Animals. Female nude BALB/c mice (around 10 weeks of age and weighing 20-25 g) were used for an animal experiment. The mice were anaesthetized with an intraperitoneal injection of hypnorm dormicum solution during cream application in order to facilitate proper application of creams. Anaesthetic Hypnorm (Janssen Pharmaceutica, Beerse, Belgium) was dissolved at amount 1:1 (vol./vol.) in physiological solution and approximately 0.1 ml of prepared solution was administered per animal. All spectroscopic measurements were performed without anaesthesia, except for the group of animals where anaesthesia was used to maintain cold skin. The mice were fed with a standard rodent diet and kept in a 12-hour on-off light cycle, except during the application of ALA and ALA-Me when they were kept in the darkness between fluorescence measurements.

ALA and ALA-Me application. ALA and ALA-Me were obtained from PhotoCure ASA (Oslo, Norway). For topical application a cream was prepared using 20% ALA or 20% ALA-Me (wt./wt.) in an ointment (Unguentum Merck, Darmstadt, Germany). Approximately 0.1 g of the freshly prepared cream was then applied topically on mouse skin. The application area was covered with a transparent dressing (OpSite Flexigrid, Smith & Nephew Medical Ltd., Hull, England). The patch area of ALA-cream on the mouse flank was about 1 cm2 after the dressing had been applied.

Temperature control. Temperature of mouse skin was controlled by a thermocouple connected to a temperature measuring device (model 457 XP, Kane-May Ltd., Swallowfield, UK). Skin temperature was varied by applying different procedures. Application of a cotton coat on mice resulted in an increase of skin temperature for up to 38oC (defined as warmed skin).  Injection of anaesthetics (0.05 ml per animal) in such a way that animals were slightly active resulted in a moderate decrease of skin temperature down to 28-32oC (defined as cold skin).  Injection of anaesthetics (0.1 ml per animal) in such a way that animals were not active and application of a flask containing ice resulted in an average skin temperature around 14-16oC (defined as cooled skin). Continuous anaesthesia was maintaining for one animal for up to 21 h after administration of high dose (0.5 ml) of anaesthetic (skin temperature around 22oC). Normal skin of mice (temperature around 37oC) that were not affected neither by external temperature nor by anaesthesia was used as control.

Fluorescence measurements. The amount of ALA-induced porphyrins in the skin was determined by fluorescence measurements. A Perkin Elmer LS-50B luminescence spectrometer (Norwalk, CT, USA) equipped with a photomultiplier tube R928 (Hamamatsu, Japan) and a non-invasive fibre-optic probe assembled for fluorescence measurements in vivo was used. The output signal of the luminescence spectrometer was calibrated with a LS-1-CAL calibrated light source (Ocean Optics, Inc., Dunedin, FL, USA) and fluorescence emission spectra are corrected to the spectral sensitivity of the instrument. Fluorescence excitation wavelength was set at 407 nm, which corresponds to the maximum of the fluorescence excitation spectrum of PpIX in the skin. Skin autofluorescence background, i.e. fluorescence spectrum of mouse skin measured before application of ALA and ALA-Me, is subtracted from fluorescence spectrum. Fluorescence kinetics were measured at the PpIX fluorescence maximum (637 nm). Skin autofluorescence intensity corresponding to 637 nm, which was around 0.4 rel. u., is subtracted from the fluorescence kinetics. During experiments of continuous ALA application, creams and transparent dressings were continuously present during the measurements. This did not interfere with the fluorescence readings. During experiments of short ALA and ALA-Me application, creams and dressings were removed from mice after 10 min. or 3 h of application.