Kevin R. Diamond

Quantification of fluorophore concentration in tissue-simulating media by fluorescence measurements with a single optical fiber

Quantifying fluorescent compounds in turbid media such as tissue is made difficult by the effects of multiple scattering and absorption of the excitation and emission light. The approach that we used was to measure fluorescence using a single 200-microm optical fiber as both the illumination source and the detector. Fluorescence of aluminum phthalocyanine tetrasulfonate (AlPcS4) was measured over a wide range of fluorophore concentrations and optical properties in tissue-simulating phantoms. A root-mean-square accuracy of 10.6% in AlPcS4 concentration was attainable when fluorescence was measured either interstitially or at the phantom surface. The individual effects of scattering, absorption, and the scattering phase function on the fluorescence signal were also studied by experiments and Monte Carlo simulations.

Noninvasive measurement of fluorophore concentration in turbid media with a simple fluorescence/reflectance ratio technique

Measurement of the concentration of fluorescent compounds in turbid media is difficult because the absorption and multiple scattering of excitation and emission of light has a large effect on the detected fluorescence. For surface measurements with optical fibers we demonstrate by experiments and numerical simulation that this effect can be minimized by measurement of the fluorescence at one source-detector distance, the diffusely reflected excitation light at a second distance, and with the ratio of these two signals as an indicator of fluorophore concentration. For optical properties typical of soft tissue in the red and the near infrared the optimum performance is obtained by measurement of fluorescence at 0.65 mm and reflectance at 1.35 mm. This choice reduces the rms error in fluorophore concentration to 14.6% over a wide range of absorption and scattering coefficients.

Measurement of fluorophore concentrations and fluorescence quantum yield in tissue-simulating phantoms using three diffusion models of steady-state spatially resolved fluorescence

Steady-state diffusion theory models of fluorescence in tissue have been investigated for recovering fluorophore concentrations and fluorescence quantum yield. Spatially resolved fluorescence, excitation and emission reflectance Carlo simulations, and measured using a multi-fibre probe on tissue-simulating phantoms containing either aluminium phthalocyanine tetrasulfonate (AlPcS4), Photofrin meso-tetra-(4-sulfonatophenyl)-porphine dihydrochloride The accuracy of the fluorophore concentration and fluorescence quantum yield recovered by three different models of spatially resolved fluorescence were compared. The models were based on: (a) weighted difference of the excitation and emission reflectance, (b) fluorescence due to a point excitation source or (c) fluorescence due to a pencil beam excitation source. When literature values for the fluorescence quantum yield were used for each of the fluorophores, the fluorophore absorption coefficient (and hence concentration) at the excitation wavelength (mu(a,x,f)) was recovered with a root-mean-square accuracy of 11.4% using the point source model of fluorescence and 8.0% using the more complicated pencil beam excitation model. The accuracy was calculated over a broad range of optical properties and fluorophore concentrations. The weighted difference of reflectance model performed poorly, with a root-mean-square error in concentration of about 50%. Monte Carlo simulations suggest that there are some situations where the weighted difference of reflectance is as accurate as the other two models, although this was not confirmed experimentally. Estimates of the fluorescence quantum yield in multiple scattering media were also made by determining mu(a,x,f) independently from the fitted absorption spectrum and applying the various diffusion theory models. The fluorescence quantum yields for AlPcS4 and TPPS4 were calculated to be 0.59 +/- 0.03 and 0.121 +/- 0.001 respectively using the point source model, and 0.63 +/- 0.03 and 0.129 +/- 0.002 using the pencil beam excitation model. These results are consistent with published values.

