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BACKGROUND Mucous membranes may be affected by various diseases and the diagnostic accessibility is limited. Multiphoton laser tomography (MPT) is a useful tool for in vivo evaluation of superficial skin structures and might also be useful for the imaging of mucosa. OBJECTIVES In order to investigate the suitability of MPT for the evaluation of mucous membranes, tissue samples of different donors and anatomical localizations have been imaged. METHODS Human mucosa samples from the urinary bladder, palatine tonsil and ocular conjunctiva were investigated by MPT and subsequently compared with conventional histology. RESULTS Horizontal images of the epithelium and the underlying connective tissue were obtained using the autofluorescence and second harmonics generation signals by MPT. The stratification of multilayered epithelium was consistently reproduced. Also inflammatory changes like lymphocytic infiltrates and widened intercellular spaces were imaged in some cases. CONCLUSION Mucous tissue samples were obtained and MPT imaging was successfully performed. In the direct comparison with histology of the same tissue samples, the characteristic morphology of the epithelia was found. Further studies are necessary in order to compare images of healthy and diseased mucosal epithelium and the use of MPT for its evaluation in vivo.

Background Multiphoton laser tomography (MPT) is a non-invasive technique that allows imaging of skin in vivo with very high spatial resolution and contrast. Previous work of our group has demonstrated that known morphological changes due to erythematogenic ultraviolet B (UVB) irradiation may be imaged in vivo by MPT. The present work investigated if morphological skin changes known from experimental erythematogenic UVB irradiation are also demonstrable in the course of a standard phototherapy regime that implies suberythematogenic doses of narrow band UVB. Methods Sixteen patients with psoriasis vulgaris receiving a narrow band phototherapy were included. A test field and a light-protected control field were measured with the multiphoton tomograph DermaInspect® at four time points: at baseline, the next day, after 3 days and at the day of the last exposure. Results In the course of the UVB phototherapy, spongiosis and pleomorphy as parameters of inflammation and cellular damage did not show significant changes. By contrast, an adaptive skin reaction with significant changes of keratosis and pigmentation was observed. Conclusion MPT is a suitable technique for the investigation of qualitative and quantitative skin changes after UVB irradiation. After suberythematogenic UVB irradiation, photoadaptive skin changes, but no cellular damage can be observed with MPT.

Background: As the sequential inflammatory changes are the same in erythema elevatum diutinum (EED) and granuloma faciale (GF), histopathologic distinction may be difficult. Methods: All available cases from 1998 to 2009 with the diagnosis of EED and GF were collected and reviewed, both clinically and histopathologically. Nine cases of EED and 41 cases of GF were reviewed in a blinded fashion using a checklist of 26 histopathologic criteria. Results: Only four of the evaluated criteria showed differences between GF and EED. High density of the infiltrate was noted in 97% of cases of GF but only in 56% of cases of EED. Eosinophils were the predominant cell type in 59% of cases of GF but in none of the cases of EED. Plasma cells were more frequent in GF (64%) than in EED (22%), and granulomas were never found in GF but in 22% of EED. A zone of perijunctional sparing (Grenz zone) was observed in about three quarters of the cases in both the groups. Conclusions: The histopathology of GF and EED is very similar and overlapping. The presence of a Grenz zone and patterned fibrosis does not distinguish the two diseases. However, granulomatous nodules are only seen in EED, and a predominance of eosinophils in the infiltrate favors a diagnosis of GF. Ziemer M, Koehler MJ, Weyers W. Erythema elevatum diutinum – a chronic leukocytoclastic vasculitis microscopically indistinguishable from granuloma faciale?

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.

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. Methods: In a study group of 30 healthy volunteers aged 21–82 years multiphoton depth-resolved measurements of autofluorescence and second harmonics have been performed in order to evaluate the dermal matrix composition. Results: Characteristic intensity curves depending on the penetration depth were derived and differences between age groups were found. Conclusion: With the present work we provide evidence for the accuracy of the measurement of dermal matrix composition by MLT and give detailed advice for the measurement procedure. Furthermore, we propose the use of depth-dependent emission intensity curves for monitoring of anti-aging treatment.

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.

