Significance: 5-Aminolevulinic acid (5-ALA)-based fluorescence guidance in conventional neurosurgical microscopes is limited to strongly fluorescent tumor tissue. Therefore, more sensitive, intrasurgical 5-ALA fluorescence visualization is needed.

Aim: Macroscopic fluorescence lifetime imaging (FLIM) was performed ex vivo on 5-ALA-labeled human glioma tissue through a surgical microscope to evaluate its feasibility and to compare it to fluorescence intensity imaging.

Approach: Frequency-domain FLIM was integrated into a surgical microscope, which enabled parallel wide-field white-light and fluorescence imaging. We first characterized our system and performed imaging of two samples of suspected low-grade glioma, which were compared to histopathology.

Results: Our imaging system enabled macroscopic FLIM of a 6.5  ×  6.5  mm² field of view at spatial resolutions <20  μm. A frame of 512  ×  512  pixels with a lifetime accuracy <1  ns was obtained in 65 s. Compared to conventional fluorescence imaging, FLIM considerably highlighted areas with weak 5-ALA fluorescence, which was in good agreement with histopathology.

Conclusions: Integration of macroscopic FLIM into a surgical microscope is feasible and a promising method for improved tumor delineation.

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.