The effect of fluence rate on the acute response of vessel diameter and red blood cell velocity during topical 5-aminolevulinic acid photodynamic therapy.

BACKGROUND: In a previous study it is shown that for topically applied ALA-PDT, PpIX concentration correlates with vascular changes including vasoconstriction and/or vascular leakage of small vessels and arterioles in the mouse epidermis and dermis. In this study we report on vascular responses induced by ALA-PDT for different fluence rates, including both changes in vessel diameter and dynamics in RBC velocity in arterioles, imaged using intra-vital confocal microscopy in skinfold chambers in hairless mice. Our interest is in the dynamics of vascular changes in the early stages of illumination. METHODS: We have determined the total PDT dose to be relatively low, 13 J cm(-2), and fluence rates of 26, 65 and 130 mW cm(-2) were investigated. Local vascular effects occurred very soon after the start of the therapeutic illumination in ALA-PDT. RESULTS: In this study, we did not find a significant difference between fluence rates. Arterioles were particularly sensitive to vasoconstriction during low dose PDT, often resulting in complete vasoconstriction. When we observed complete vasoconstriction, this coincided with changes in RBC velocity. CONCLUSION: Since the therapeutic effects of PDT are dependent on a fine balance between the need for oxygen during illumination and disruption of the vasculature, the results of the present study add to our understanding of acute vascular effects during ALA-PDT and aid our efforts to optimize PDT using porphyrin pre-cursors.

The effect of light fractionation with a 2-h dark interval on the efficacy of topical hexyl-aminolevulinate photodynamic therapy in normal mouse skin.

BACKGROUND: Light fractionation with a 2-h dark interval increases the efficacy of topical aminolevulinic acid (ALA) photodynamic therapy (PDT). Hexyl-aminolevulinate (HAL) is the hexyl ester of ALA. Both HAL and ALA lead to protoporphyrin IX (PpIX) accumulation in endothelial cells and to vascular effects, which are important for light fractionation. We investigated light fractionation for HAL-PDT in a mouse skin model and compared this with ALA. METHODS: Three illumination schemes were studied: (a) 100 J cm(-2) in a single illumination; (b) 50+50 J cm(-2) in a twofold illumination; (c) a small first light fraction until 50% of PpIX was photobleached (ca. 3 J cm(-2)), followed by 97 J cm(-2) 2h later. PpIX fluorescence was measured continuously during illumination. Efficacy was evaluated by daily visual skin damage scoring up to 7 days after PDT. RESULTS: Light fractionation showed a trend towards increased efficacy for HAL-PDT. Both the initial PpIX synthesis and the PpIX resynthesis during the dark interval were higher for ALA, but these were not correlated with efficacy. Single HAL-PDT was more effective than single ALA-PDT. Photobleaching rates of HAL and ALA were similar indicating similar biodistributions at depth. CONCLUSION: Our results provide evidence to support that light fractionation may be beneficial for HAL-PDT. We are cautious because we found only a non-significant increase in response. However, combining our results with literature data suggest that the illumination scheme may be further optimized for HAL-PDT to potentially enhance the effect of light fractionation.

Topical hexylaminolevulinate and aminolevulinic acid photodynamic therapy: complete arteriole vasoconstriction occurs frequently and depends on protoporphyrin IX concentration in vessel wall.

Vascular responses to photodynamic therapy (PDT) may influence the availability of oxygen during PDT and the extent of tumor destruction after PDT. However, for topical PDT vascular effects are largely unknown. Arteriole and venule diameters were measured before and after hexylaminolevulinate (HAL) and aminolevulinic acid (ALA) PDT and related to the protoporphyrin IX (PpIX) concentration in the vessel wall. A mouse skin fold chamber model and an intravital confocal microscope allowed direct imaging of the subcutaneous vessels underlying the treated area. In both HAL and ALA groups over 60% of arterioles constricted completely, while venules generally did not respond, except for two larger veins that constricted partially. Arteriole vasoconstriction strongly correlated with PpIX fluorescence intensity in the arteriole wall. Total PpIX fluorescence intensity was significantly higher for HAL than ALA for the whole area that was imaged but not for the arteriole walls. In conclusion, complete arteriole vasoconstriction occurs frequently in both HAL and ALA based topical PDT, especially when relatively high PpIX concentrations in arteriole walls are reached. Vasoconstriction will likely influence PDT effect and should be considered in studies on topical HAL and ALA-PDT. Also, our results may redefine the vasculature as a potential secondary target for topical PDT.

