Photodynamic therapy (PDT) is an anticancer combination therapy, which requires a photosensitiser, which tends to accumulate preferentially in the tumour, and light. Historically large, complex lasers have been used to carry out PDT treatment. Nowadays there is a wide range of coherent and non-coherent sources that can be used. This paper considers the important characteristics of light sources for PDT, including dye lasers pumped by argon or metal vapour lasers and frequency-doubled Nd:YAG lasers. Non-laser sources including tungsten filament, xenon arc, metal halide and fluorescent lamps are also discussed. New exciting developments such as LEDs and femtosecond lasers are also reviewed. The relative merits of laser and non-laser sources are critically examined.

In vivo and ex vivo tissue autofluorescence (endogenous fluorescence) have been employed to investigate the presence of markers that could be used to detect tissue abnormalities and/or malignancies. We present a study of the autofluorescence of normal skin and tumor in vivo, conducted on 18 patients diagnosed with nonmelanoma skin cancers (NMSC). We observed that both in basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) the endogenous fluorescence due to tryptophan residues was more intense in tumor than in normal tissue, probably due to epidermal thickening and/or hyperproliferation. Conversely, the fluorescence intensity associated with dermal collagen crosslinks was generally lower in tumors than in the surrounding normal tissue, probably because of degradation or erosion of the connective tissue due to enzymes released by the tumor. The decrease of collagen fluorescence in the connective tissue adjacent to the tumor loci was validated by fluorescence imaging on fresh-frozen tissue sections obtained from 33 NMSC excised specimens. Our results suggest that endogenous fluorescence of NMSC, excited in the UV region of the spectrum, has characteristic features that are different from normal tissue and may be exploited for noninvasive diagnostics and for the detection of tumor margins.

We investigated the use of attenuated total reflection-Fourier transform infrared spectroscopy as a method to study differences in the molecular components of human stratum corneum in vivo. These variations as a function of the anatomic site and of the depth into its layered structure are important to understand the biology and physiology of the tissue. In this preliminary study we have investigated spectroscopic changes in 18 healthy individuals. Total reflection-Fourier transform infrared spectroscopy represents a potentially powerful tool to study biophysical properties of surfaces. We observed that, in vivo, biophysical parameters of the stratum corneum (such as hydration, lipid composition, and conformation of the aliphatic chains) are indeed dependent on the anatomic site. As in total reflection-Fourier transform infrared spectroscopy experiments the penetration depth of the evanescent field into the stratum corneum is comparable with the thickness of a layer of corneocytes, this technique can be used to follow the distribution of lipids, water, and proteins as a function of depth into the tissue. We found that, in vivo, these molecular components are non-uniformly distributed, in agreement with the presence of water and lipid reservoirs as observed with ex vivo ultrastructural investigations. Composition and conformational order of lipids are also a function of depth into the stratum corneum. Finally we compared the in vivo superficial hydration measured using the infrared absorption of the OH stretch of water, with the hydration measured using the Skicon hygrometer. Our results indicate that total reflection-Fourier transform infrared spectroscopy might be useful to measure important chemical and biophysical parameters of stratum corneum in vivo.

We have investigated the in vivo fluorescence of human skin with UV excitation and the effect of UV irradiation on the UV fluorescence. A particular chromophore was found to be very sensitive to suberythemogenic UV radiation. This chromophore has the spectral characteristics of tryptophan residues in proteins and is characterized by a fluorescence excitation maximum at 295 nm. The fluorescence of this chromophore in human epidermis has an excitation maximum that is coincident with the maximum of the action spectrum of most UV-induced photobiologic responses to human skin. The fluorescence of the chromophore was found to increase with UV exposure. The action spectrum was determined by following the increase of the emission at 345 nm with excitation at 295 nm as a function of skin exposure to a number of wavelengths in the UV region of the spectrum. The results show that irradiation in the UVB (290-320 nm) is more effective in producing the change in the fluorescence of tryptophan. Irradiation in the UVA (320-380 nm) was found to be capable of producing the increase but to a smaller extent. Whereas tryptophan fluorescence is found in both the epidermis and the dermis, it is only the epidermal component that increases with UV exposure. The change in 295 nm fluorescence with UV exposure was determined to be oxygen dependent. The fluorescence of tryptophan moieties measured in situ was found to increase when epidermal proliferation increases. This was verified by inducing epidermal repair after mechanical insult (tape stripping). The results suggest two possible scenarios for the UV-induced increase of the fluorescence: a prompt photooxidation of tryptophan moieties or a fast proliferation response to the insult created by UV irradiation.

In vivo and ex vivo tissue autofluorescence (endogenous fluorescence) have been employed to investigate the presence of markers that could be used to detect tissue abnormalities and/or malignancies. We present a study of the autofluorescence of normal skin and tumor in vivo, conducted on 18 patients diagnosed with nonmelanoma skin cancers (NMSC). We observed that both in basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) the endogenous fluorescence due to tryptophan residues was more intense in tumor than in normal tissue, probably due to epidermal thickening and/or hyperproliferation. Conversely, the fluorescence intensity associated with dermal collagen crosslinks was generally lower in tumors than in the surrounding normal tissue, probably because of degradation or erosion of the connective tissue due to enzymes released by the tumor. The decrease of collagen fluorescence in the connective tissue adjacent to the tumor loci was validated by fluorescence imaging on fresh‐frozen tissue sections obtained from 33 NMSC excised specimens. Our results suggest that endogenous fluorescence of NMSC, excited in the UV region of the spectrum, has characteristic features that are different from normal tissue and may be exploited for noninvasive diagnostics and for the detection of tumor margins.

We have investigated the binding of two porphyrins, meso-tetrakis ( p-sulfonatophenyl) porphyrin (TSPP) and protoporphyrin IX (PPIX), to tubulin alpha,beta-heterodimers. TSPP had been shown to directly target microtubules in cells. A comparative study between TSPP and PPIX was carried out because the latter is used in clinical applications and is hydrophobic, in comparison with the water soluble TSPP. The results presented in this manuscript show that both porphyrins bind tubulin with nearly identical stoichiometry but with different affinity (1.76 x 10 (5) M (-1) for PPIX; 1.1 x 10 (6) M (-1) for TSPP). The combination of spectroscopic data and molecular simulations suggests that both porphyrins bind as monomers and that their binding site is in proximity of one (or more) Trp residues but do not overlap with the binding site of other well characterized ligands. Molecular simulations also show that the sites that yield the lower energy minima place the porphyrins near the surface of the protein. In the case of TSPP, binding is favored by replacing the ion-dipole interaction of monodispersed TSPP in water with ion-ion interactions provided by two basic residues (His and Lys) at the location of the binding site. Although preliminary, the data show that porphyrin binding could be used to explain some of the effects that photosensitizers may directly produce on protein targets.

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