Application Of 5-aminolevulinic Acid Research Articles

The application of 5-aminolevulinic acid-induced fluorescence for cystoscopic diagnosis and treatment of bladder carcinoma

To investigate the clinical application of 5-aminolevulinic acid (ALA) assisted fluorescence cystoscopy for early detection of bladder carcinoma and completely transurethral resection. ALA-assisted fluorescence cystoscopy was performed on 51 cases of anodynia haematuria. Fluorescence cystoscopy was performed with a D-Light system 2-3h after intravesical instillation of 5-ALA. Biopsy was performed in fluorescence positive regions and/or the fluorescence negative regions with an abnormal gross appearance under a white light. Thirty-two biopsies were subjected to frozen sectioning and the fluorescence distributions were analyzed with a confocal fluorescence microscopy and PpIX fluorescence contents in different tissues were quantified. Transurethral resection was performed in fluorescence positive regions. Red fluorescence (fluorescence positive) was found in 47 cases, among them 40 urothelial carcinomas and seven benign lesions confirmed by pathological examination; fluorescence negative was found in four cases of benign lesions. The sensitivity of fluorescence cystoscope for the diagnosis of bladder carcinoma was 97.1% (68/70) with a false-positive rate of 23.6% (21/89). Micro-fluorescence positive regions were found near the tumor or at distant region in 19 cases of transitional cell carcinoma with two carcinoma in situ unidentifiable with white light, and were confirmed of the existence of malignancy by biopsy. ALA-induced PpIX fluorescence was mainly distributed on the surface layer of the tissue and PpIX fluorescence was fairly weak in the deep tissue. Fluorescence quantization showed that the fluorescence intensity of the surface layer of the tissue was 5-10 fold higher than deep tissue, which was correlated with the grade of tumor (G3>G1-2), but the mean fluorescence intensity in tumor tissue was significantly higher than that of benign lesions. ALA-assisted fluorescence cystoscope offers a simple photodynamic diagnosis approach for the diagnosis of bladder carcinoma, in particular, flat and small malignant lesions.

Relation between intracellular location and photodynamic efficacy of 5-aminolevulinic acid-induced protoporphyrin IX in vitro. Comparison between human glioblastoma cells and other cancer cell lines

A promising clinical application of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PP IX) is fluorescence detection and photodynamic treatment of residual tumour tissue during surgical resection of high grade malignant glioma. U373 MG human glioblastoma cells were used as a model system to study the relation between intracellular location and photodynamic efficacy of 5-ALA-induced PP IX in more detail. Therefore, ultra-sensitive fluorescence microscopy, using either optical excitation of whole cells or selective excitation of the plasma membrane by an evanescent electromagnetic field, was combined with quantitative measurements of intracellular porphyrin amount and phototoxicity. Glioblastoma cells accumulated PP IX to a moderate extent as compared to T47D breast cancer cells (high accumulation) or OV2774 ovarian cancer cells (low accumulation). Although photodynamic inactivation of the different cell lines (decreasing in the order T47D > U373 MG > OV2774) seemed to be directly related to PP IX accumulation, examination of the data in more detail revealed that photodynamic efficacy per photosensitizer molecule (PE) was about two times higher in glioblastoma and ovarian cancer cells as compared to breast cancer cells. The different photodynamic efficacy of PP IX was related to the different intracellular location. In contrast to breast cancer cells where PP IX fluorescence was localized in small granules, PP IX fluorescence in glioblastoma cells and ovarian cancer cells originated mainly from cellular membranes. Thus, the intracellular location of PP IX in a predominantly lipophilic environment, characterized by a comparably high photostability (probed by photobleaching and photoproduct formation) and a lower degree of porphyrin aggregation (probed previously by fluorescence decay kinetics), seems to be the key factor for high photodynamic efficacy of 5-ALA-induced PP IX. In the case of OV2774 ovarian cancer cells, however, a low PP IX accumulation limited cell inactivation upon irradiation, whereas the results obtained for glioblastoma cells are encouraging to develop PDT to an additional therapeutic option for the treatment of tumour margins in patients who underwent fluorescence-guided resection of high grade malignant glioma after 5-ALA administration.