In vivo quantification of the scattering properties of tissue using multi-diameter single fiber reflectance spectroscopy.

Multi diameter single fiber reflectance (MDSFR) spectroscopy is a non-invasive optical technique based on using multiple fibers of different diameters to determine both the reduced scattering coefficient (µs') and a parameter γ that is related to the angular distribution of scattering, where γ = (1-g2)/(1-g1) and g1 and g2 the first and second moment of the phase function, respectively. Here we present the first in vivo MDSFR measurements of µs'(λ) and γ(λ) and their wavelength dependence. MDSFR is performed on nineteen mice in four tissue types including skin, liver, normal tongue and in an orthotopic oral squamous cell carcinoma. The wavelength-dependent slope of µs'(λ) (scattering power) is significantly higher for tongue and skin than for oral cancer and liver. The reduced scattering coefficient at 800 nm of oral cancer is significantly higher than of normal tongue and liver. Gamma generally increases with increasing wavelength; for tumor it increases monotonically with wavelength, while for skin, liver and tongue γ(λ) reaches a plateau or even decreases for longer wavelengths. The mean γ(λ) in the wavelength range 400-850 nm is highest for liver (1.87 ± 0.07) and lowest for skin (1.37 ± 0.14). Gamma of tumor and normal tongue falls in between these values where tumor exhibits a higher average γ(λ) (1.72 ± 0.09) than normal tongue (1.58 ± 0.07). This study shows the potential of using light scattering spectroscopy to optically characterize tissue in vivo.

Optimizing ALA-PDT in the management of non-melanoma skin cancer by fractionated illumination.

The aim of this review was to describe briefly the mechanism and history of photodynamic therapy (PDT). The achieved preclinical and clinical results in Rotterdam are discussed in the light of a search to optimize aminolevulinic acid-photodynamic therapy (ALA-PDT). As the incidence of skin cancer is rising, an optimized treatment in non-melanoma skin cancer is needed.

Laser speckle imaging of dynamic changes in flow during photodynamic therapy.

We present a study investigating the use of laser speckle imaging (LSI) for monitoring blood flow during photodynamic therapy (PDT) utilizing the therapeutic illumination radiation. The coherent nature of a laser source, often used in PDT, offers the possibility of obtaining information on the blood flow without interrupting treatment. We have found that in the rat skin-fold observation chamber, it is possible to monitor the vasculature response to PDT in individual arteries, veins and in tumour microvasculature with significantly higher spatial and temporal resolution than current methods. This illustrates the potential for LSI for monitoring PDT, in particular for vascular-localizing photosensitizers, where current non-invasive methods are difficult because of high absorption due to blood and the specific localization of photosensitizer within the vasculature. However, critical problems need to be further investigated and solved, like the influence of tissue sampling volume, changing of optical properties and movement artefacts from other vessels on the LSI signal. Until then, the real potential of LSI for monitoring blood flow remains of limited value.

Fractionated illumination after topical application of 5-aminolevulinic acid on normal skin of hairless mice: the influence of the dark interval.

We have previously shown that light fractionation during topical aminolevulinic acid based photodynamic therapy (ALA-PDT) with a dark interval of 2h leads to a significant increase in efficacy in both pre-clinical and clinical PDT. However this fractionated illumination scheme required an extended overall treatment time. Therefore we investigated the relationship between the dark interval and PDT response with the aim of reducing the overall treatment time without reducing the efficacy. Five groups of mice were treated with ALA-PDT using a single light fraction or the two-fold illumination scheme with a dark interval of 30 min, 1, 1.5 and 2h. Protoporphyrin IX fluorescence kinetics were monitored during illumination. Visual skin response was monitored in the first seven days after PDT and assessed as PDT response. The PDT response decreases with decreasing length of the dark interval. Only the dark interval of 2h showed significantly more damage compared to all the other dark intervals investigated (P<0.05 compared to 1.5h and P<0.01 compared to 1h, 30 min and a single illumination). No relationship could be shown between the utilized PpIX fluorescence during the two-fold illumination and the PDT response. The rate of photobleaching was comparable for the first and the second light fraction and not dependent of the length of dark interval used. We conclude that in the skin of the hairless mouse the dark interval cannot be reduced below 2h without a significant reduction in PDT efficacy.

In vivo nonlinear spectral imaging in mouse skin.

