Sensitivity and Specificity of Multiphoton Laser Tomography for In Vivo and Ex Vivo Diagnosis of Malignant Melanoma

Abstract

The incidence of malignant melanoma has shown a dramatic increase over the past three decades. Patient outcome and curability depend on early diagnosis. In vivo multiphoton laser tomography represents a recently developed diagnostic tool that allows non-invasive tissue imaging. We aim to demonstrate the application of multiphoton laser tomography for the in vivo differentiation and diagnosis of melanoma. Laser radiation in the near infrared spectrum was used to image endogenous fluorophores by multiphoton excitation. Eighty-three melanocytic skin lesions have been investigated. The results showed distinct morphological differences in melanoma compared with melanocytic nevi. In particular, six characteristic features of malignant melanoma were specified and statistically evaluated. Sensitivity values up to 95% (range: 71-95%) and specificity values up to 97% (range: 69-97%) were achieved for diagnostic classification. Logistic regression analysis was performed to identify the most significant diagnostic criteria. We found that architectural disarray of the epidermis, poorly defined keratinocyte cell borders as well as the presence of pleomorphic or dendritic cells were of prime importance. By means of this procedure accuracy values up to 97% were reached. These findings underline the potential applicability of multiphoton laser tomography in melanoma diagnosis of melanocytic skin lesions.

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.…”

Three‐dimensional multiphoton/optical coherence tomography for diagnostic applications in dermatology

“…Meanwhile, BCC displayed features such as plug-like signal intense structures and signal-free cavities in OCT [20,48]. Melanoma was characterized in MPT by features such as large intercellular distances, pleomorphic cells and the presence of dendritic cells [12]. In OCT, malignant melanoma was differentiated from benign lesions by its marked architectural disarray and presence of vertical, icicle-shaped structures [49].…”

“…The reduced coenzymes, NADH and NAD(P)H, located mainly in mitochondria, are fluorescent only when reduced. These mitochondrial coenzymes have been widely used for analyzing cellular metabolic state [12,13]. On the contrary, flavin adenine dinucleotide (FAD) fluoresces in its oxidative state.…”

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.…”