Cell-Substrate Topology upon ALA-PDT Using Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Because of the low penetration depth of an evanescent electromagnetic field, total internal reflection fluorescence microscopy (TIRFM) proved to be a powerful technique to examine fluorescent dyes or photosensitizers in close vicinity to the plasma membrane of living cells. In addition, on variation of the angle of incidence of exciting laser light, the penetration depth is varied, so that cell-substrate topology can be examined with nanometer resolution. Using a specific illumination device for TIRFM and a highly sensitive electron multiplying (EM) CCD camera, fluorescence of the photosensitizer protoporphyrin IX (PPIX) was studied in human cancer cells after application of 5-aminolevulinic acid (5-ALA) prior to and after irradiation with sublethal light doses (635 nm, 4 J/cm2). For cells growing on microscope cover slides, cell-substrate distances varied between approximately 20 and 250 nm with a mean distance of approximately 120 nm. On light exposure, these distances generally decreased, and a mean value below 100 nm was attained. Moreover, focal contacts visualized with a fusion protein of yellow fluorescent protein and focal adhesion kinase were maintained on light exposure, i.e., light-induced detachment of cells from their substrate was not likely to occur.

Inhibition of lycopene cyclase results in accumulation of chlorophyll precursors

Free porphyrins and their magnesium complexes, including chlorophylls, are potent photo-sensitizers. Plants usually accumulate these compounds bound to proteins together with protective compounds like carotenoids. Besides their protective role, carotenoids can play a structural role in these complexes. To analyze the effect of impaired carotenogenesis on plastid membranes we applied to barley seedlings the bleaching herbicide 2-(4-chlorophenylthio)triethylamine (CPTA) as a specific inhibitor for the cyclization of lycopene. To avoid interference with photo-oxidation, the essential experiments were performed on seedlings grown in darkness. While the amount of total carotenoids decreased, we found accumulation of more 6-carotene than lycopene in darkness clearly showing that CPTA inhibits the lycopene beta-cyclase more effectively than the lycopene epsilon-cyclase. The CPTA treatment resulted in accumulation of non-photoactive protochlorophyllide a; the amount of photoactive protochlorophyllide and NADPH:protochlorophyllide oxidoreductase remained constant. Further, the level of Mg protophorphyrin and its monomethyl ester increased to an extent similar to that obtained by application of 5-aminolevulinic acid (ALA). The perturbation of the ultrastructure of etioplast inner membranes, observed after CPTA-treatment, was not found after ALA-treatment; this excluded the accumulated tetrapyrroles as responsible for the perturbation. By contrast, the down-regulation of Lhcb and RbcS genes found after CPTA-treatment was compatible with the presumed role of Mg protophorphyrin as "plastid signal" for regulation of nuclear gene expression. Possible mechanisms for enhancement of tetrapyrrole accumulation by non-cyclic carotenoids are discussed.

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 2 h 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 2 h. 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 2 h showed significantly more damage compared to all the other dark intervals investigated ( P < 0.05 compared to 1.5 h and P < 0.01 compared to 1 h, 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 2 h without a significant reduction in PDT efficacy.

Fluorescence-Guided Minimally Invasive Parathyroidectomy: A Novel Surgical Therapy for Secondary Hyperparathyroidism

Secondary hyperparathyroidism (SHPT) is a severe and frequent complication in patients with advanced chronic kidney disease, characterized by hyperplasia of all parathyroid glands and elevated serum parathyroid hormone levels. When surgery is required to prevent cardiovascular consequences, bone pain, osteoporosis, or even soft-tissue calcifications, detection of the enlarged glands often can be difficult because of their variability in number and location. A novel surgical technique, fluorescence-guided minimally invasive parathyroidectomy, may facilitate intraoperative localization of parathyroid glands. A 52-year-old woman with SHPT underwent minimally invasive videoscopic-assisted parathyroidectomy after photosensitization with aminolevulinic acid (ALA): Under special fluorescence illumination by D-Light (Karl Storz Co, Tuttlingen, Germany), bilateral neck exploration was performed. All enlarged parathyroid glands were identified because of their ALA-induced intense red fluorescence. Such surrounding structures as thyroid, lymph nodes, and soft tissue remained nonfluorescent and could be distinguished easily from parathyroid glands. Total parathyroidectomy with autotransplantation into the sternocleidoid muscle was performed. In patients with SHPT, exploration of all parathyroid glands during surgery is mandatory. However, to date, there is no convincing technical aid for the surgeon to facilitate this procedure. The ALA-induced fluorescence technique represents an innovative visual detection method for intraoperative identification of parathyroid glands. The technique serves as an additional tool requiring only moderate technical and clinical expenditure.

