Bastiaan Kruijt

Monitoring ALA-induced PpIX Photodynamic Therapy in the Rat Esophagus Using Fluorescence and Reflectance Spectroscopy

The presence of phased protoporphyrin IX (PpIX) bleach kinetics has been shown to correlate with esophageal response to 5‐aminolevulinic acid‐based photodynamic therapy (ALA‐PDT) in animal models. Here we confirm the existence of phased PpIX photobleaching by increasing the temporal resolution of the fluorescence measurements using the therapeutic illumination and long wavelength fluorescence detection. Furthermore fluorescence differential pathlength spectroscopy (FDPS) was incorporated to provide information on the effects of PpIX and tissue oxygenation distribution on the PpIX bleach kinetics during illumination. ALA at a dose of 200 mg kg−1 was orally administered to 15 rats, five rats served as control animals. PDT was performed at an in situ measured fluence rate of 75 mW cm−2 using a total fluence of 54 J cm−2. Forty‐eight hours after PDT the esophagus was excised and histologically examined for PDT‐induced damage. Fluence rate and PpIX photobleaching at 705 nm were monitored during therapeutic illumination with the same isotropic probe. A new method, FDPS, was used for superficial measurement on saturation, blood volume, scattering characteristics and PpIX fluorescence. Results showed two‐phased PpIX photobleaching that was not related to a (systematic) change in esophageal oxygenation but was associated with an increase in average blood volume. PpIX fluorescence photobleaching measured using FDPS, in which fluorescence signals are only acquired from the superficial layers of the esophagus, showed lower rates of photobleaching and no distinct phases. No clear correlation between two‐phased photobleaching and histologic tissue response was found. This study demonstrates the feasibility of measuring fluence rate, PpIX fluorescence and FDPS during PDT in the esophagus. We conclude that the spatial distribution of PpIX significantly influences the kinetics of photobleaching and that there is a complex interrelationship between the distribution of PpIX and the supply of oxygen to the illuminated tissue volume.

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.

In vivo quantification of chromophore concentration using fluorescence differential path length spectroscopy

We present an optical method based on fluorescence spectroscopy for measuring chromophore concentrations in vivo. Fluorescence differential path length spectroscopy (FPDS) determines chromophore concentration based on the fluorescence intensity corrected for absorption. The concentration of the photosensitizer m-THPC (Foscan) was studied in vivo in normal rat liver, which is highly vascularized and therefore highly absorbing. Concentration estimates of m-THPC measured by FDPS on the liver are compared with chemical extraction. Twenty-five rats were injected with 0.3 mg kg m-THPC. In vivo optical concentration measurements were performed on tissue 3, 24, 48, and 96 h after m-THPC administration to yield a 10-fold variation in tissue concentration. After the optical measurements, the liver was harvested for chemical extraction. FDPS showed good correlation with chemical extraction. FDPS also showed a correlation between m-THPC fluorescence and blood volume fraction at the two shortest drug-light intervals. This suggests different compartmental localization of m-THPC for different drug-light intervals that can be resolved using fluorescence spectroscopy. Differences in measured m-THPC concentration between FDPS and chemical extraction are related to the interrogation volume of each technique; approximately 0.2 mm(3) and approximately 10(2) mm(3), respectively. This indicates intra-animal variation in m-THPC distribution in the liver on the scale of the FDPS sampling volume.

Ex vivo quantification of mTHPC concentration in tissue: influence of chemical extraction on the optical properties.

A method for the quantification of the concentration of the photosensitizer meso-tetra(hydroxyphenyl) chlorin (mTHPC) in tissue samples is presented. The technique is an extension of a previously published method based on alkaline hydrolysis of tissue, using Solvable as a tissue solubilizer. mTHPC quantification was achieved by subsequent fluorescence spectroscopy. Since the original extraction method involved multiple steps in which water dilution of the sample was implemented, we studied the spectral characteristics of mTHPC in different Solvable/water mixtures. Using UV-VIS absorption and fluorescence spectroscopy, it was demonstrated that the spectral characteristics of mTHPC vary for different Solvable concentrations. In the range of 20-100% Solvable, the fluorescence intensity of mTHPC did not change, while dramatic changes in the mTHPC fluorescence intensity were observed for lower Solvable concentrations (< 20%) due to increasing hydrophilicity of the environment, combined with pH alterations. We also demonstrated that the absorption and fluorescence spectra of the dissolved tissue were time-dependent. Longer incubation of the samples resulted in a significant increase of the native tissue chromophore fluorescence. This implies that for the correct quantification of photosensitizer concentrations, the fluorescence of native tissue chromophores must be accounted for.

