Experimental decorrelation requirement for robust estimation of fluorophores using multiple-wavelength excitation fluorescence spectroscopy

Abstract

Protoporphyrin IX (PpIX) is a fluorophore being currently used to localize tumoral tissues. The tissue is usually excited at one wavelength, e.g., 405 nm, and the fluorescence signal is used to estimate the amount of PpIX during surgery. However, other fluorophores (baseline) whose emission spectra are close to the one of PpIX impair the quantification of PpIX and consequently the tissue pathological status classification. An efficient multi-excitation wavelengths method, free from any a priori on the baseline shape, has been proposed to cope with this issue. This method requires decorrelated measurements in the range of PpIX emission at multiple excitation wavelengths. We investigated the influence of the source bandwith on this decorelation by comparing two experimental setups using either LED or laser diode sources. The experimental setup using laser diodes for excitation increases the decorrelation by 35.3 % compared to the one using LEDs in the spectral range of PpIX emission.