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

The first-ever application of high-frequency ultrasound combined with multiphoton tomography (MPT) and dermoscopy in a clinical trial is reported. 47 patients with different dermatoses such as benign and malign skin cancers, connective tissue diseases, inflammatory skin diseases, and autoimmune bullous skin diseases have been investigated with (i) state-of-the-art and highly sophisticated ultrasound systems for dermatology, (ii) the femtosecond laser multiphoton tomograph and (iii) dermoscopes. Dermoscopy provides two-dimensional color images of the skin surface with a magnification up to 70 x. Depending on the ultrasonic frequencies from 7.5 MHz to 100 MHz, the signal depth varies from about 1 mm to 80 mm. Vertical ultrasound wide-field images provide fast information on depth and volume of the lesion. The 100 MHz ultrasound allows imaging with resolutions down to 16 µm (axial) and 32 µm (lateral). Multiphoton tomography provides 0.36 x 0.36 x 0.001 mm³ horizontal optical sections of a particular region of interest with submicron resolution down to 200 µm tissue depth. The autofluorescence of mitochondrial coenzymes, keratin, melanin, and elastin as well as the network of collagen structures can be imaged. The combination of ultrasound and MPT opens novel synergistic possibilities in diagnostics of skin diseases with a special focus on the early detection of skin cancer as well as the evaluation of treatments.

Multiphoton tissue imaging using high-NA microendoscopes and flexible scan heads for clinical studies and small animal research.

Multiphoton tomographs based on femtosecond laser and GRIN lens technology in combination with flexible scan heads have been developed for clinical high-resolution tissue imaging and small animal research. The novel tissue tomograph possesses a 0.5 m long flexible mirror arm in combination with piezodriven focusing optics and multiple single photon counting PMT detectors. The photodetectors are in particular useful to obtain information on the extracellular matrix by the simultaneous measurement of the two-photon autofluorescence of elastin as well as the second harmonic generation of collagen. A major application is the in vivo determination of the skin age index. The rigid two-photon microendoscope with a high numerical aperture of 0.8 is based on a combination of silver-doped 1.0 mm rod-shaped GRIN lens with a hemispheric front optics. It was used in combination with the multiphoton tomograph for clinical studies as well as for inner organ and eye imaging of small animals.

MEL4B3, a novel mRNA is induced in skin tumors and regulated by TGF-beta and pro-inflammatory cytokines.

Tumor-stroma interactions play a decisive role in the growth and metastasis of solid tumors, and involve signalling either by soluble mediators or direct cell-cell interaction. Here, we report the isolation and characterisation of a novel cDNA (MEL4B3), which is induced in cultured dermal fibroblasts exposed to supernatants of melanoma cell lines. MEL4B3 shares high homology with two predicted cDNA sequences for which no activity has so far been described. In situ hybridisation revealed the expression of MEL4B3 in malignant melanoma increasing with tumor depth; in basal cell carcinoma and in squamous cell carcinoma. MEL4B3 was barely detectable in normal skin or non-malignant melanocytic naevi. Furthermore, MEL4B3 was expressed at high level in the epidermis of psoriatic skin. In vitro, the expression of MEL4B3 was found to be induced by the exposure of human dermal fibroblasts to melanoma cell culture supernatants or to transforming growth factor-beta, interleukin-1 and tumor necrosis factor-alpha. The expression MEL4B3 therefore reflects closely cell activation occurring during tumor growth, metastasis and inflammation.