We report on two-photon autofluorescence and second harmonic spectral imaging of live mouse tissues. The use of a high sensitivity detector and ultraviolet optics allowed us to record razor-sharp deep-tissue spectral images of weak autofluorescence and short-wavelength second harmonic generation by mouse skin. Real-color image representation combined with depth-resolved spectral analysis enabled us to identify tissue structures. The results show that linking nonlinear deep-tissue imaging microscopy with autofluorescence spectroscopy has the potential to provide important information for the diagnosis of skin tissues.

Monitoring in situ dosimetry and protoporphyrin IX fluorescence photobleaching in the normal rat esophagus during 5-aminolevulinic acid photodynamic therapy.

Experimental therapies for Barrett's esophagus, such as 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT), aim to ablate the premalignant Barrett's epithelium. However, the reproducibility of the effects should be improved to optimize treatment. Accurate irradiation with light of a proper wavelength (633 nm), fluence and fluence rate has shown to be critical for successful ALA-PDT. Here, we have used in situ light dosimetry to adjust the fluence rate measured within the esophagus for individual animals and monitored protoporphyrin IX (PpIX) fluorescence photobleaching simultaneously. Rats were administered 200 mg kg-1 ALA (n = 14) or served as control (n = 7). Animals were irradiated with an in situ measured fluence rate of 75 mW cm-2 and a fluence of 54 J cm-2. However, this more accurate method of light dosimetry did not decrease the variation in tissue response. Large differences were also observed in the dynamics of PpIX fluorescence photobleaching in animals that received the same measured illumination parameters. We found that higher PpIX fluorescence photobleaching rates corresponded with more epithelial damage, whereas lower rates corresponded with no response. A two-phased decay in PpIX fluorescence could be identified in the response group, with a rapid initial phase followed by a slower rate of photobleaching. Non-responders did not show the rapid initial decay and had a significantly lower rate of photobleaching during the second phase of the decay (P = 0.012).

In vivo pharmacokinetics of protoporphyrin IX accumulation following intracutaneous injection of 5-aminolevulinic acid.

Photodynamic therapy with 5-aminolevulinic acid (ALA) derived protoporphyrin IX (PpIX) as photosensitizer is a promising treatment for basal cell carcinomas. Until now ALA has been administered topically as an oil-in-water cream in most investigations. The disadvantage of this administration route is insufficiënt penetration in deeper, nodular tumours. Therefore we investigated intracutaneous injection of ALA as an alternative administration route. ALA was administered in 6-fold in the normal skin of three 6-week-old female Dutch pigs by intracutaneous injection of an aqueous solution of ALA (pH 5.0) in volumes of 0.1-0.5 ml and concentrations of 0.5-2% and by topical administration of a 20% ALA cream. During 8 h fluorescence of ALA derived PpIX was measured under 405 nm excitation. For the injection the measured fluorescence was shown to be dose dependent. All injected doses of 3 mg ALA or more lead to a faster initial increase rate of PpIX synthesis and significantly greater fluorescence than that measured after topical administration of ALA. Irradiation (60 Jcm(-2) for 10 min) of the spots was performed at 3.5 h after ALA administration. After 48 and 96 h visual damage scores were evaluated and biopsies were taken for histopathological examination. After injection of 2 mg ALA or more the PDT damage after illumination was shown to be significantly greater than after topical application of 20% ALA. An injected dose of 10 mg ALA (0.5 ml of a 2% solution) resulted in significantly more tissue damage after illumination than all other injected doses.

Protoporphyrin IX fluorescence kinetics and localization after topical application of ALA pentyl ester and ALA on hairless mouse skin with UVB-induced early skin cancer.

In order to improve the efficacy of 5-aminolevulinic acid-based (ALA) photodynamic therapy (PDT), different ALA derivatives are presently being investigated. ALA esters are more lipophilic and therefore may have better skin penetration properties than ALA, possibly resulting in enhanced protoporphyrin IX (PpIX) production. In previous studies it was shown that ALA pentyl ester (ALAPE) does considerably enhance the PpIX production in cells in vitro compared with ALA. We investigated the in vivo PpIX fluorescence kinetics after application of ALA and ALAPE to hairless mice with and without UVB-induced early skin cancer. ALA and ALAPE (20% wt/wt) were applied topically to the mouse skin and after 30 min, the solvent was wiped off and PpIX fluorescence was followed in time with in vivo fluorescence spectroscopy and imaging. At 6 and 12 h after the 30 min application, skin samples of visible lesions and adjacent altered skin (UVB-exposed mouse skin) and normal mouse skin were collected for fluorescence microscopy. From each sample, frozen sections were made and phase contrast images and fluorescence images were recorded. The in vivo fluorescence kinetics showed that ALAPE induced more PpIX in visible lesions and altered skin of the UVB-exposed mouse skin, but not in the normal mouse skin. In the microscopic fluorescence images, higher ALAPE-induced PpIX levels were measured in the stratum corneum, but not in the dysplastic layer of the epidermis. In deeper layers of the skin, PpIX levels were the same after ALA and ALAPE application. In conclusion, ALAPE does induce higher PpIX fluorescence levels in vivo in our early skin cancer model, but these higher PpIX levels are not located in the dysplastic layer of the epidermis.

