278  2001 American Society for Photobiology 0031-8655/00 $5.00+0.00 Photochemistry and Photobiology, 2001, 73(3): 278–282 The Application of a Compact Multispectral Imaging System with Integrated Excitation Source to In vivo Monitoring of Fluorescence During Topical Photodynamic Therapy of Superficial Skin Cancers ¶ Jacqueline Hewett* 1 , Valerie Nadeau 2 , James Ferguson 3 , Harry Moseley 3 , Sally Ibbotson 3 , John W. Allen 1 , Wilson Sibbett 1 and Miles Padgett 2 1 School of Physics and Astronomy, University of St. Andrews, St. Andrews, UK; 2 Department of Physics and Astronomy, University of Glasgow, Glasgow, UK and 3 Photobiology Unit, Department of Dermatology, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK Received 2 August 2000; accepted 20 November 2000 ABSTRACT A novel, compact and low-cost multispectral fluorescence imaging system with an integrated excitation light source is described. Data are presented demonstrating the ap- plication of this method to in vivo monitoring of fluores- cence before, during and after topical 5-aminolevulinic acid photodynamic therapy of superficial skin cancers. The excitation source comprised a fluorescent tube with the phosphor selected to emit broadband violet light cen- tered at 394 nm. The camera system simultaneously cap- tured spectrally specific images of the fluorescence of the photosensitizer, protoporphyrin IX, the illumination pro- file and the skin autofluorescence. Real-time processing enabled images to be manipulated to create a composite image of high contrast. The application and validation of this method will allow further detailed studies of the characteristics and time-course of protoporphyrin IX fluorescence, during topical photodynamic therapy in hu- man skin in vivo. INTRODUCTION Optical imaging techniques for noninvasive detection of fluorescence for the diagnosis of cancer have developed rap- idly over the past decade (1). These techniques are based on the detection of fluorescence of endogenous species, such as collagen and nicotinamide adenine dinucleotide, which con- tribute partly to broadband autofluorescence in the blue/ green spectral region (2), or the fluorescence of exogenous photosensitizers or their precursors. The photosensitizers are fluorescent and have been shown to accumulate preferentially in many types of cancer in comparison with the surrounding healthy tissue (3). This phenomenon is the foundation on ¶Posted on the website on 8 January 2001. *To whom correspondence should be addressed at: School of Phys- ics and Astronomy, University of St. Andrews, St. Andrews KY16 9SS, UK. Fax: 44-1334-463104; e-mail: jh38@at-and.ac.uk which both photodetection (PD)² and photodynamic therapy (PDT) are based. With advances in technology, the avail- ability of compact light sources and delivery systems, high- speed frame grabbers and sensitive cameras has facilitated the development of real-time fluorescence detection systems, with the potential to increase diagnostic accuracy and com- plement the subjective forms of visual assessment in vivo. Nonmelanoma skin cancer is the most common cancer in Caucasian populations and is a major cause of morbidity and demand on health resources. Early detection and delineation of tumor borders, or of dysplastic precursors, is often diffi- cult using the naked eye. PD, using a low concentration of photosensitizer, has emerged as a promising method for the early diagnosis of superficial nonmelanoma skin cancers or areas of dysplasia. Using this method, accurate demarcation of tumor margins may improve not only the diagnostic ac- curacy, but also allow for a tissue sparing approach to treat- ment. Topical PDT of nonmelanoma skin cancers was intro- duced by Kennedy in 1990 (4) and employs the naturally occurring molecule 5-aminolevulinic acid (5-ALA) as the photosensitizer precursor. 5-ALA is not itself a photosensi- tizing agent, but is converted to a highly efficient photosen- sitizer, protoporphyrin IX (PpIX), by enzymatic conversion in the endogenous heme biosynthetic pathway. Under nor- mal conditions, PpIX is present at very low levels in all nucleated cells and tight regulation of the heme cycle en- sures that the concentration of PpIX does not result in pho- tosensitization. Administration of exogenous 5-ALA bypass- es the rate-limiting step of ALA synthase activity in the heme biosynthetic pathway (Fig. 1), resulting in the accu- mulation of excess PpIX and photosensitization, as required for PD and PDT (4). PpIX has a broad absorption spectrum centered at 405 nm, and a characteristic fluorescence emission with a dominant peak at 635 nm. After 5-ALA application, the enhanced red fluorescence of PpIX in neoplastic tissue has been shown to ² Abbreviations: 5-ALA, 5-aminolevulinic acid; BD, Bowens dis- ease; PDT, photodynamic therapy; PpIX, protoporphyrin IX; PD, photodetection; SBCC, superficial basal cell carcinoma.