Abstract
.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.
Highlights
In a recent study by our group, we observed that morphological features characteristic to melanoma metastasis can be discerned in lymph node (LN) tissues using multiphoton laser scanning microscopy (MPM),[25] but improved contrast would be required to facilitate diagnostics
The acquired lifetimes in both spectral channels most likely correspond to signals from NADH and flavin adenine dinucleotide (FAD), but they are difficult to separate due to spectral cross-talk.[29]
Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Biomedical-Optics on 08 Nov 2021 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use distribution >2000 ps in channel 2 [Fig. 2(f)] most likely corresponds to FAD, while the lifetimes in the range 300 to 2000 ps probably originate from NADH,[29,30,31] which is observed in channel 1
Summary
Recent advancements in optical techniques such as multiphoton laser scanning microscopy (MPM),[1,2] confocal reflectance microscopy,[3] and optical coherence tomography[4] have strengthened ongoing research targeting the development of optical tools that enable intravital tissue biopsy.[5,6] MPM has been explored in the field of skin cancer research for the last few years and has demonstrated potential for use in facilitating diagnostics of primary malignant melanoma.[7,8] Fluorescence lifetime imaging microscopy (FLIM) is a time-correlated fluorescence technique that generates images based on the fluorescence lifetime rather than intensity.[9,10,11] FLIM has been applied to mapping the local environment of fluorophores such as ion concentration and pHJournal of Biomedical OpticsJuly 2020 Vol 25(7)James et al.: Report on fluorescence lifetime imaging using multiphoton laser scanning microscopy. . .sensing in various samples, since fluorescence lifetime depends on the molecular environment.[12,13] FLIM can enable studies of metabolic activities in cellular environments by identifying the bound and unbound forms of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), two of the most common metabolic markers.[14,15]According to the Global Burden of Disease Study in 2015,16 malignant melanoma processes a global incidence of 0.3 million, making it one of the most widely spreading skin cancers across the world. Recent advancements in optical techniques such as multiphoton laser scanning microscopy (MPM),[1,2] confocal reflectance microscopy,[3] and optical coherence tomography[4] have strengthened ongoing research targeting the development of optical tools that enable intravital tissue biopsy.[5,6] MPM has been explored in the field of skin cancer research for the last few years and has demonstrated potential for use in facilitating diagnostics of primary malignant melanoma.[7,8] Fluorescence lifetime imaging microscopy (FLIM) is a time-correlated fluorescence technique that generates images based on the fluorescence lifetime rather than intensity.[9,10,11] FLIM has been applied to mapping the local environment of fluorophores such as ion concentration and pH. We propose employing MPM combined with FLIM for investigation of primary melanoma lesions.[8,26,27]
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