Monitoring Of Photodynamic Therapy Research Articles

Tumor fluorescence and oxygenation monitoring during photodynamic therapy with chlorin e6 photosensitizer

BackgroundThe study is aimed at developing a method for monitoring photodynamic therapy (PDT) of a tumor using chlorin-type photosensitizers (PSs). Lack of monitoring of chlorin e6 (Cе6) photobleaching, hemoglobin oxygenation and blood flow during light exposure can limit the PDT effectiveness. Materials and methodsPhototheranostics includes spectral-fluorescence diagnostics of Ce6 distribution in the NIR range and PDT with simultaneous assessment of hemoglobin oxygenation and tumor blood flow. Fluorescence diagnostics and PDT were performed using the single laser λexc=660 ± 5 nm. ResultsCombined spectroscopic PDT monitoring method allowed simultaneous estimation of Ce6 photobleaching, hemoglobin oxygenation and tumor vascular thrombosis during PDT without interrupting the therapeutic light exposure. ConclusionThe developed method of tumor phototheranostics using chlorin-type PSs may make it possible to personalize the duration of therapeutic light exposure during PDT.

Time-gated luminescent probes for lysosomal singlet oxygen: Synthesis, characterizations and bioimaging applications

BackgroudSingle oxygen (1O2), the molecular oxygen at its excited state, plays a crucial role in the photodynamic therapy (PDT) of some diseases owing to its strong oxidizing property to destroy malignant cells. Although the fluorescent probe technique has proven its powerful application abilities for detection of 1O2 in biological systems, most of the reported fluorescent probes suffered from the interference of background autofluorescence of biological samples. It is clear that the real-time and in situ, background-free fluorescent detection of 1O2 generated in live cells, especially in some organelles, is of great significance for understanding the action mechanism of PDT drugs. ResultsBy introducing a lysosome-anchoring motif, a morpholine moiety, into a 1O2-specifically-reactive terpyridine polyacid ligand, [4’-(9-anthryl)-2,2’:6′,2″-terpyridine-6,6″-diyl] bis(methylenenitrilo) tetrakis (acetic acid) (ATTA), and chelating with lanthanide ions (Eu3+ or Tb3+), two lanthanide complex-based “turn-on” luminescent probes that can be used for the background-free time-gated luminescent (TGL) detection of lysosomal 1O2, Lyso-ATTA-Eu3+ and Lyso-ATTA-Tb3+, have been developed. The probes exhibit fast luminescence responses (within 2.5 min) towards 1O2 with high selectivity and sensitivity (<0.75 μM) in a wide pH range (4–11). And the excellent lysosome-localization performance of the probes allowed them to be used for the monitoring of endogenous 1O2 in lysosomes, which enabled the variability of lysosomal-1O2 concentrations induced by different photosensitizers to be successfully discriminated. Furthermore, by doping Lyso-ATTA-Eu3+ into the polyethylene glycol (PEG) hydrogel, the smart luminescent sensor film, PEG-Lyso-ATTA-Eu3+, was prepared, and successfully used for the detection of the on-site 1O2 production during the PDT process of psoriatic disease in model mice. SignificantTwo lysosome-targetable background-free TGL probes for 1O2 were firstly reported. The developed smart luminescent sensor film could be a powerful tool for the clinical monitoring of PDT on skin diseases without using sophisticated and expensive instruments.

Photo and spectral fluorescence analysis of the spinal cord injury area in animal models

The purpose of the work is to follow the dynamics of changes in fluorescent signals in the near-surface layers of tissue of injured areas of the back of laboratory animals, which will allow, by indirect evidence, to evaluate the information content of fluorescence diagnosis for subsequent possible diagnostic monitoring of photodynamic therapy of the spinal cord. The model animals were Wistar rats. Two types of contusions were modeled: pneumo-contusion and contusion by a falling load. Methylene blue and indocyanine green were used as photosensitizers. Fluorescence measurements were carried out by imaging and spectrometric methods. A stroboscopic fluorescence imager with an excitation wavelength of 630 nm was used to acquire fluorescence images. The LESA-01-BIOSPEC spectrometer with a He-Ne laser excitation allowed to obtain spectra. It was shown that both methods make it possible to estimate the fluorescence value of methylene blue and indocyanine green in the tissues under study. Moreover, the photographic method also allows to obtain the spatial distribution of fluorescence. The general trend found in the data is a more intense and uniform fluorescence of the dorsal region of rats with methylene blue and a less intense, but more contrasting distribution of indocyanine green. The presented methods are non-invasive, which makes them attractive for diagnostic use. However, due to the shallow depth of signal reception, the condition of the spine can be determined only indirectly, by the condition of the near-surface layers of tissue that accumulate the photosensitizer.

