Michel Fortin

Reference optical phantoms for diffuse optical spectroscopy. Part 1 – Error analysis of a time resolved transmittance characterization method

Development, production quality control and calibration of optical tissue-mimicking phantoms require a convenient and robust characterization method with known absolute accuracy. We present a solid phantom characterization technique based on time resolved transmittance measurement of light through a relatively small phantom sample. The small size of the sample enables characterization of every material batch produced in a routine phantoms production. Time resolved transmittance data are pre-processed to correct for dark noise, sample thickness and instrument response function. Pre-processed data are then compared to a forward model based on the radiative transfer equation solved through Monte Carlo simulations accurately taking into account the finite geometry of the sample. The computational burden of the Monte-Carlo technique was alleviated by building a lookup table of pre-computed results and using interpolation to obtain modeled transmittance traces at intermediate values of the optical properties. Near perfect fit residuals are obtained with a fit window using all data above 1% of the maximum value of the time resolved transmittance trace. Absolute accuracy of the method is estimated through a thorough error analysis which takes into account the following contributions: measurement noise, system repeatability, instrument response function stability, sample thickness variation refractive index inaccuracy, time correlated single photon counting system time based inaccuracy and forward model inaccuracy. Two sigma absolute error estimates of 0.01 cm(-1) (11.3%) and 0.67 cm(-1) (6.8%) are obtained for the absorption coefficient and reduced scattering coefficient respectively.

Design and modeling of a prototype fiber scanning CARS endoscope

An endoscope capable of Coherent Anti-Stokes Raman scattering (CARS) imaging would be of significant clinical value for improving early detection of endoluminal cancers. However, developing this technology is challenging for many reasons. First, nonlinear imaging techniques such as CARS are single point measurements thus requiring fast scanning in a small footprint if video rate is to be achieved. Moreover, the intrinsic nonlinearity of this modality imposes several technical constraints and limitations, mainly related to pulse and beam distortions that occur within the optical fiber and the focusing objective. Here, we describe the design and report modeling results of a new CARS endoscope. The miniature microscope objective design and its anticipated performance are presented, along with its compatibility with a new spiral scanningfiber imaging technology developed at the University of Washington. This technology has ideal attributes for clinical use, with its small footprint, adjustable field-of-view and high spatial-resolution. This compact hybrid fiber-based endoscopic CARS imaging design is anticipated to have a wide clinical applicability.

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.

Effect of liposomal confinement on photothermal and photo-oximetric fluorescence lifetimes of photosensitizers with varying hydrophilicity

The time-resolved fluorescence of photosensitizers (PSs) of varying hydrophobicities, di-and tetrasulfonated Al phthalocyanines (Al-2 and Al-4), and Photochlor (HPPH), was investigated in liposomes used as cell-mimetic models. Using frequency-and time-domain apparatus, the fluorescence lifetime, tau(fluo), was compared for PSs free in aqueous solution and in a liposome-associated state at varied temperatures (25 to 78 degrees C) and oxygen concentrations (0-190 microM). The analysis of tau(fluo) revealed different decay behaviors for the free-solution and liposome-confined PSs, most significantly for the lipophilic HPPH. Hydrophilic PS drugs (Al-4, Al-2) were less affected by the liposomal confinement, depending on the relative hydrophilicity of the compound and the consequent localization in liposomes. Changes in the emission decay due to confinement were detected as differences in the lifetime between the bulk solution and the liposome-localized PS in response to heating and deoxygenation. Specifically, hydrophilic Al-4 produced an identical lifetime trend as a function of temperature both in solu and in a liposome-confined state. Hydrophobic HPPH exhibited a fundamental transformation in its fluorescence decay kinetics, transitioning from a multiexponential (in free solution) to single-exponential (in liposome) decay. Deoxygenation resulted in a ubiquitous tau(fluo) increase for all PSs in free solution, while the opposite, a tau(fluo) decrease, occurred in all liposomal PSs.

