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.Key words: melanoma -melanocytic skin lesions -multiphoton laser tomography -selective melanin imaging -spectral fluorescence lifetime imaging Please cite this paper as: Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis.

In vivo non‐invasive monitoring of collagen remodelling by two‐photon microscopy after micro‐ablative fractional laser resurfacing

“…In conclusion, for the first time non-linear microscopy was used in vivo with the final goal of monitoring a laser-based treatment. On the basis of the results described here, and of several other successful dermatological applications experienced in vivo by means of non-linear microscopy in recent years [35][36][37][38][58][59][60], we believe that in the near future non-linear laser scanning microscopy will find a stable place in the clinical dermatological setting.…”

“…Among them, laser scanning confocal microscopy [15][16][17][18] offers an optical solution for in vivo skin imaging, and it has already been used for monitoring post-treatment wound healing related to laser fractional resurfacing [19]. A more powerful imaging modality is represented by two-photon excited fluorescence (TPF) microscopy [20], which is a high-resolution laser scanning imaging technique enabling deep optical imaging of tissues, as demonstrated by studies performed on ex vivo tissue samples [21][22][23][24][25], fresh biopsies [26][27][28][29][30][31], and also in vivo on both animals [32] and humans [33][34][35][36][37][38]. Additional morphological information can be provided by second-harmonic generation (SHG) microscopy [39][40][41][42][43][44][45][46][47][48][49], which can be combined with TPF microscopy using the same laser source.…”

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

“…The ageing skin of young and old people show a significant difference with the semi-quantitative evaluation of dermal collagen and elastin fibre network assessed in vivo by the multiphoton excitation imaging (Koehler et al, 2006). Procedures of selective imaging as well as spectral fluorescence lifetime analysis by means of multiphoton laser tomography support diagnostic decisions and may improve the process of non-invasive early detection of melanoma (König et al, 2007;Dimitrow et al, 2009) (Fig. 9) and non-melanoma skin cancer (Paoli et al, 2008).…”

“…Fluorescence spectra of healthy skin, melanocytic nevus and melanoma were detected in different epidermal layers. (Reproduced from Dimitrow et al, 2009. The Wiley publisher is acknowledged.)…”

Clinical application of multiphoton tomography in combination with high‐frequency ultrasound for evaluation of skin diseases

“…The MPT resolution is better than the optical resolution of the microscopes used by pathologists (VIS, NA < 1) to analyze the HE-stained 7 mm thick vertical sections obtained from biopsies (gold standard). Currently, 15 MPT systems are operating in Europe, Asia, and Australia [13] for the early detection of melanoma [14,15] and other skin diseases [16], to track in vivo sunscreen nanoparticles [17] and pharmaceutical compounds as well as to evaluate skin aging [18][19][20][21][22], and the efficiency of anti-aging products [23]. Ultrasonic images cover larger areas and provide information from deeper tissue areas than MPT.…”