Clinical assessment of a low-cost, hand-held, smartphone-attached intraoral imaging probe for 5-aminolevulinic acid photodynamic therapy monitoring and guidance.

India has one of the highest rates of oral squamous cell carcinoma (OSCC) in the world, with an incidence of 15 per 100,000 and more than 70,000 deaths per year. The problem is exacerbated by a lack of medical infrastructure and routine screening, especially in rural areas. New technologies for oral cancer detection and timely treatment at the point of care are urgently needed. Our study aimed to use a hand-held smartphone-coupled intraoral imaging device, previously investigated for autofluorescence (auto-FL) diagnostics adapted here for treatment guidance and monitoring photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence (FL). A total of 12 patients with 14 buccal mucosal lesions having moderately/well-differentiated micro-invasive OSCC lesions ( diameter and depth) were systemically (in oral solution) administered three doses of ALA (total ). Lesion site PpIX and auto-FL were imaged using the multichannel FL and polarized white-light oral cancer imaging probe before/after ALA administration and after light delivery (fractionated, total of 635nm red LED light). The handheld device was conducive for access to lesion site images in the oral cavity. Segmentation of ratiometric images in which PpIX FL is mapped relative to auto-FL enabled improved demarcation of lesion boundaries relative to PpIX alone. A relative FL (-value) threshold of 1.4 was found to segment lesion site PpIX production among the patients with mild to severe dysplasia malignancy. The segmented lesion size is well correlated with ultrasound findings. Lesions for which -value was at the time of treatment were associated with successful outcomes. These results indicate the utility of a low-cost, handheld intraoral imaging probe for image-guided PDT and treatment monitoring while also laying the groundwork for an integrated approach, combining cancer screening and treatment with the same hardware.

Agar phantoms of biological tissue for fluorescence monitoring of photodynamic therapy

An approach to fabricating agar phantoms mimicking spectral optical properties of biological tissues with fluorescent inclusions is proposed, which allows one to imitate the problem of optical visualisation of superficial biological tissues after the administration of a chlorin-based photosensitiser. The different arrangement of a fluorescent layer within a phantom makes it possible to simulate biological tissue in the cases of both topical application and intravenous injection of a photosensitiser. It is shown that absorption and scattering spectra of phantoms are in good agreement with the spectra of real biological tissues in the wavelength range of 500 – 800 nm. Changes in spectra of absorption and scattering coefficients of phantoms, as well as in their fluorescent properties induced by the addition of a fluorescent marker (chlorin-based photosensitiser) are demonstrated.

Dual-Wavelength Fluorescence Monitoring of Photodynamic Therapy: From Analytical Models to Clinical Studies.

Simple SummaryFluorescence imaging is an efficient tool in monitoring photodynamic therapy procedures allowing us to track accumulation and photobleaching of a photosensitizer (PS). Chlorin-based PSs feature high absorption in the red and blue bands of visible spectrum. Due to spectral dispersion of light penetration depth in biotissues, fluorescence signals registered upon excitation by red or blue light are formed in different measurement volumes. We present analytical and numerical models of dual-wavelength fluorescence imaging for evaluation of PS localization depth in the cases of topical administration and intravenous injection. The results of analytical and numerical simulations are in good agreement with the phantom experiments, and are translated to the in vivo imaging, which allows to interpret experimental observations in animal trials, human volunteers, and clinical studies. The proposed approach allows us to noninvasively estimate typical accumulation depths of PS localization which are consistent with the morphologically expected values.Fluorescence imaging modalities are currently a routine tool for the assessment of marker distribution within biological tissues, including monitoring of fluorescent photosensitizers (PSs) in photodynamic therapy (PDT). Conventional fluorescence imaging techniques provide en-face two-dimensional images, while depth-resolved techniques require complicated tomographic modalities. In this paper, we report on a cost-effective approach for the estimation of fluorophore localization depth based on dual-wavelength probing. Owing to significant difference in optical properties of superficial biotissues for red and blue ranges of optical spectra, simultaneous detection of fluorescence excited at different wavelengths provides complementary information from different measurement volumes. Here, we report analytical and numerical models of the dual-wavelength fluorescence imaging of PS-containing biotissues considering topical and intravenous PS administration, and demonstrate the feasibility of this approach for evaluation of the PS localization depth based on the fluorescence signal ratio. The results of analytical and numerical simulations, as well as phantom experiments, were translated to the in vivo imaging to interpret experimental observations in animal experiments, human volunteers, and clinical studies. The proposed approach allowed us to estimate typical accumulation depths of PS localization which are consistent with the morphologically expected values for both topical PS administration and intravenous injection.

