Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis

Abstract:  Multiphoton excited tissue fluorescence summarises the emission of all naturally occurring endogenous fluorescent bio-molecules with their often overlapping fluorescence spectra. Common fluorescence intensity measurements could not be utilised to distinguish between different fluorophores or metabolic states. To overcome this limitation, we investigated new procedures of selective melanin imaging and spectral fluorescence lifetime imaging in combination with high resolution multiphoton laser tomography. Overall 46 melanocytic lesions of human skin were analysed. We suggested that fluorescence light, detected in such a way, may yield additional information for melanoma diagnostics. Remarkable differences in lifetime behaviour of keratinocytes in contrast to melanocytes were observed. Fluorescence lifetime distribution was found in correlation with the intracellular amount of melanin. Spectral analysis of melanoma revealed a main fluorescence peak around 470 nm in combination with an additional peak close to 550 nm throughout all epidermal layers. Excitation at 800 nm shows a selectively observable fluorescence of melanin containing cells and offers the possibility of cell classification. Procedures of selective imaging as well as spectral fluorescence lifetime imaging by means of multiphoton laser tomography support diagnostic decisions and may improve the process of non-invasive early detection of melanoma.     

In vivo characterization of minipig skin as a model for dermatological research using multiphoton microscopy

Minipig skin is one of the most widely used non-rodent animal skin models for dermatological research. A thorough characterization of minipig skin is essential for gaining deeper understanding of its structural and functional similarities with human skin. In this study, three-dimensional (3-D) in vivo images of minipig skin was obtained non-invasively using a multimodal optical imaging system capable of acquiring two-photon excited fluorescence (TPEF) and fluorescence lifetime imaging microscopy (FLIM) images simultaneously. The images of the structural features of different layers of the minipig skin were qualitatively and quantitatively compared with those of human skin. Label-free imaging of skin was possible due to the endogenous fluorescence and optical properties of various components in the skin such as keratin, nicotinamide adenine dinucleotide phosphate (NAD(P)H), melanin, elastin and collagen. This study demonstrates the capability of optical biopsy techniques, such as TPEF and FLIM, for in vivo non-invasive characterization of cellular and functional features of minipig skin, and the optical image-based similarities of this commonly utilized model of human skin. These optical imaging techniques have the potential to become promising tools in dermatological research for developing a better understanding of animal skin models, and for aiding in translational pre-clinical to clinical studies.     

A pilot study of fluorescence diagnosis of basal cell carcinoma using a digital flash light-based imaging system

Background/purpose: Fluorescence diagnosis (FD) is a promising method for the non‐invasive detection of tumor boundaries. We aimed to investigate the clinical utility of a flash light‐based fluorescence imaging system. Methods: Sixteen patients with basal cell carcinomas were included in this pilot study. FD was performed using a 20% 5‐aminolevulinic acid (ALA) cream. The FD tumor area was determined 3 h after the ALA application using a digital flash light‐based fluorescence imaging system and an image analysis system. The tumor area was also assessed by means of the clinical diagnosis (CD) based on the normal colored picture. The tumors were excised following the FD procedure, but according to the clinically diagnosed lesional area. Results: Though there was a very strong correlation between the tumor areas assessed by FD and CD, the mean tumor area that was visualized by FD was significantly smaller than the tumor area determined by CD. The mean±SD FD/CD ratio was 0.78±0.11 in total. In 81.3% of cases, complete tumor excision was achieved. Conclusion: This pilot study indicates that the FD technology may be less sensitive than CD, and refinements of the technique along with larger systematic trials on the sensitivity and specificity of FD are required.