Influence of Physiological Parameters on the Production of Protoporphyrin IX in Human Skin by Topical Application of 5-Aminolevulinic Acid and its Hexylester

Formation of protoporphyrin IX (PpIX) after topical application of 5-aminolevulinic acid (ALA) and its hexylester derivative (ALA-Hex) was studied on healthy human skin. Temperature, density of hai ...

Detection of peritoneal endometriotic lesions by autofluorescence laparoscopy

This study was undertaken to evaluate the feasibility of autofluorescence laparoscopy in the diagnosis of endometriotic lesions. Prospective analysis of 83 consecutive patients undergoing laparoscopy for suspected endometriosis under white light illumination and autofluorescence diagnosis. The study measured total number of endometriotic lesions diagnosed under white light illumination and with autofluorescence diagnosis. The biopsy-based sensitivity of white light diagnosis alone and white light illumination and autofluorescence for detecting nonpigmented peritoneal endometriotic lesions was 65% compared with 92% (1.42-fold increase). The corresponding specificity was 68% as opposed to 84%. Occult areas of endometriosis were discovered using autofluorescence diagnosis. Statistical analysis was performed with chi2 test and McNemar test. Combination of white light illumination and autofluorescence is significantly superior to white light illumination alone in detecting nonpigmented endometriotic lesions. Autofluorescence diagnosis of nonpigmented endometriotic lesions may become an alternative to fluorescence diagnosis after application of 5-aminolevulinic acid, especially because of no side effects.

Fluorescence staining of human ovarian cancer tissue following application of 5‐aminolevulinic acid: Fluorescence microscopy studies

Application of 5-aminolevulinic acid (ALA) for fluorescence-guided second-look laparoscopy has been shown to be a promising new procedure in the early diagnosis of ovarian carcinoma metastases. However, for assessing the reliability of this method, information on the microscopic distribution of protoporphyrin IX (PP IX) in the tissue is needed. Additionally, the selectivity of PP IX uptake is essential for a potential photodynamic therapy (PDT) of ovarian cancer metastases. Thirty-six patients with epithelial ovarian cancer and two patients suffering from fallopian tube carcinoma underwent a laparoscopic second-look procedure 5 hours after the application of ALA. In 17 patients 36 fluorescence-guided biopsies were taken from fluorescing and non-fluorescing tissues for further evaluation. Fluorescence microscopy and digital image processing were utilized to determine the presence of PP IX fluorescence. A specificity of 88% and a sensitivity of 100% with a negative predictive value of 100% and a positive predictive value of 91% were calculated for PP IX fluorescence on a microscopic level as marker for ovarian cancer metastases. On a microscopic scale, ALA-induced PP IX fluorescence is confined to ovarian cancer tumor tissue sparing stromal tissues.

In vitro percutaneous absorption and in vivo protoporphyrin IX accumulation in skin and tumors after topical 5-aminolevulinic acid application with enhancement using an erbium:YAG laser

5-Aminolevulinic acid (ALA) is used as a precursor of protoporphyrin IX (PpIX) for photodynamic therapy (PDT) of superficial skin cancers and subcutaneous metastases of internal malignancies. The permeability of ALA across intact skin is always low, making it difficult to achieve the desired therapeutic benefits. Hence new methods for enhancing ALA permeation are urgently needed. The aim of this study was to determine the in vivo kinetics of PpIX generation in mouse tissues after topical ALA application enhanced by an erbium (Er):yttrium-aluminum-garnet (YAG) laser. The in vitro permeation of ALA was also used to screen the optimal method for the in vivo study. The efficacy of the improved drug delivery was determined as a function of various laser fluences and cancer models. ALA applied to laser-treated skin produced a higher accumulations of PpIX within superficial skin and subcutaneous tumors as compared to those of the non-treated group (t-test, p<0.05). The enhancement ratios (ER) of laser-treated skin ranged from 1.7 to 4.9 times as compared to the control depending to the fluences used. The enhanced PpIX level of laser-treated skin was generally more pronounced in normal and lesional skin than in subcutaneous nodular tumors. Confocal laser scanning microscopy (CLSM) of laser-treated skin revealed intense red fluorescence within the epidermis and upper dermis, and a much-weaker fluorescence within the bottom layers of the skin. On the other hand, the fluorescence intensity of the control group was much lower than that of laser-treated group. The barrier properties of the skin irradiated by the laser had completely recovered within 3days. Pretreatment of skin using an Er:YAG laser was useful in increasing the amount of Pp IX within skin tumors.

