Significance: Sentinel lymph node (SLN) biopsy is an important method for metastasis staging in, e.g., patients with malignant melanoma. Tools enabling prompt histopathological analysis are expected to facilitate diagnostics; optical technologies are explored for this purpose.

Aim: The objective of this exploratory study was to investigate the potential of adopting multiphoton laser scanning microscopy (MPM) together with fluorescence lifetime analysis (FLIM) for the examination of lymph node (LN) tissue ex vivo.

Approach: Five LN tissue samples (three metastasis positive and two negative) were acquired from a biobank comprising tissues from melanoma patients. Tissues were deparaffinized and subjected to MPM-FLIM using an experimental MPM set-up equipped with a time correlated single photon counting module enabling FLIM.

Results: The data confirm that morphological features similar to conventional histology were observed. In addition, FLIM analysis revealed elevated morphological contrast, particularly for discriminating between metastatic cells, lymphocytes, and erythrocytes.

Conclusions: Taken together, the results from this investigation show promise for adopting MPM-FLIM in the context of SLN diagnostics and encourage further translational studies on fresh tissue samples.