Development of optical phantoms for use in fluorescence-based imaging

We fabricated permanent solid polyurethane-based phantoms in which fluorophores were homogeneously incorporated. For this study, fluorophores of three different families were used: Cyanines, Alexa Fluor and Quantum Dots. The goal of this study was to evaluate the impact of casting the fluorophores in a polyurethane matrix on their optical properties, more specifically the absorbance, molecular extinction coefficient, emission of fluorescence and the resultant fluorescence intensity. All measurements were carried out with 5 concentrations of each fluorophores embedded in polyurethane and in solution. Stability over time was also monitored for a three months period. The casting of fluorophores affects the optical properties of the three dyes under study. The max absorbance, the fluorescence emission and intensity along with the molar extinction coefficient were all affected. Quantum dots behave differently to the cyanine and Alexa Fluor dyes. It was also observed that the incorporation of dyes enables long-term stability of the fluorescence signal.

Time-resolved luminescence measurements of the magnetic field effect on paramagnetic photosensitizers in photodynamic reactions

The development of multimodal molecular probes and photosensitizing agents for use in photodynamic therapy (PDT) is vital for optimizing and monitoring cytotoxic responses. We propose a combinatorial approach utilizing photosensitizing molecules that are both paramagnetic and luminescent with multimodal functionality to perturb, control, and monitor molecular-scale reaction pathways in PDT. To this end, a time-domain single photon counting lifetime apparatus with a 400 nm excitation source has been developed and integrated with a variable low field magnet (0- 350mT). The luminescence lifetime decay function was measured in the presence of a sweeping magnetic field for a custom designed photosensitizing molecule in which photoinduced electron transfer was studied The photosensitizer studied was a donor-acceptor complex synthesized using a porphyrin linked to a fullerene molecule. The magneto-optic properties were investigated for the free-base photosensitizer complex as well as those containing either diamagnetic (paired electron) or paramagnetic (unpaired electron) metal centers, Zn(II) and Cu(II). The magnetic field was employed to affect and modify the spin states of radical pairs of the photosensitizing agents via magnetically induced hyperfine and Zeeman effects. Since the Type 1 reaction pathway of an excited triplet state photosensitizer involves the production of radical species, lifetime measurements were conducted at low dissolved oxygen concentration (0.01ppm) to elucidate the dependence of the magnetic perturbation on the photosensitization mechanistic pathway. To optimize the magnetic response, a solvent study was performed examining the dependence of the emission properties on the magnetic field in solutions of varying dielectric constants. Lastly, the cytotoxicity in murine tumor cell suspensions was investigated for the novel porphyrin-fullerene complex by inducing photodynamic treatments and determining the associated cell survival.

The effects of self-absorption and detection geometry on fluorescence intensity and decay lifetime

This paper presents a theoretical model of the effect of the geometry of illumination and collection in fluorescent media, which exhibit self-absorption at sufficiently high concentrations. In order to derive a relation between the incident excitation intensity and the fluorescence emission intensity, we consider the series of paths and transformations that light takes between the source and the detector. The preliminary supporting experiments were conducted on non-turbid liquid fluorescent samples using classical right-angle detection scheme, based on Time-Correlated Single Photon Counting (TCSPC). The fluorescent dyes tested in these experiments (Coumarins 1, 314 and 343) were chosen because they all are excitable at 405 nm, and exhibit varying Stokes shifts. The results suggest that the geometry of the illumination and collection, as well as the self-absorption process, should be taken into account in time-resolved and intensity fluorescence measurements.

Uncertainty analysis of time resolved transmittance characterization of solid tissue phantoms

Solid tissue phantom are the preferred tool for the development, validation, testing and calibration of photon migration instrument. Accuracy, or trueness, of the optical properties of reference phantoms is of the utmost importance as they will be used as the conventional true value against which instrument errors will be evaluated. A detailed quantitative analysis of the uncertainty of time-resolved transmittance characterization of solid optical tissue phantom is presented. Random error sources taken into account are Poisson noise of the photon counting process, additive dark count noise and instrument response function stability. Systematic error sources taken into account are: phantom thickness uncertainty, refractive index uncertainty, time correlated single photon counting system time base calibration uncertainty. Correction procedures for these systematic errors are presented whenever a correction is possible.

Use of Magnetic Fields to Probe and Alter Photodynamic Processes in Photosensitizers

Spin states of Type 1 photosensitizer radicals are perturbed using weak magnetic fields (<200mT) to affect their luminescence, measured using time-domain photon counting. Magneto-photosensitization effects on photodynamic pathways in liposome cell phantoms are examined.