Analytical model enabling the correction of absorption and scattering in dual-color ratiometric multiphoton microscopy of brain tissue.

SIGNIFICANCE: Multiphoton microscopy with fluorescent indicators enables the monitoring of biomarker dynamics through changes in fluorescence intensity. However, quantitative interpretation remains challenging because fluorescence signals are highly sensitive to experimental conditions. Dual-color ratiometric approaches improve quantification, yet wavelength-dependent absorption and scattering of emitted photons can still introduce significant bias. AIM: This study aimed to develop a correction method for absorption and scattering effects to achieve unbiased dual-color ratiometric measurements in multiphoton microscopy. APPROACH: We developed an analytical model that incorporates the spectral optical properties of biological tissues and fluorophores. The model was applied to the rodent cerebral cortex parenchyma and to the lumen of individual blood vessels. Its predictions were evaluated using simulations and in vivo two-photon microscopy experiments. RESULTS: In the cerebral cortex parenchyma, absorption and scattering bias dual-color ratiometric measurements by up to ∼ 100 % at depths of approximately ∼ 400    μ m . Our model accurately describes and corrects these effects. Within the vessel lumen, however, the situation is more complex: the model effectively corrects the biases in veins, but not in arterioles. CONCLUSIONS: Overall, the proposed model enhances the accuracy of biomarker quantification in multiphoton microscopy and provides practical tools for correcting optical biases in biological tissues.