High optical-throughput spectroscopic singlet oxygen and photosensitizer luminescence dosimeter for monitoring of photodynamic therapy.

We report a high light-throughput spectroscopic dosimeter system that is able to noninvasively measure luminescence signals of singlet oxygen (1 O2 ) produced during photodynamic therapy (PDT) using a CW (continuous wave) light source. The system is based on a compact, fiber-coupled, high collection efficiency spectrometer (>50% transmittance) designed to maximize optical throughput but with sufficient spectral resolution (~7nm). This is adequate to detect 1 O2 phosphorescence in the presence of strong luminescence background in vivo. This system provides simultaneous acquisition of multiple spectral data points, allowing for more accurate determination of luminescence baseline via spectral fitting and thus the extraction of 1 O2 phosphorescence signal based solely on spectroscopic decomposition, without the need for time-gating. Simultaneous collection of photons at different wavelengths improves the quantum efficiency of the system when compared to sequential spectral measurements such as filter-wheel or tunable-filter based systems. A prototype system was tested during in vivo PDT tumor regression experiments using benzoporphyrin derivative (BPD) photosensitizer. It was found that the treatment efficacy (tumor growth inhibition rate) correlated more strongly with 1 O2 phosphorescence than with PS fluorescence. These results indicate that this high photon-collection efficiency spectrometer instrument may offer a viable option for real-time 1 O2 dosimetry during PDT treatment using CW light.

Combined Application of Dual-Wavelength Fluorescence Monitoring and Contactless Thermometry during Photodynamic Therapy of Basal Cell Skin Cancer.

The aim of the study was to assess the capabilities of combined application of dual-wavelength fluorescence visualization and contactless skin thermometry during photodynamic therapy monitoring (PDT) of basal cell cancer.Materials and Methods.The study was performed at the University Clinic of Privolzhsky Research Medical University (Nizhny Novgorod). Nine clinically, dermatoscopically, and histologically verified foci of basal cell skin cancer were exposed to PDT sessions (wavelength of 662 nm, light dose density of 150 J/cm2) with systemic application of chlorin-based photosensitizer Fotoditazin. A semiconductor laser system Latus-T (Russia) was employed for irradiation. Dual-wavelength fluorescence visualization and contactless thermometry with an IR pyrometer were used to monitor the PDT sessions.Results.The PDT sessions of nine foci of basal cell cancer were carried out under the control of fluorescence imaging and contactless thermometry. Photosensitizer photobleaching in all foci amounted to 40% signifying a percent of photosensitizer involved in the photodynamic reaction. It has been shown that the combined employment of dual-wavelength fluorescence monitoring and contactless thermometry during the PDT of basal cell skin cancer allows oncologists to control simultaneously the degree of photosensitizer photobleaching and the depth of the photodynamic effect in tissues, the extent of involving the mechanisms associated with hyperthermia as well as the correctness of the procedure conducting. In the course of 9-month dynamic follow-up after the treatment, no clinical and dermatoscopic signs of recurrence were found.Conclusion.A bimodal control of PDT enables the assessment of the correctness and efficacy of the procedure performance. The contactless control of tissue heating allows ensuring the temperature mode for hyperthermia realization, while the fluorescence monitoring makes it possible to evaluate the accumulation of the photosensitizer in the tumor and the depth of the PDT action as well as to predict the procedure efficacy based on the photobleaching data. The complementary use of these techniques allows the adjustment of the mode directly in the course of the PDT procedure. The acquisition of the sufficient statistical data on the combined monitoring will result in the development of a novel PDT protocol.