Topical 5-aminolevulinic acid-photodynamic therapy of hairless mouse skin using two-fold illumination schemes: PpIX fluorescence kinetics, photobleaching and biological effect.

Light fractionation with dark periods of the order of hours has been shown to considerably increase the efficacy of 5-aminolevulinic acid-photodynamic therapy (ALA-PDT). Recent investigations have suggested that this increase may be due to the resynthesis of protoporphyrin IX (PpIX) during the dark period following the first illumination that is then utilized in the second light fraction. We have investigated the kinetics of PpIX fluorescence and PDT-induced damage during PDT in the normal skin of the SKH1 HR hairless mouse. A single illumination (514 nm), with light fluences of 5, 10 and 50 J cm-2 was performed 4 h after the application of 20% ALA, to determine the effect of PDT on the synthesis of PpIX. Results show that the kinetics of PpIX fluorescence after illumination are dependent on the fluence delivered; the resynthesis of PpIX is progressively inhibited following fluences above 10 J cm-2. In order to determine the influence of the PpIX fluorescence intensity at the time of the second illumination on the visual skin damage, 5 + 95 and 50 + 50 J cm-2 (when significantly less PpIX fluorescence is present before the second illumination), were delivered with a dark interval of 2 h between light fractions. Each scheme was compared to illumination with 100 J cm-2 in a single fraction delivered 4 or 6 h after the application of ALA. As we have shown previously greater skin damage results when an equal light fluence is delivered in two fractions. However, significantly more damage results when 5 J cm-2 is delivered in the first light fraction. Also, delivering 5 J cm-2 at 5 mW cm-2 + 95 J cm-2 at 50 mW cm-2 results in a reduction in visual skin damage from that obtained with 5 + 95 J cm-2 at 50 mW cm-2. A similar reduction in damage is observed if 5 + 45 J cm-2 are delivered at 50 mW cm-2. PpIX photoproducts are formed during illumination and subsequently photobleached. PpIX photoproducts do not dissipate in the 2 h dark interval between illuminations.

Protoporphyrin IX fluorescence photobleaching during ALA-mediated photodynamic therapy of UVB-induced tumors in hairless mouse skin.

Fluorescence photobleaching of protoporphyrin IX (PpIX) during superficial photodynamic therapy (PDT), using 514 nm excitation, was studied in UVB-induced tumor tissue in the SKH-HR1 hairless mouse. The effects of different irradiance and light fractionation regimes upon the kinetics of photobleaching and the PDT-induced damage were examined. Results show that the rate of PpIX photobleaching (i.e., fluorescence intensity vs fluence) and the PDT damage both increase with decreasing irradiance. We have also detected the formation of fluorescent PpIX photoproducts in the tumor during PDT, although the quantity recorded is not significantly greater than generated in normal mouse skin, using the same light regime. The subsequent photobleaching of the photoproducts also occurs at a rate (vs fluence) that increases with decreasing irradiance. In the case of light fractionation, the rate of photobleaching increases upon renewed exposure after the dark period, and there is a corresponding increase in PDT damage although this increase is smaller than that observed with decreasing irradiance. The effect of fractionation is greater in UVB-induced tumor tissue than in normal tissue and the damage is enhanced when fractionation occurs at earlier time points. We observed a variation in the distribution of PDT damage over the irradiated area of the tumor: at high irradiance a ring of damage was observed around the periphery. The distribution of PDT damage became more homogeneous with both lower irradiance and the use of light fractionation. The therapeutic dose delivered during PDT, calculated from an analysis of the fluorescence photobleaching rate, shows a strong correlation with the damage induced in normal skin, with and without fractionation. The same correlation could be made with the data obtained from UVB-induced tumor tissue using a single light exposure. However, there was no such correlation when fractionation schemes were employed upon the tumor tissue.

Improvement of systemic 5-aminolevulinic acid-based photodynamic therapy in vivo using light fractionation with a 75-minute interval.