Quantification of fluorophore concentration in vivo using two simple fluorescence-based measurement techniques

The effect of photodynamic therapy treatments depends on the concentration of photosensitizer at the treatment site; thus a simple method to quantify concentration is desirable. This study compares the concentration of a fluorophore and sensitizer, aluminum phthalocyanine tetrasulfonate (AlPcS4), measured by two simple fluorescence-based techniques in vivo to post mortem chemical extraction and fluorometric assay of those tissues: skin, muscle, fascia, liver, and kidney (cortex and medulla). Fluorescence was excited and detected by a single optical fiber, or by an instrument that measured the ratio of the fluorescence and excitation reflectance. The in vivo measurements were compared to calibration measurements made in tissue-simulating phantoms to estimate the tissue concentrations. Reasonable agreement was observed between the concentration estimates of the two instruments in the lighter colored tissues (skin, muscle, and fascia). The in vivo measurements also agreed with the chemical extractions at low (< 0.6 microg/g) tissue concentrations, but underestimated higher tissue concentrations. Measurements of fluorescence lifetime in vivo demonstrated that AlPcS4 retains its mono-exponential decay in skin, muscle, and fascia tissues with a lifetime similar to that measured in aqueous tissue-simulating phantoms. In liver and kidney an additional short lifetime component was evident.

Characterization of Fluorescence Lifetime of Photofrin and Delta-Aminolevulinic Acid Induced Protoporphyrin IX in Living Cells Using Single- and Two-Photon Excitation

Photodynamic therapy (PDT) is an effective treatment option for various types of invasive tumors. The efficacy of PDT treatment depends strongly on selective cell uptake and selective excitation of the tumor. The characterization of fluorescence lifetimes of photosensitizers localized inside living cells may provide the basis for further investigation of in vivo PDT dosage measurements using time-domain spectroscopy and imaging. In this communication, we investigated the fluorescence lifetime of localized Photofrin and delta-aminolevulinic acid (ALA) induced protoporphyrin IX (PpIX) in living MAT-LyLu (MLL) rat prostate adenocarcinoma cells. The MLL cells were incubated with the photosensitizers, and then treated with light under well-oxygenated conditions using a two-photon fluorescence lifetime imaging microscope (FLIM). Fluorescence lifetime images of these cells were recorded with average lifetimes of 5.5 plusmn 1.2 ns for Photofrin and 6.3 plusmn 1.2 ns for ALA-induced PpIX. When localized in cells, the lifetimes of both photosensitizers were found to be significantly shorter than those measured in organic solutions. The result for PpIX is consistent with literature values, while the lifetime of Photofrin is shorter than what has been reported. These results suggest that time-domain methods measuring photosensitizer lifetime changes may be good candidates for in vivo PDT dosage monitoring.

Monitoring photosensitizer uptake using two photon fluorescence lifetime imaging microscopy.

Photodynamic Therapy (PDT) provides an opportunity for treatment of various invasive tumors by the use of a cancer targeting photosensitizing agent and light of specific wavelengths. However, real-time monitoring of drug localization is desirable because the induction of the phototoxic effect relies on interplay between the dosage of localized drug and light. Fluorescence emission in PDT may be used to monitor the uptake process but fluorescence intensity is subject to variability due to scattering and absorption; the addition of fluorescence lifetime may be beneficial to probe site-specific drug-molecular interactions and cell damage. We investigated the fluorescence lifetime changes of Photofrin® at various intracellular components in the Mat-LyLu (MLL) cell line. The fluorescence decays were analyzed using a bi-exponential model, followed by segmentation analysis of lifetime parameters. When Photofrin® was localized at the cell membrane, the slow lifetime component was found to be significantly shorter (4.3 ± 0.5 ns) compared to those at other locations (cytoplasm: 7.3 ± 0.3 ns; mitochondria: 7.0 ± 0.2 ns, p < 0.05).

The use of magnetic field effects on photosensitizer luminescence as a novel probe for optical monitoring of oxygen in photodynamic therapy

The effect of a magnetic field on the steady-state and time-resolved optical emission of a custom fullerene-linked photosensitizer (PS) in liposome cell phantoms was studied at various oxygen concentrations (0.19-190 microM). Zeeman splitting of the triplet state and hyperfine coupling, which control intersystem crossing between singlet and triplet states, are altered in the presence of low magnetic fields (B < 320 mT), perturbing the luminescence intensity and lifetime as compared to the triplet state at B = 0. Measurements of the luminescence intensity and lifetime were performed using a time-domain apparatus integrated with a magnet. We propose that by probing magnet-affected optical emissions, one can monitor the state of oxygenation throughout the course of photodynamic therapy. Since the magnetic field effect (MFE) operates primarily by affecting the radical ion pairs related to type I photodynamic action, the enhancement or suppression of the MFE can be used as a measure of the dynamic equilibrium between the type I and II photodynamic pathways. The unique photo-initiated charge-transfer properties of the PS used in this study allow it to serve as both cytotoxic agent and oxygen probe that can provide in situ dosimetric information at close to real time.