Photodynamic treatment (ALA-PDT) suppresses the expression of the oncogenic Bcr-Abl kinase and affects the cytoskeleton organization in K562 cells

K562 is the chronic myelogenous leukemia (CML)-derived cell line that expresses high levels of chimeric oncoprotein Bcr-Abl. The deregulated (permanent) kinase activity of Bcr-Abl leads to continuous proliferation of K562 cells and their resistance to the apoptosis promotion by conventional drugs. The photodynamic treatment (PDT) based on the application of 5-aminolevulinic acid (ALA) and irradiation with blue light (ALA-PDT) resulted in the suppression of K562 cells proliferation. It was followed by a necrosis-like cell death [K. Kuželová, D. Grebeňová, M. Pluskalová, I. Marinov, Z. Hrkal, J. Photochem. Photobiol. B 73 (2004) 67–78]. ALA-PDT led to the perturbation of the Hsp90/p23 multichaperone complex of which the Bcr-Abl is the client protein. Bcr-Abl protein was suppressed whereas the bcr-abl mRNA level was not affected. Further on, we observed several changes in the cytoskeleton organization. We detected ALA-PDT-mediated disruption of filamental actin structure using FITC-Phalloidin staining. In connection with this we uncovered certain cytoskeleton organizing proteins involved in the cell response to the treatment. Among these proteins, Septin2, which plays a role in maintaining actin bundles, was suppressed. Another one, PDZ-LIM domain protein 1 (CLP36) was altered. This protein acts as an adaptor molecule for LIM-kinase which phosphorylates and thus inactivates cofilin. Cofilin was indeed dephosphorylated and could thus be activated and operate as an actin-depolymerizing factor. We propose the scheme of molecular response of K562 cells to ALA-PDT.

Long-Term Efficacy of ALA Photodynamic Therapy of AKs

Topical aminolevulinic acid photodynamic therapy (ALA-PDT) has been approved by the FDA for the treatment of actinic keratoses (AKs) of the face and scalp. Treatment involves application of aminolevulinic acid to the lesions, followed 18 hours later by exposure to 417-nm blue light. Although this therapy has been shown to produce short-term regression of thin to moderately thick AKs, the long-term efficacy has not been determined. Investigators in …

Correlation Between Macroscopic Fluorescence and Protoporphyrin IX Content in Psoriasis and Actinic Keratosis Following Application of Aminolevulinic Acid

In fluorescence diagnosis with 5-aminolevulinic acid (ALA)-induced porphyrins (FDAP), protoporphyrin IX (PpIX) accumulation can be macroscopically visualized. Interpretation of these data is still problematic because of the low reproducibility of the procedure and poor understanding of the mechanisms involved in PpIX tumor selectivity. In this study, PpIX accumulation is investigated in patients with psoriasis and actinic keratosis (AK) following FDAP. For this purpose, desquamated lesional and non-lesional skin were incubated with 20% ALA ointment for 3 h, FDAP was performed, and highly fluorescing lesional skin and non-lesional skin were biopsied. In extracts from these biopsies, PpIX, protein, and dsDNA were quantified by spectrofluorometry. Digital images acquired with FDAP were analyzed using image analysis software. PpIX per biopsy in lesional skin in both psoriasis and AK was significantly higher than in non-lesional skin (p < 0.05). When corrected for epidermal involvement, only lesional psoriatic skin showed significantly higher PpIX levels than non-lesional skin. The PpIX-ratio lesional:non-lesional skin (mean(pmol per mL)+/-SEM) was 4.12+/-0.91 in psoriasis and 1.96+/-0.24 in AK. In FDAP, the ratio of lesional:non-lesional skin was 1.77+/-0.06 in psoriasis and 1.37+/-0.07 in AK. Macroscopic fluorescence and PpIX content appeared to be well correlated (r = 0.73), thus making FDAP a good predictor of PpIX content.

Photosensibilisierung und Photoprotektion bei der topischen Photodynamischen Therapie mit Aminolävulinsäure und Methyl-amino-oxo-pentanoat

The period of photosensitization and subsequent photoprotection after fluorescence diagnosis and photodynamic therapy with aminolevulinic acid (ALA) or methylaminooxopentanoat (MAOP) is not well understood. During the last years many in vitro and in vivo studies were performed to gain more insight into this matter. The data of the reviewed studies are not very valid but first conclusions can be drawn. In general, the duration of the photosensitivity lasts for 24 - 36 hours. ALA-derivatives seem to induce higher and earlier photosensitization with higher phototoxicity in the tumour tissue than ALA itself. More rapid penetration, homogeneous distribution and higher selectivity is discussed as the main reason for the different characteristics of ALA and ALA-derivatives. In addition, it could be shown, that also the healthy skin can be highly photosensitized after application of ALA or MAOP. Therefore, all areas (neoplastic and healthy skin) treated with a photosensitizer should be kept protected from light after photodynamic therapy and especially fluorescence diagnosis. Otherwise, severe and unwanted phototoxic reactions induced by UV and also visible light may occur up to 36 hours after application of the photosensitizer. For protection light impermeable clothes and aluminium foil containing dressings are recommended.