We report on the first clinical study based on optical coherence tomography (OCT) in combination with multiphoton tomography (MPT) and dermoscopy. 47 patients with a variety of skin diseases and disorders such as skin cancer, psoriasis, hemangioma, connective tissue diseases, pigmented lesions, and autoimmune bullous skin diseases have been investigated with (i) state-of-the-art OCT systems for dermatology including multibeam swept source OCT, (ii) the femtosecond laser multiphoton tomograph, and (iii) dermoscopes. Dermoscopy provides two-dimensional color images of the skin surface. OCT images reflect modifications of the intratissue refractive index whereas MPT is based on nonlinear excitation of endogenous fluorophores and second harmonic generation. A stack of cross-sectional OCT “wide field” images with a typical field of view of 5 × 2 mm2 gave fast information on the depth and the volume of the lesion. Multiphoton tomography provided 0.36 × 0.36 mm2 horizontal/diagonal optical sections within seconds of a particular region of interest with superior submicron resolution down to a tissue depth of 200 μm. The combination of OCT and MPT provides a unique powerful optical imaging modality for early detection of skin cancer and other skin diseases as well as for the evaluation of the efficiency of treatments. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Background: Morphological changes in the dermal collagen and elastin fibre network are characteristic for skin ageing and for pathological skin conditions of the dermis. Objectives: To characterize pathological and physiological conditions by multiphoton laser scanning tomography (MLT) in vivo, it is necessary to investigate and identify morphological alterations related to ageing. Methods: In vivo MLT was used to image two-photon excited autofluorescence (AF) and second harmonics generation (SHG) in human dermis of 18 volunteers of different ages. Criteria for the evaluation of age-dependent morphological changes in MLT images were fibre tension and morphology, network pattern, clot formation and image homogeneity. These criteria were weighted and a score was calculated. Results: The resulting MLT-based Dermis Morphology Score is correlated with age (R2 = −0.90) and with the previously published SHG to AF Ageing Index of Dermis (R2 = 0.66). The two groups of young (age 21–38) and old (age 66–84) volunteers showed a significant difference in MLT score values (P

Background In vivo multiphoton tomography (MPT) of human skin has become a valuable tool for non-invasive examination of morphological and biophysical skin properties and their alterations. So far, skin changes after UVB irradiation were mainly evaluated clinically and histologically. The present study aimed at non-invasive imaging of histological changes during acute UVB irradiation by multiphoton laser tomography. Methods In 10 volunteers, five areas were irradiated once with an erythematous UVB dose. Multiphoton measurements were performed four times, i.e. before irradiation (baseline), and 24, 48 and 72 h after irradiation, respectively. The data were evaluated for changes of epidermal pleomorphy, spongiosis, pigmentation and thickness. Results The four parameters were altered significantly by acute UVB irradiation, i.e. epidermal pleomorphy, spongiosis, pigmentation and thickness increased within 72 h after irradiation. Conclusion Thus, the study has shown that typical epidermal changes induced by acute UVB irradiation can be evaluated by MPT.

In recent years, there has been an increasing interest in non-invasive imaging techniques in clinical and investigational dermatology. Besides the growing number of biophysical methods for the assessment of skin properties, novel imaging tools have emerged and classical imaging methods are substantially improved.This review focuses on well established and some of the most promising imaging techniques for application in dermatology and cutaneous research. These comprise dermoscopy, sonography, confocal microscopy, multiphoton tomography and optical coherence tomography. Non-invasive imaging techniques are presented concerning their functional principles, the history of their development and their current clinical and research application. Advantages and limitations of each of the methods are discussed.Available non-invasive imaging techniques in dermatology show substantial differences concerning their limitations and opportunities, potential clinical applicability and practicability. Also, the current data available differ largely between these methods due to different levels of experience in research and clinical application. Future research will not only aim at improving current technical limitations, but also investigate the potential synergistic effects of combining two or more techniques in order to enhance their diagnostic impact.

Necrobiotic xanthogranuloma (NXG) usually shows a stereotypical histopathologic presentation. However, few unusual cases have been published. We present a patient with NXG showing exceptional histopathologic features. NXG in our patient presents with exclusively dermal granulomatous inflammation mimicking interstitial granuloma annulare. Not only subcutaneous involvement, but also, evident zones of degenerated collagen, foam cells, and cholesterol clefts were missing. Moreover, the case shows overlaps with recently published granulomatous scleromyxedema. Some common clinical and histopathologic features of NXG and scleromyxedema might be based on shared underlying paraproteinemia.