Production, review, and impact of technical quality control guidelines in a national context

A close partnership between the Canadian Partnership for Quality Radiotherapy (CPQR) and the Canadian Organization of Medical Physicists (COMP) Quality Assurance and Radiation Safety Advisory Committee (QARSAC) has resulted in the development of a suite of Technical Quality Control (TQC) guidelines for radiation treatment equipment; they outline specific performance objectives and criteria that equipment should meet in order to assure an acceptable level of radiation treatment quality. The adopted framework for the development and maintenance of the TQCs ensures the guidelines incorporate input from the medical physics community during development, measures the workload required to perform the QC tests outlined in each TQC, and remain relevant (i.e., “living documents”) through subsequent planned reviews and updates. The framework includes consolidation of existing guidelines and/or literature by expert reviewers, structured stages of public review, external field‐testing, and ratification by COMP. This TQC development framework is a cross‐country initiative that allows for rapid development of robust, community‐driven living guideline documents that are owned by the community and reviewed to keep relevant in a rapidly evolving technical environment. Community engagement and uptake survey data shows 70% of Canadian centers are part of this process and that the data in the guideline documents reflect, and are influencing, the way Canadian radiation treatment centers run their technical quality control programs. For a medium‐sized center comprising six linear accelerators and a comprehensive brachytherapy program, we evaluate the physics workload to 1.5 full‐time equivalent physicists per year to complete all QC tests listed in this suite. PACS number(s): 87.55.Qr, 87.56.Fc, 87.56.‐v

Effect of liposomal confinement on photochemical properties of photosensitizers with varying hydrophilicity

Preferential tumor localization and the aggregation state of photosensitizers (PSs) can depend on the hydrophilic/hydrophobic nature of the molecule and affect their phototoxicity. In this study, three PSs of different hydrophilicity are introduced in liposomes to understand the structure-photochemistry relationship of PSs in this cellular model system. Absorbance and fluorescence spectra of amphiphilic aluminum (III) phthalocyanine disulfonate chloride adjacent isomer (Al-2), hydrophilic aluminum (III) phthalocyanine chloride tetrasulfonic acid (Al-4), and lipophilic 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH) are compared in a liposomal confined state with free PS in bulk solution. For fluorescence measurements, a broad range of concentrations of both bulk and liposomal confined PSs are examined to track the transition from monomers to dimers or higher order aggregates. Epifluorescence microscopy, absorbance, and fluorescence measurements all confirm different localization of the PSs in liposomes, depending on their hydrophilicity. In turn, the localization affects the aggregation of molecules inside the liposome cell model. Data obtained with such cellular models could be useful in optimizing the photochemical properties of photosensitizing drugs based on their structure-dependent interactions with cellular media and subcellular organelles.

Evaluation of a ferrous benzoic xylenol orange transparent PVA cryogel radiochromic dosimeter

A stable cryogel dosimeter was prepared using ferrous benzoic xylenol orange (FBX) in a transparent poly-(vinyl alcohol) (PVA) cryogel matrix. Dose response was evaluated for different numbers of freeze-thaw cycles (FTCs), different concentrations of PVA, and ratios of water/dimethyl sulfoxide. Linear relationships between dose and absorbance were obtained in the range of 0-1000 cGy for all formulations. Increasing the concentration of PVA and number of FTCs resulted in increased absorbance and sensitivity. The effects of energy and dose rate were also evaluated. No significant dose rate dependence was observed over the range 1.05 to 6.33 Gy min(-1). No energy response was observed over photon energies of 6, 10, and 18 MV.