Towards Bimodal Optical Monitoring of Photodynamic Therapy with Targeted Nanoconstructs: A Phantom Study

Increase of the efficiency of photodynamic therapy (PDT) requires the development of advanced protocols employing both novel photosensitizer (PS) carriers and aids for online monitoring. Nanoconstructs may be comprised of a photosensitizer, chemotherapy drugs, or inhibitors of molecular pathways that support cancer growth. In this paper, we analyze the efficiency of a bimodal approach involving fluorescence and optoacoustic imaging in monitoring drug distribution and photobleaching. The study evaluates typical sensitivities of these techniques to the presence of the two key moieties of a nanoconstruct: benzoporphyrin derivatives (BPD) serving as a PS, and IRDye800 acting as a contrast agent. Both imaging modalities employ dual-wavelength probing at the wavelengths corresponding to absorption peaks of BPD and IRDye800, which enables their separate detection. In an experiment on a tissue-mimicking phantom with inclusions containing separate BPD and IRDye800 solutions, fluorescence imaging demonstrated higher contrast as compared to optoacoustic imaging for both components, though strong light scattering in the surrounding media restricted accurate localization of the markers. It was also sensitive to photobleaching, which is a measure of PDT efficiency. Optoacoustic imaging demonstrated sufficient sensitivity to both components, though less than that of fluorescence imaging, however, it enabled depth-resolved detection of an absorber and estimation of its relative content. Employment of the bimodal approach in monitoring of PS photobleaching adds to its potential in intraprocedural PDT monitoring.

Complementary bimodal approach to monitoring of photodynamic therapy with targeted nanoconstructs: numerical simulations

We propose a new approach to monitoring photodynamic therapy (PDT) of glioblastoma with the use of targeted nanoconstructs containing a photosensitiser (PS) [benzoporphyrin derivative (BPD)] and IRDye800 dye, antibodies for efficient accumulation of the drug in a tumour, and a chemotherapeutic agent for combined effect on tumour cells. Application of IRDye800 is determined by the shift of its absorption and fluorescence spectra to the near-IR range with respect to BPD that ensures a higher imaging depth. Monitoring of PDT is based on the simultaneous fluorescence and optoacoustic (OA) imaging. Fluorescence imaging provides visualisation of fluorescence agents with high molecular sensitivity and monitoring of the effectiveness of PDT by PS photobleaching. OA allows one to examine the vascular pattern of the tumour environment, as well as assess the depth of the tumour. IRDye800 is a better contrast agent in comparison to BPD due to red-shifted spectral characteristics and a higher fluorescence quantum yield. Monte Carlo simulations combined for OA simulations with K-wave modelling allowed the feasibility of the complementary approach to be studied and demonstrated that this combination allows one to localise a tumour with a size of 2 mm at depths from 100 μm to 2 mm.

To question of increasing of efficiency of photodynamic therapy of tumors on the basis of multispectral treatment and diagnostic device

Damage to tumor cells as a result of photodynamic therapy is a multi-stage process, the effectiveness of which depends on a number of factors, and in particular on the properties of the photosensitizer used and the parameters of light irradiation of the tissues that it has colored. Nevertheless, the experience gained over decades of using photodynamic therapy in the clinic indicates a high heterogeneity of the tumor response to photodynamic therapy. Thus, there is an urgent need to create hardware complexes capable of real-time record- ing the effectiveness of photodynamic therapy monitoring by monitoring its key parameters, and, if possible, automatically adjust the scheme of the procedure, contributing to the creation of an individualized approach in the treatment of neoplasms. The paper considers a method for increasing the efficiency of the photodynamic therapy method by devel- oping a prototype of such a diagnostic and treatment unit based on a combination of the principles of optical exposure and optical diagnostics. The device provides the possibility of non-invasive monitoring of the accu- mulation kinetics of photosensitizer in the tumor zone; determine the contrast of its accumulation relative to healthy tissues; control of tumor oxygenation at all stages of photodynamic therapy ; and also the effect on the tumor by optical radiation with a wavelength corresponding to the absorption peak used by the photosensitizer. From the values of these parameters, the course of the final cascade of cytotoxic and inflammatory reactions in the tumor and the mechanism of restoring damage after therapy are mainly dependent.

