Phasor-FLIM and SHG imaging for quantitative analysis of lung cancer autofluorescence.

Histopathology using hematoxylin and eosin (H&E) staining remains the gold standard for tumor diagnosis. However, extracting quantitative data from stained slides is challenging, limiting the ability to obtain objective biomarkers for disease progression. Tissue autofluorescence provides an alternative by exploiting endogenous fluorophores, such as collagen, elastin, and NAD(P)H, which provide optical signatures of tissue pathology. Fluorescence Lifetime Imaging Microscopy (FLIM), when combined with phasor-based analysis, enables quantitative, fit-free assessment of metabolic and structural changes in tissues, simplifying data interpretation and enhancing diagnostic accuracy. In this study, we applied a phasor-FLIM approach to systematically analyze two distinct histotypes of non-small cell lung cancer (NSCLC): adenocarcinoma (ADC) and squamous cell carcinoma (SQC). Our findings revealed significant elastin deposition (elastosis) in tumor tissues. By integrating FLIM with second harmonic generation (SHG) imaging, we characterized the fiber compositions in healthy versus tumor tissues, distinguishing between collagen and elastin autofluorescence signatures. This combined imaging strategy allowed for precise discrimination of tumor regions in unstained biopsy sections, demonstrating the potential of autofluorescence-based techniques for enhanced cancer diagnostics. These results highlight the advantages of FLIM and phasor analysis in providing quantitative insights into tumor microenvironments, facilitating the histopathological assessments in unstained tissue slices.