We have studied different single and fractionated illumination schemes after systemic administration of 5-aminolevulinic acid (ALA) to Improve the response of nodular tumors to ALA-mediated photodynamic therapy. Tumors transplanted on the thigh of female WAG/Rij rats were transdermally illuminated with red light (633 nm) after systemic ALA administration (200 mg/kg). The effectiveness of each treatment scheme was determined from the tumor volume doubling time. A single illumination (100 J/cm2 at 100 mW/cm2, 2.5 h after ALA administration) yielded a doubling time of 6.6+/-1.2 days. This was significantly different from the untreated control (doubling time, 1.7+/-0.1 days). The only treatment scheme that yielded a significant improvement compared to all other schemes studied was illumination at both 1 and 2.5 h after ALA administration (both 100 J/cm2 at 100 mW/cm2) and resulted in a tumor volume doubling time of 18.9+/-2.9 days. A possible mechanism to explain this phenomenon is that the protoporphyrin IX formed after administration of ALA is photodegraded by the first illumination. In the 75-min interval, new porphyrin is formed enhancing the effect of the second illumination.

Photodynamic effectiveness and vasoconstriction in hairless mouse skin after topical 5-aminolevulinic acid and single- or two-fold illumination.

Several options were investigated to increase the efficacy of photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5-aminolevulinic acid (ALA). Hairless mice with normal skin or UVB-light-induced skin changes were used as a model. In the first part of the study animals were illuminated immediately (t = 4) or 6 h (t = 10, PpIX fluorescence maximum) after the end of a 4 h ALA application. A total incident light fluence of 100 J/cm2 (514.5 nm) was delivered at a fluence rate of 100 or 50 mW/cm2. The PDT-induced damage to normal skin was more severe after treatment at t = 10 than at t = 4. Illumination at 50 mW/cm2 caused significantly more visible damage than the same light fluence given at 100 mW/cm2. For UVB-illuminated skin, different intervals or fluence rates made no significant difference in the severity of damage, although some qualitative differences occurred. In situ fluence rate measurements during PDT indicated vasoconstriction almost immediately after the start of the illumination. A fluorescein exclusion assay after PDT demonstrated vasoconstriction that was more pronounced in UVB-treated skin than in normal skin. The second part of the study examined the effect of two illuminations. The first illumination bleaches the PpIX fluorescence. At the start of the second illumination, new PpIX had been formed. Light of 514.5 nm was delivered at 100 mW/cm2 to a total incident light fluence of 200 J/cm2 at t = 4 (single illumination) or 100 J/cm2 at t = 4 plus 100 J/cm2 at t = 10. There was no visual difference in skin damage between 100 and 200 J/cm2 single illumination. Two-fold illumination (100 + 100 J/cm2) caused significantly more skin damage, indicating a potentially successful option for increasing the efficacy of topical ALA-PDT.

Fluorescence photobleaching of ALA-induced protoporphyrin IX during photodynamic therapy of normal hairless mouse skin: the effect of light dose and irradiance and the resulting biological effect.

The photobleaching of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) was investigated during superficial photodynamic therapy (PDT) in normal skin of the SKH HR1 hairless mouse. The effects of light dose and fluence rate on the dynamics and magnitude of photobleaching and on the corresponding PDT-induced damage were examined. The results show that the PDT damage cannot be predicted by the total light dose. Photobleaching was monitored over a wide range of initial PpIX fluorescence intensities. The rate of PpIX photobleaching is not a simple function of fluence rate but is dependent on the initial concentration of sensitizer. Also, at high fluence rates (50-150 mW/cm2, 514 nm) oxygen depletion is shown to have a significant effect. The rate of photobleaching with respect to light dose and the corresponding PDT damage both increase with decreasing fluence rate. We therefore suggest that the definition of a bleaching dose as the light dose that causes a 1/e reduction in fluorescence signal is insufficient to describe the dynamics of photobleaching and PDT-induced damage. We have detected the formation of PpIX photoproducts during the initial period of irradiation that were themselves subsequently photobleached. In the absence of oxygen, PpIX and its photoproducts are not photobleached. We present a method of calculating a therapeutic dose delivered during superficial PDT that demonstrates a strong correlation with PDT damage.

Photobleaching during and re-appearance after photodynamic therapy of topical ALA-induced fluorescence in UVB-treated mouse skin.

Photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5-aminolevulinic acid (ALA) seems a promising alternative for the treatment of superficial non-melanoma skin cancer and actinic keratosis. In this study, the kinetics of new PpIX fluorescence arising after a PDT treatment that had photobleached the original fluorescence were determined. Our purpose was to examine the feasibility of multiple irradiations, following a single topical ALA application, to increase PDT efficacy. In addition, photobleaching during PDT and the fluorescence spectra during and after PDT were studied. As a model we used hairless mice with and without UVB-induced skin lesions. ALA was applied to the skin for 4 hr. An illumination was delivered either immediately after application or 6 hr after the end of the application (at interval of maximum fluorescence). During PDT, the fluorescence of normal skin decreased at a faster rate than the fluorescence of the skin lesions. In the fluorescence study after PDT, the areas treated immediately post-application showed a fluorescence increase over time similar to that in non-treated areas on the same mice. A remarkable result was that the fluorescence of areas treated at maximum fluorescence increased, whereas the fluorescence of non-treated areas did not increase over time. With both treatment intervals the new fluorescence showed a characteristic PpIX spectrum. Our results demonstrate that a second illumination, when new PpIX fluorescence has been formed, may increase PDT efficacy after topical ALA application. This finding has been demonstrated previously for systemic ALA administration.

Evidence for an important role of neutrophils in the efficacy of photodynamic therapy in vivo.

To investigate the role of neutrophils in the efficacy of photodynamic therapy (PDT) in rhabdomyosarcoma-bearing rats, the number of these circulating phagocytes was decreased or increased before interstitial PDT by use of rabbit anti-rat neutrophil serum or granulocyte colony-stimulating factor, respectively. After administration of the antiserum, the number of circulating neutrophils decreased by 99.9%. However, the number of monocytes, lymphocytes, and platelets decreased as well (by 100%, 80%, and 25%, respectively). Under these conditions, PDT did not retard tumor growth at all. However, after cessation of the antiserum treatment 5 days after PDT, a striking decrease in the growth rate occurred subsequent to an increase above the normal range of the number of circulating neutrophils. Administration of the granulocyte colony-stimulating factor led to a specific 4-fold increase in the number of circulating neutrophils. In these rats, the tumor growth at day 2 after PDT was retarded as compared with PDT-treated rats that received saline only. Statistical evaluation of both experimental conditions showed that the efficacy of PDT, expressed as the percentage of change in tumor volume at day 2 after treatment, was dependent on the number of circulating neutrophils present at the day of PDT (P = 0.001; r2 = 0.482). Apparently, neutrophils are indispensable for successful PDT in vivo.

Kinetics and localisation of PpIX fluorescence after topical and systemic ALA application, observed in skin and skin tumours of UVB-treated mice.

In this study the kinetics and localisation of protoporphyrin IX (PpIX) fluorescence in skin and skin tumours were examined after topically (20% for 4h) or systemically (200 mg/kg,i.p.) administered 5-aminolaevulinic acid (ALA). As a model we used hairless mice with skin lesions (actinic keratoses and squamous cell carcinoma), which were induced by daily UVB irradiation. The epidermis of the skin surrounding the tumours (T) was altered (AS); owing to the UVB irradiation, the epidermis was thicker and less elastic. Therefore, non-UVB-irradiated mice were used to assess fluorescence of normal skin (NS). Light from a halogen lamp was used to excite at 500 +/- 20 nm and fluorescence was detected through a filter that passes light of 670 +/- 50 nm. Maximal fluorescence following i.p. ALA was observed 2 h post injection (p.i.) and was three times less than after topically applied ALA. Furthermore, after i.p. ALA a lower T selectively (T/NS) could be obtained than after topically applied ALA. Maximal fluorescence following topically applied ALA was achieved 6 h after the end of the 4 h application time. At that interval fluorescence of T was twice as high as directly after the application period. Furthermore, T selectivity (T/NS) after topical ALA at the interval of maximal fluorescence was higher than at the interval directly after application. With fluorescence cryomicroscopy localisation of fluorescence in the skin at the interval of maximal fluorescence was determined after both administration routes. For both cases fluorescence was mainly located in T, epidermis and hair follicles. Fluorescence in subcutis could only be observed at 2 h post i.p. ALA and a 6 h post topical ALA. No fluorescence could be observed in muscle. We conclude that, in this model and with these ALA doses, a higher fluorescence intensity and selectivity (T/NS) was achieved after topically applied ALA than after systemically administered ALA. These results make topically applied ALA more favourable for ALA-PDT of superficial skin tumours in this model. In general these results imply that by optimising the time after ALA application the efficacy and selectivity of topical ALA-PDT for skin tumours may be improved.