Accurate and rapid evaluation of lymph node metastasis is required in tumor staging and the decision of treatment strategy.
General intraoperative pathological evaluation, however, takes at least a few tens of minutes or longer for metastasis
diagnosis. 5-aminolevulinic acid (5-ALA)-based fluorescence diagnosis is a solution for accurate and ultrarapid diagnosis
of malignant lesions. 5-ALA-based diagnosis evaluates fluorescence intensity of a fluorescent metabolite of 5-ALA,
protoporphyrin IX (PPIX); however, the fluorescence of PPIX is often affected by autofluorescence of tissue
chromophores, such as collagen and flavins. To enhance the accuracy of the diagnosis of malignant lesions based on the
PPIX fluorescence, elimination of the autofluroescence is required. In this study, we proposed and experimentally
demonstrated background-free PPIX fluorescence estimation method by simplified and optimized multispectral imaging.
To realize background-free PPIX fluorescence estimation, we computationally optimized observation wavelength regions
in terms of minimizing prediction error of PPIX fluorescence intensity in the presence of typical chromophores, collagen
and flavins. We verified the fundamental detection capability of our method by using known-chemical mixtures.
Furthermore, we applied our method to lymph node metastasis, and successfully realized background-free
histopathological evaluation of metastatic lesions of lymph node metastasis. Our results confirmed the potential of the
background-free estimation method of PPIX fluorescence for 5-ALA-based fluorescence diagnosis of malignant lesions,
and we expect this method to be beneficial for intraoperative and rapid cancer diagnosis.