Kinetics of Protoporphyrin IX Formation in Rat Oral Mucosa and Skin After Application of 5‐Aminolevulinic Acid and its Methylester¶

ABSTRACTThe kinetics of accumulation of protoporphyrin IX (PpIX) after topical application of 5‐aminolevulinic acid (ALA) and its methylester (5‐aminolevulinic acid methylester [ALA‐Me]) was studied on rat oral mucosa. The accumulation of PpIX in mucosa and skin after intravenous injection of ALA and ALA‐Me was also studied. The elimination rate of PpIX was dependent on drug and dose as well as on administration route. Application of ALA on rat oral mucosa and skin caused a systemic effect with PpIX building up in remote skin sites not exposed to the drugs. No such systemic effect was seen after application of ALA‐Me either in mucosa or on skin. Intravenous injection of the drugs (0.2 g/kg) leads to more fluorescence in the skin than topical application of the drug (20%). For mucosa, the opposite is true. Maximal PpIX fluorescence appeared later after application of high concentrations of the drugs (around 8 h for 5% and 20% wt/wt) than after application of low concentrations (around 3–5 h for 1% and 2% wt/wt).

ABCG2-mediated transport of photosensitizers: Potential impact on photodynamic therapy

In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered after which the photosensitizer is activated by exposure to a light source of a given wavelength. This, in turn, generates reactive oxygen species that induce cellular apoptosis and necrosis in tumor tissue. Based on our earlier finding that the photosensitizer pheophorbide a is an ABCG2 substrate, we explored the ability of ABCG2 to transport photosensitizers with a structure similar to that of pheophorbide a. ABCG2-overexpressing NCI-H1650 MX50 bronchoalveolar carcinoma cells were found to have reduced intracellular accumulation of pyropheophorbide a methyl ester and chlorin e6 compared to parental cells as measured by flow cytometry. The ABCG2 inhibitor fumitremorgin C was found to abrogate ABCG2-mediated transport. Intracellular fluorescence of hematoporphyrin IX, meso-tetra(3- hydroxyphenyl)porphyrin, and meso-tetra(3-hydroxyphenyl)chlorin was not substantially affected by ABCG2. ABCG2-overexpressing cells also displayed decreased intracellular fluorescence of protoporphyrin IX generated by exogenous application of 5-aminolevulinic acid. Mutations at amino acid 482 in the ABCG2 protein known to affect substrate specificity were not found to impact transport of the photosensitizers. In cytotoxicity assays, ABCG2-transfected HEK-293 cells were 11-fold, 30-fold, 4-fold, and >7-fold resistant to PDT with pheophorbide a, pyropheophorbide a methyl ester, chlorin e6, and ALA, respectively. ABCG2- transfected cells were not resistant to PDT with meso-tetra(3-hydroxyphenyl) chlorin. Neither MRP1 expression nor Pgp expression appreciably decreased the intracellular fluorescence of any of the photosensitizers as measured by flow cytometry. The results presented here implicate ABCG2 as a possible cause for cellular resistance to photodynamic therapy.

Update on the role of laparoscopy in the treatment of gynaecological malignancy

To update the available information and to report on how the recent literature has better defined the role of laparoscopy for the management of gynaecological malignancies. When compared with laparotomy, laparoscopy provides a similar outcome with a shorter hospitalization, an earlier recovery, and an improved quality of life for the treatment of endometrial cancer. Recent reports in the literature on cervical cancer management now include follow-up data; however, only one study included a control group. These studies confirm the feasibility of radical hysterectomy by laparoscopy. The 2-year disease-free and overall survivals were similar in patients treated by laparoscopy and laparotomy in the study that included a control group. The role of laparoscopy for early ovarian cancer is limited by the absence of available data on upstaging. For advanced ovarian carcinoma, new applications of laparoscopy, such as laparoscopic fluorescence detection after intraperitoneal application of 5-aminolevulinic acid, have been reported but the real utility needs further investigation. One of the challenges for the development of laparoscopic surgery is the difficulty for physicians of acquiring advanced laparoscopic surgical skills. The feasibility and safety of laparoscopy for most of the surgical procedures that are used for gynaecological malignancies are now established from cohort or case-control analytical studies. The absence of large phase III studies needs to be balanced by the relatively low incidence of cervical and ovarian cancer.

