of fluorescent marker or simply an artifact from light not reaching the center of the organoid.