Exploring the Interplay of Wavelength, Quantum Yield, and Penetration Depth in In Vivo Fluorescence Imaging.

The intricate interplay between the irradiation wavelength, the fluorophore quantum yield (QY) and penetration depth profoundly influences the efficacy of in vivo fluorescence imaging in various applications. Understanding the complex behavior of fluorescence in vivo, specifically how variations in wavelength affect the QY of commonly used dyes and the depth of imaging is crucial for optimizing fluorescence imaging techniques, as it directly impacts the accuracy and efficiency of imaging in biological tissues. In our study, we explore these dynamics through Monte Carlo simulations conducted under conditions reflective of wide-field fluorescence imaging, examining how variations in wavelength impact the dye's QY and depth of imaging, and consequently, the fluorescence behavior. A transition in the exponential decay of the emission depth exponent is observed around the 500-600 nm range, indicating varying degrees of influence of depth on the fluorescence emission. The analysis of the fluorophore's QY reveals wavelength-dependent variations, with the most significant impact observed in the 600-700 nm range. Moreover, we continued our investigation to explore multiplexing, unveiling insights into the spacing between identical spots in multiplexing images across various depths and wavelengths.