Monitoring Interstitial m-THPC-PDT In Vivo Using Fluorescence and Reflectance Spectroscopy

In order to understand the mechanisms of photodynamic therapy (PDT) it is important to monitor parameters during illumination that yield information on deposited PDT dose. The aim of this study is to investigate the possibility of monitoring implicit parameters, such as photobleaching, in addition to monitoring explicit parameters (fluence (rate), oxygenation, photosensitizer concentration) directly or indirectly. These parameters are monitored during PDT without interrupting the therapeutic illumination.

Increase in protoporphyrin IX after 5-aminolevulinic acid based photodynamic therapy is due to local re-synthesis

Protoporphyrin IX (PpIX) fluorescence that is bleached during aminolevulinic acid (ALA) mediated photodynamic therapy (PDT) increases again in time after treatment. In the present study we investigated if this increase in PpIX fluorescence after illumination is the result of local re-synthesis or of systemic redistribution of PpIX. We studied the spatial distribution of PpIX after PDT with and without cooling using the skin-fold observation chamber model. We were unable to show a correlation between the local PpIX fluorescence increase and the distance from a blood vessel. The spatial distribution of PpIX fluorescence within normal tissue or tumour is not changed in response to the illumination. These observations suggest that there is no diffusion of PpIX into the treated tissue. Cooling the tissue to 12 degrees C, a temperature at which PpIX synthesis is inhibited, inhibited the PpIX fluorescence increase normally observed after illumination. We also found a strong correlation between local PpIX photobleaching during illumination and the fluorescence intensity 1 h after illumination similar to what we have observed in patients treated with ALA-PDT. Therefore we conclude that the increase in PpIX fluorescence after illumination is due to local cellular re-synthesis.

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.

Optical Spectroscopy to Guide Photodynamic Therapy of Head and Neck Tumors

In contrast to other interstitial applications of photodynamic therapy (PDT), optical guidance or monitoring in the head and neck is at a very early stage of development. The present paper reviews the use of optical approaches, in particular optical spectroscopy, that have been used or have the potential to guide the application of PDT. When considering the usefulness of these methods, it is important to consider the volume over which these measurements are acquired, the influence of differences in and changes to the background optical properties, the implications for these effects on the measured parameters and the difficulty of incorporating these types of measurements in clinical practice in head and neck PDT. To illustrate these considerations, we present an application of a recently developed technique, which we term fluorescence differential path length spectroscopy for monitoring meta-tetra(hydroxyphenyl)-chlorin or Foscan-PDT of interstitial head and neck cancer.

Light fractionation increases the efficacy of ALA-PDT but not of MAL-PDT: What is the role of (vascular) endothelial cells?

Photodynamic therapy (PDT) using protoporpyrin IX (PpIX) precursors like 5-aminolevulinic acid (ALA) or methyl-aminolevulinate (MAL) has shown to be effective in the treatment of various skin diseases. Using ALA we have shown in numerous studies a significantly improved efficacy by applying light fractionation with a long dark interval. In contrast, in the hairless mouse model, the PDT efficacy using MAL is unaffected by adopting this approach. More acute edema is found after ALA-PDT suggesting a difference in response of endothelial cells to PDT. To investigate the role of endothelial cells, cryo-sections of hairless mouse skin after 4 hours of topical MAL or ALA application were stained with a fluorescent endothelial cell marker (CD31). Co-localization of this marker with the PpIX fluorescence was performed using the spectral imaging function of the confocal microscope. We have also used intra-vital confocal microscopy to image the PpIX fluorescence distribution in correlation with the vasculature of live mouse skin. Our results show PpIX fluorescence at depth in cryo-sections of mouse skin after 4 hours of topical application. Co-localization has shown to be difficult due to the changes in tissue organization caused by the staining procedure. As expected we found high PpIX fluorescence levels in the epidermis after both MAL and ALA application using intra-vital microscopy. After ALA application more PpIX fluorescence was found deep in the dermal layer of the skin than after MAL. Furthermore we detected localized fluorescence in unidentified structures that could not be correlated to blood vessels or nerves.

In vivo quantification of mTHPC fluorescence in skinfold observation chamber using excitation and detection towards the near infrared region

In this study, a ratiometric quantification method is developed and applied to monitor mesotetra(hydroxyphenyl) chlorin (mTHPC) pharmacokinetics in the rat skin-fold observation chamber. The method employs a combination of dual-wavelength excitation and dual-wavelength detection. The excitation and detection wavelengths were selected in close to NIR. The first excitation wavelength was used to excite the mTHPC and autofluorescence and the second to excite only autofluorescence, so that this could be substracted. Subsequently the difference was divided by the autofluorescence. Since the method applies division of signal with no mTHPC fluorescence, theory suggests on linear dependency of the method on photosensitizer concentration.