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.

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. Objectives: In the present study, an in vivo measurement of four different morphometric epidermal parameters is performed. Methods: Thirty healthy volunteers aged 21–82 years were included in the study after informed consent and approval of the local ethics committee. At the dorsal forearm and the dorsum of the hand, the thicknesses of the total epidermis, viable epidermis and stratum corneum and the depth of the papillary dermis were calculated from depth-resolved intensity curves after correlation with multiphoton images. Results: We have shown consistently that in all age groups, the four morphometric parameters are significantly higher at the hand compared with the forearm, while there were no differences between age groups. This is consistent with most previous findings. Conclusion: The method presented here provides a novel in vivo investigation tool for the measurement of epidermal morphometric parameters that may be useful for the observation of epidermal changes over time in skin disorders, therapy side effects or in cosmetic science.

The first clinical trial of optical coherence tomography (OCT) combined with multiphoton tomography (MPT) and dermoscopy is reported. State-of-the-art (i) OCT systems for dermatology (e.g. multibeam swept source OCT), (ii) the femtosecond laser multiphoton tomograph DermaInspectTM, and (iii) digital dermoscopes were applied to 47 patients with a diversity of skin diseases and disorders such as skin cancer, psoriasis, hemangioma, connective tissue diseases, pigmented lesions, and autoimmune bullous skin diseases. Dermoscopy, also called 'epiluminescent microscopy', provides two-dimensional color images of the skin surface. OCT imaging is based on the detection of optical reflections within the tissue measured interferometrically whereas nonlinear excitation of endogenous fluorophores and the second harmonic generation are the bases of MPT images. OCT cross sectional "wide field" image provides a typical field of view of 5 x 2 mm 2 and offers fast information on the depth and the volume of the investigated lesion. In comparison, multiphoton tomography presents 0.36 x 0.36 mm 2 horizontal or diagonal sections of the region of interest within seconds with submicron resolution and down to a tissue depth of 200 μm. The combination of OCT and MPT provides a synergistic optical imaging modality for early detection of skin cancer and other skin diseases.

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. Methods: Young healthy volunteers using indoor tanning facilities and aged people were compared with appropriate controls by measurements of skin elasticity with the Cutometer and the Reviscometer and by semi-quantitative evaluation of the dermal matrix composition by the multiphoton laser tomograph DermaInspect. Results: We found differences between the sun-protected volar forearm and the dorsal side as well as between young and old test persons with all three methods. No significant differences were found between the skin of indoor-tanned test persons and control. Also, gender had no influence on the severity of skin aging. Conclusion: The most consistent results were obtained with the DermaInspect. The considerable inter-individual variation due to the cross-sectional design of the study may have disguised the factual skin damage caused by tanning beds.

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.

Erysipelas carcinomatosum is a rare but severe manifestation of metastatic breast cancer. Although its diagnosis can generally be made unambiguously from the medical history, clinical appearance, and histopathologic picture, the therapy of inflammatory breast cancer remains challenging. Nevertheless, early diagnosis, management, and referral to an oncologist are most important for management of recurrent disease.

The influence of female sex hormones on skin aging has repeatedly been investigated with contradictory results. In our study, the skin roughness of eight women receiving hormone therapy decreased significantly by approximately 15% in 12 months. Our results provide new evidence of the antiaging effect of female sex hormones.

Changes of dermal collagen and elastin content are characteristic for skin aging as well as for pathological skin conditions. To evaluate these changes, we used in vivo multiphoton laser tomography to measure two-photon excited autofluorescence (AF) and second harmonic generation (SHG). We tested 18 patients of all ages and calculated the SHG-to-AF aging index of dermis (SAAID). We observed a negative relationship between the SAAID and age, which was accelerated for the female (n=7) subgroup. The current findings are the first in vivo demonstration of this relationship, and they show that specific characteristics of aged skin such as the ratio of extracellular matrix components collagen and elastin can be evaluated by in vivo A-F and SHG measurements using near-IR femtosecond laser pulses.