Photodynamic therapy monitoring with optical coherence angiography

Photodynamic therapy (PDT) is a promising modern approach for cancer therapy with low normal tissue toxicity. This study was focused on a vascular-targeting Chlorine E6 mediated PDT. A new angiographic imaging approach known as M-mode-like optical coherence angiography (MML-OCA) was able to sensitively detect PDT-induced microvascular alterations in the mouse ear tumour model CT26. Histological analysis showed that the main mechanisms of vascular PDT was thrombosis of blood vessels and hemorrhage, which agrees with angiographic imaging by MML-OCA. Relationship between MML-OCA-detected early microvascular damage post PDT (within 24 hours) and tumour regression/regrowth was confirmed by histology. The advantages of MML-OCA such as direct image acquisition, fast processing, robust and affordable system opto-electronics, and label-free high contrast 3D visualization of the microvasculature suggest attractive possibilities of this method in practical clinical monitoring of cancer therapies with microvascular involvement.

Fluorescent Monitoring of Photodynamic Therapy for Skin Cancer in Clinical Practice

The aim of the investigation was to assess the fluorescent imaging capabilities to monitor photodynamic therapy (PDT) of non-melanoma skin cancer, and study the correlation of PS (photosensitizer) fluorescence value (accumulation and photobleaching rate) and efficacy of the treatment provided. Materials and Methods. The study was conducted in the Nizhny Novgorod Regional Oncologic Hospital. We analyzed fluorescent images and PDT outcomes in 226 patients with non-melanoma skin carcinomas. Results. The assessment of short-term treatment results revealed the relationship between PS photobleaching and tumor complete response rate: a complete response was found in 89% cases in complete photobleaching, in 87% cases — in partial photobleaching, and in 81% — in no photobleaching (p>0.05). However, we found no effect of PS accumulation rate on complete response rate. The analysis of long-term results with significant difference (p=0.044) showed tumor recurrence rate in low PS concentration (tumor/norm) to be 9.5%, while in moderate and high concentrations the recurrence rate appeared to be 4.1%. There was revealed the tendency for recurrence rate increase — 10.4% with no PS photobleaching versus 4.4% in PS complete and partial photobleaching (p=0.051). The patients with high accumulation rate and complete PS photobleaching had the best clinical findings, the observation period being from 4 to 40 months. Conclusion. Fluorescent monitoring enables to maintain noninvasive control of PS accumulation and photobleaching that can contribute to the selection of individual laser exposure parameters. It is reasonable to develop multimodal bioimaging for follow-up real-time monitoring of basic photodynamic reactions and treatment results.

Noninvasive monitoring of photodynamic therapy on skin neoplastic lesions using the optical attenuation coefficient measured by optical coherence tomography.

Photodynamic therapy (PDT) has become a promising alternative for treatment of skin lesions such as squamous cell carcinoma. We propose a method to monitor the effects of PDT in a noninvasive way by using the optical attenuation coefficient (OAC) calculated from optical coherence tomography (OCT) images. We conducted a study on mice with chemically induced neoplastic lesions and performed PDT on these lesions using homemade photosensitizers. The response of neoplastic lesions to therapy was monitored using, at the same time, macroscopic clinical visualization, histopathological analysis, OCT imaging, and OCT-based attenuation coefficient measurement. Results with all four modalities demonstrated a positive response to treatment. The attenuation coefficient was found to be 1.4 higher in skin lesions than in healthy tissue and it decreased after therapy. This study shows that the OAC is a potential tool to noninvasively assess the evolution of skin neoplastic lesions with time after treatment.

Imaging a photodynamic therapy photosensitizerin vivowith a time-gated fluorescence tomography system

We report the tomographic imaging of a photodynamic therapy (PDT) photosensitizer, 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH) in vivo with time-domain fluorescence diffuse optical tomography (TD-FDOT). Simultaneous reconstruction of fluorescence yield and lifetime of HPPH was performed before and after PDT. The methodology was validated in phantom experiments, and depth-resolved in vivo imaging was achieved through simultaneous three-dimensional (3-D) mappings of fluorescence yield and lifetime contrasts. The tomographic images of a human head-and-neck xenograft in a mouse confirmed the preferential uptake and retention of HPPH by the tumor 24-h post-injection. HPPH-mediated PDT induced significant changes in fluorescence yield and lifetime. This pilot study demonstrates that TD-FDOT may be a good imaging modality for assessing photosensitizer distributions in deep tissue during PDT monitoring.