Kinetics of Protoporphyrin IX Formation in Rat Oral Mucosa and Skin After Application of 5-Aminolevulinic Acid and its Methylester¶

The kinetics of accumulation of protoporphyrin IX (PpIX) after topical application of 5-aminolevulinic acid (ALA) and its methylester (5-aminolevulinic acid methylester [ALA-Me]) was studied on rat oral mucosa. The accumulation of PpIX in mucosa and skin after intravenous injection of ALA and ALA-Me was also studied. The elimination rate of PpIX was dependent on drug and dose as well as on administration route. Application of ALA on rat oral mucosa and skin caused a systemic effect with PpIX building up in remote skin sites not exposed to the drugs. No such systemic effect was seen after application of ALA-Me either in mucosa or on skin. Intravenous injection of the drugs (0.2 g/kg) leads to more fluorescence in the skin than topical application of the drug (20%). For mucosa, the opposite is true. Maximal PpIX fluorescence appeared later after application of high concentrations of the drugs (around 8 h for 5% and 20% wt/wt) than after application of low concentrations (around 3-5 h for 1% and 2% wt/wt).

Technique d’imagerie par fluorescence : intérêt diagnostique et thérapeutique en gynécologie

Fluorescence techniques are presently used by several medical and surgical disciplines (dermatology, pneumology, urology, gastrointestinal surgery) for the diagnosis of pre-cancerous and cancerous lesions. The technique is based on the application of 5-aminolevulinic acid (5-ALA) which induces the production of an endogen photosensitizer: protoporphyrin IX (PpIX). Fluorescence detection of the infraclinical dysplasia lesion is based on the contrast between fluorescent pathological tissue and non-fluorescent healthy tissue. Moreover, there is a correlation between the degree of tissue dysplasia and fluorescence intensity. Fluorescence imaging could allow easier detection of infraclinical lesions enabling more well-targeted treatment. In gynecology, many experimental and clinical studies have been conducted on the detection and treatment of cervical dysplasia and the diagnosis and treatment of vaginal and vulvar diseases as well as the diagnosis of peritoneal micrometastasis from ovarian cancer, the diagnosis and treatment of endometrial alterations, and the treatment of locoregional recurrent skin breast cancer. The aim of this work is to present the fundamental principles of fluorescence imaging technical and to expose the diagnostic and therapeutic prospects in gynecology.

Dose Response of Combination Photorejuvenation Using Intense Pulsed Light–Activated Photodynamic Therapy and Radiofrequency Energy

To report the results of a dose-response study using a novel photorejuvenation regimen consisting of intense pulsed light-activated photodynamic therapy and radiofrequency energy in patients with Fitzpatrick skin types I through III. A combination intense pulsed light and radiofrequency device (Syneron Medical Ltd, Yokneam, Israel) was used in combination with the topical photosensitizer aminolevulinic acid (ALA) (Levulan Kerastick; DUSA Pharmaceuticals Inc, Wilmington, Mass). Duplicate titration trials were performed on the dorsal forearm skin of 3 patients with Fitzpatrick skin types I through III. Multiple treatments, with varying times of application of ALA (30 minutes and 1, 2, and 3 hours) and intense pulsed light fluence (24-30 J/cm(2)), were completed to determine a minimal erythema dose and a maximal tolerated dose based on epidermal reaction (erythema and crusting) and patient discomfort. Radiofrequency energy levels were constant in all treatment groups. The ALA application time of 1 to 2 hours for all skin types and fluence levels of 26 to 28 and 24 to 26 J/cm(2) for Fitzpatrick skin types I/II and III, respectively, were determined to be the minimal erythema dose. The ALA application times of 2 to 3 hours and fluence levels of 28 to 30 and 26 to 28 J/cm(2) for skin types I/II and III, respectively, were determined to be the maximal tolerated dose and resulted in severe erythema and crusting. The presence of severe erythema and discomfort precluded longer ALA exposure and higher fluence levels. No epithelial breakdown was observed at any treatment levels. The dose-response results defining the minimal erythema dose and the maximal tolerated dose of intense pulsed light-activated photodynamic therapy and radiofrequency energy have led to the development of 2 photorejuvenation protocols based on skin type and severity of photodamage.