Clinical application of multiphoton tomography in combination with confocal laser scanning microscopy for in vivo evaluation of skin diseases Martin Johannes Koehler 1 , Marco Speicher 2 , Susanne Lange-Asschenfeldt 3 , Eggert Stockfleth 3 , Susanne Metz 1 , Peter Elsner 1 , Martin Kaatz 1,4 and Karsten Ko¨ nig 2,5,6 1 Department of Dermatology, University Hospital Jena, Jena, Germany; 2 JenLab GmbH, Schillerstrasse 1, Jena, Germany; 3 Department of Dermatology, Venerology and Allergy, Charite´ Universita¨tsmedizin, Berlin, Germany; 4 Department of Dermatology, SRH Waldklinikum Gera, Gera, Germany; 5 Department of Biophotonics and Laser Technology, Saarland University, Saarbru¨cken, Germany; 6 Beckman Laser Institute and Medical Clinic, University of California Irvine, CA, USA Correspondence: Martin Johannes Koehler, MD, Department of Dermatology, University Hospital Jena, Erfurter Str. 35, 07743 Jena, Germany, Tel.: +49-3641-937836, Fax: +49-3641-937343, e-mail: johannes.koehler@med.uni-jena.de 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. Key words: actinic keratosis – biophotonics – Confocal laser scanning microscopy – imaging methods – melanoma – multiphoton tomography – pemphigus vulgaris – psoriasis – scar – seborrheic keratosis Accepted for publication 31 January 2011 Introduction There is a broad spectrum of diseases affecting the skin and in many cases, by visual inspection is not sufficient for exact diagno- sis. For most dermatoses, dermatopathological evaluation of an excised skin specimen is still the diagnostic gold standard. The disadvantages of this procedure are evident: besides the discomfort for the patient and the general risks of a surgical intervention including scar formation, prolonged bleeding and wound infec- tion. Lastly, histological processing is time-consuming and irre- versibly alters the tissue under investigation. Therefore, there is increasing interest for fast, non-invasive imaging techniques allow- ing dermatological diagnosis. Because of their different strengths and weaknesses of available imaging methods, a combination of two or more techniques could have synergistic effects, ideally add- ing their advantages and compensating their disadvantages. Multiphoton tomography (MPT) is an in vivo imaging tech- nique with very high spatial resolution and efforts are made to combine MPT with other non-invasive imaging methods. Recently, the combination of MPT with optical coherence tomog- raphy (OCT) and ultrasound was reported (1,2). In this work, we present the results from a clinical study combining in vivo MPT with in vivo confocal laser scanning microscopy (CLSM). We also evaluated the lesions by visual inspection and by dermoscopy because these are the ‘classical’ in vivo imaging methods. The study goals were (i) description of the features of different dermatological entities as seen in MPT and CLSM, since to date only very few disorders have been characterized using these meth- ods; (ii) comparison of these two novel techniques and the ‘classi- cal’ diagnostic measures visual inspection, dermoscopy and histology; (iii) discussion of strenghts and weaknesses of the dif- ferent methods and the potential benefit from the combination of CLSM and MPT. Dermoscopy Dermoscopy or epiluminescence microscopy is a non-invasive technique for in vivo evaluation of skin lesions. It is mainly used as a hand-held device with an internal light source. The system employs polarized light and the objective requires optical coupling by an immersion medium such as oil, water or gel to make the stratum corneum translucent. In addition, digital colour cameras Abbreviations: CLSM, confocal laser scanning microscopy; FLIM, fluorescence lifetime imaging; MPT, multiphoton tomography; OCT, optical coherence tomography; NIR, near infrared; PMT, photo multiplier tube; SHG, second harmonics generation. DOI:10.1111/j.1600-0625.2011.01279.x www.blackwellpublishing.com/EXD Original Article ª 2011 John Wiley & Sons A/S, Experimental Dermatology, 20, 589–594 589