Spatial photosensitizer fluorescence emission predictive analysis for photodynamic therapy monitoring applied to a skin disease

The development of Photodynamic Therapy (PDT) predictive models has become a valuable tool for an optimal treatment planning, monitoring and dosimetry adjustment. A few attempts have achieved a quite complete characterization of the complex photochemical and photophysical processes involved, even taking into account superficial fluorescence in the target tissue. The present work is devoted to the application of a predictive PDT model to obtain fluorescence tomography information during PDT when applied to a skin disease. The model takes into account the optical radiation distribution, a non-homogeneous topical photosensitizer distribution, the time dependent photochemical interaction and the photosensitizer fluorescence emission. The results show the spatial evolution of the photosensitizer fluorescence emission and the amount of singlet oxygen produced during PDT. The depth dependent photosensitizer fluorescence emission obtained is essential to estimate the spatial photosensitizer concentration and its degradation due to photobleaching. As a consequence the proposed approach could be used to predict the photosensitizer fluorescence tomographic measurements during PDT. The singlet oxygen prediction could also be employed as a valuable tool to predict the short term treatment outcome.

Non-invasive monitoring of photodynamic therapy of oral cancers by fluorescence differential path-length spectroscopy

Non-invasive monitoring of photodynamic therapy of oral cancers by fluorescence differential path-length spectroscopy

PL10 Violet (‘blue’) laser acupuncture research and computer-based heart rate and heart rate variability analysis

The objective of this study was to develop an applicator for delivery of light and monitoring of photodynamic therapy (PDT) in the anal cavity for treatment of anal intraepithelial neoplasia grade III (AIN III), which can progress to invasive anal cancer.Forty-eight hours before treatment, patients participating in the study were injected with 0.03 (n = 2) or 0.075 (n = 2) mg kg−1 m-THPC. For light delivery and monitoring of PDT, an applicator based on standard anoscopy equipment was developed which facilitates, in addition to a light treatment fiber, fiber optic probes to monitor blood saturation, blood volume, fluorescence and fluence (rate) at two different locations in situ. Patients were given a light dose of 10–17 J cm−2 at a fluence rate of 45–50 mW cm−2 based on in situ measured light treatment parameters.We demonstrate that the applicator does not influence the fluence rate profile of the light treatment fiber. Furthermore this study shows the possibility of monitoring blood saturation, blood volume, fluorescence and fluence (rate) during therapeutic illumination without changing the light treatment protocol.

Monitoring photobleaching and hemodynamic responses to HPPH-mediated photodynamic therapy of head and neck cancer: a case report

We present initial results obtained during the course of a Phase I clinical trial of 2-1[hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH)-mediated photo-dynamic therapy (PDT) in a head and neck cancer patient. We quantified blood flow, oxygenation and HPPH drug photobleaching before and after therapeutic light treatment by utilizing fast, non-invasive diffuse optical methods. Our results showed that HPPH-PDT induced significant drug photobleaching, and reduction in blood flow and oxygenation suggesting significant vascular and cellular reaction. These changes were accompanied by cross-linking of the signal transducer and activator of transcription 3 (STAT3), a molecular measure for the oxidative photoreaction. These preliminary results suggest diffuse optical spectroscopies permit non-invasive monitoring of PDT in clinical settings of head and neck cancer patients.

A dedicated applicator for light delivery and monitoring of PDT of intra-anal intraepithelial neoplasia

The objective of this study was to develop an applicator for delivery of light and monitoring of photodynamic therapy (PDT) in the anal cavity for treatment of anal intraepithelial neoplasia grade III (AIN III), which can progress to invasive anal cancer. Forty-eight hours before treatment, patients participating in the study were injected with 0.03 (n=2) or 0.075 (n=2) mg kg(-1) m-THPC. For light delivery and monitoring of PDT, an applicator based on standard anoscopy equipment was developed which facilitates, in addition to a light treatment fiber, fiber optic probes to monitor blood saturation, blood volume, fluorescence and fluence (rate) at two different locations in situ. Patients were given a light dose of 10-17 J cm(-2) at a fluence rate of 45-50 mW cm(-2) based on in situ measured light treatment parameters. We demonstrate that the applicator does not influence the fluence rate profile of the light treatment fiber. Furthermore this study shows the possibility of monitoring blood saturation, blood volume, fluorescence and fluence (rate) during therapeutic illumination without changing the light treatment protocol.