Martin Johannes Koehler

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

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.

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.

Morphological skin ageing criteria by multiphoton laser scanning tomography: non‐invasive in vivo scoring of the dermal fibre network

Background:  Morphological changes in the dermal collagen and elastin fibre network are characteristic for skin ageing and for pathological skin conditions of the dermis.

Intrinsic, solar and sunbed‐induced skin aging measured in vivo by multiphoton laser tomography and biophysical methods

Background: Skin aging is accelerated by extrinsic factors, particularly actinic damage. Over the last decades, both clinical and pathological differences between intrinsic and actinic aging have been characterized. In this work, we aimed at quantifying skin aging by non‐invasive in vivo methods.

In vivo measurement of the human epidermal thickness in different localizations by multiphoton laser tomography.

Background: The in vivo measurement of epidermal thickness is still challenging. While ultrasound, optical coherence tomography and confocal laser microscopy are used with moderate success, this issue has not been addressed by multiphoton laser tomography.

Clinical application of multiphoton tomography in combination with confocal laser scanning microscopy for in vivo evaluation of skin diseases

Abstract:  Multiphoton tomography (MPT) is an in vivo imaging technique with very high spatial resolution and efforts are made to combine MPT with other non‐invasive imaging methods. The goals of the present study were the description of the features of different dermatological entities as seen in MPT and confocal laser scanning microscopy (CLSM) comparison of these two novel techniques and the ‘classical’ diagnostic measures visual inspection, dermoscopy and histology with respect to the strengths and weaknesses of the different methods and the potential benefit from their combined implementation. After study approval by the local Ethics Committee, 47 patients (31 male, 16 female, age range: 24–88 years) were recruited from the Department of Dermatology of the University Hospital Jena. In this work, we present an illustrative selection of eleven cases from a clinical study combining in vivo MPT with in vivo CLSM. The patients presented with a broad range of dermatological disorders including seborrheic keratoses, angioma, actinic keratoses, melanocytic nevi, malignant melanoma, psoriasis, pemphigus vulgaris and scarring. Both methods, CLSM and MPT, were found to be suitable for in vivo imaging of superficial skin layers and may therefore be useful in dermatological practice for the diagnosis of skin diseases. However, both methods differ in their technical and physical principles. Thus, despite of many similarities concerning the morphological presentation of cells and tissues, important differences are recognized. Synergies of the combination of CLSM and MPT may be obtained by combined implementation in order to benefit from the fast overview given by CLSM and the detailed imaging of skin structures by MPT.

Depth‐resolved measurement of the dermal matrix composition by multiphoton laser tomography

Background: In the last years, multiphoton laser tomography (MLT) has emerged as a promising tool for non‐invasive diagnostics in dermatology and other medical specialties. The present work is dedicated to the question to what degree the measurement depth and the thickness of the epidermis influence the evaluation of dermal matrix composition and if recommendations for future measurement procedures can be given.

Keratinocyte morphology of human skin evaluated by in vivo multiphoton laser tomography

Background: Multiphoton tomography (MPT) is a novel non‐invasive imaging method in dermatology allowing the depiction of the epidermis with sub‐cellular resolution. Here, we present a descriptive characterization of unaffected human epidermis, morphometric data on human keratinocytes and some epidermal parameters in vivo and a morphological characterization of keratinocyte changes in actinic keratoses.

Non-invasive evaluation of dermal elastosis by in vivo multiphoton tomography with autofluorescence lifetime measurements

Abstract:  The non‐invasive differentiation of dermal elastic fibres from solar elastosis in vivo is of great interest in dermatologic research, especially for efficacy testing of anti‐ageing products. To date, no studies on multiphoton excited fluorescence lifetime characteristics of human elastic fibres and solar elastosis are reported. The goal of the present work was the identification of differential criteria for elastic fibres and solar elastosis by the analysis of fluorescence decay curves acquired by time‐correlated single photon counting in vivo multiphoton tomography. For this purpose, fluorescence lifetime measurements (FLIM) were performed with 47 volunteers of different age groups at sun‐protected and sun‐exposed localizations. Bi‐exponential curve fitting was applied to the FLIM data, and characteristic differences between age groups and localizations were found in both relevant fit parameters describing the decay slope. The FLIM analyses have shown that dermal autofluorescence has different lifetimes depending on age and in part on localization.

Erythema elevatum diutinum - a chronic leukocytoclastic vasculitis microscopically indistinguishable from granuloma faciale?

Background: As the sequential inflammatory changes are the same in erythema elevatum diutinum (EED) and granuloma faciale (GF), histopathologic distinction may be difficult.