Marcia L. Vernon

Fabrication and characterization of a solid polyurethane phantom for optical imaging through scattering media

A phantom based on a polyurethane system that replicates the optical properties of tissue for use in near-infrared imaging is described. The absorption properties of tissue are simulated by a dye that absorbs in the near infrared, and the scattering properties are simulated by TiO2 particles. The scattering and absorption coefficients of the plastic were measured with a new technique based on time-resolved transmission through two slabs of materials that have different thicknesses. An image of a representative phantom was obtained from time-gated transmission.

Simple Fiber-Optic-Based Sensors for Process Monitoring: An Application in Wine Quality Control Monitoring

The goal of this research was to develop a simple and economical fiber-optic sensor technology for agrifood process monitoring. Toward this end, two fiber-optic sensors were developed to be used in combination: a single reflection V-bend sensor and a single fiber air-gap probe. The former is designed to be sensitive toward refractive index and the latter towards absorption. Experiments indicate that the micromachined V-bend fiber refractometer is most sensitive when the bend angle is centered around 140 degrees, at which angle the sensor may resolve changes in refractive index as small as 0.00015. Additionally, the V-bend sensor was found to be non-responsive toward sample absorption even in extremely absorbing solutions. The air-gap design absorption sensor, most commonly used for measurements in highly colored media, was found to be slightly sensitive towards refractive index. When the two sensors are used together, the response of the absorption sensor may be corrected for. This sensor combination is able to provide accurate measurements in situations where Beers law is not obeyed. Results are presented that show that the sensor pair was successfully used to monitor wine sugar content (Brix), and color density and hue, parameters related to the age of the wine.

Optical characterization of Pseudomonas fluorescens on meat surfaces using time-resolved fluorescence

A scanning optical system for the detection of bacteria on meat surfaces based on fluorescence lifetime and intensity measurements is described. The system detects autofluorescent light emitted by naturally occurring fluorophores in bacteria. The technique only requires minimal sample preparation and handling, thus the chemical properties of the specimen are preserved. This work presents the preliminary results obtained from a time-resolved fluorescence imaging system for the characterization of a nonpathogenic gram-negative bacteria, Pseudomonas fluorescens. Initial results indicate that the combination of fluorescence lifetime and intensity measurements provides a means for characterizing biological media and for detecting microorganisms on surfaces.

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.

Multiport time-domain laser mammography: results on solid phantoms and volunteers

A prototype for laser mammography based on a time-domain technique has been developed. The system uses a streak camera and a Titanium:sapphire laser which provides ultrashort pulses at a repetition rate of 80 MHz. A multi-port scanning head which includes optical fibers scans the breast in a point-by-point scanning procedure. Time-resolved transmission is measured at 15000 locations in 7 minutes. The breast is slightly compressed in both the cranio-caudal and the mediolateral projections. Amplitude calibration of the streak camera has been performed allowing for absolute measurement of time- resolved transmission. In addition to the shape of the time-resolved transmission, the absolute amplitude is relevant in properly evaluating the absorption and scattering coefficients. Promising results on solid phantoms and in vivo have been obtained. Both breasts of 10 volunteers have been scanned to date and a larger pilot study is planned in the near future. In addition to the usual time-gating processing, images of the scattering and absorption contributions are also extracted using an original data processing technique.

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.

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.

Frequency domain, time-resolved and spectroscopic investigations of photosensitizers encapsulated in liposomal phantoms

A broadband frequency domain fluorescence lifetime system (from ns to ms time scale) has been developed to study the photochemical and photodynamic behavior of model, well-controlled photosensitizer-encapsulating liposomes. Liposomes are known to be efficient and selective photosensitizer (PS) drug delivery vesicles, however, their chemical and physical effects on the photochemical properties of the photosensitizer have not been well characterized. The liposomes employed in this study (both blank and photosensitizer-complexed) were characterized to determine their: a) size distribution (dynamic light scattering), b) image (scanning electron microscope, confocal fluorescence microscopy), c) concentration of particles (flow cytometry), d) temperature-dependant phase transition behavior (differential scanning calorimetry, and e) spectrofluorescent spectrophotometric properties, e.g. aggregation, in the confined environment. The fluorescence decay behavior of two families of encapsulated photosensitizers, di-and tetrasulfonated metallophthalocyanines, and 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH), has been examined as a function of the liposomes physical properties (size-scale, distribution and concentration of scatterer) and the impact of the photosensitizer spatial confinement determined. It is found that the achievable size range and distribution of the PS-liposomes is controlled by the chemical nature of the PS for large liposomes (1000 nm), and is PS independent for small PS-liposomes (~140nm). The lifetime decay behavior was studied for all three photosensitizer-liposome systems and compared before and after confinement. We found the nature of the decay to be similar before and after encapsulation for the sulfonated phthalocyanines containing ionic moieties (primarily monoexponential) but not for HPPH. In the latter, the decay transitioned from multi- to monoexponential decay upon localizing lypophilic HPPH to the liposomal membrane. This behavior was confirmed by obtaining a similar change in lifetime response with an independent timedomain system. We also varied the environment in temperature and oxygen content to examine the effects on the fluorescent lifetimes of the liposomal complexes. The fluorescence decay of all three PS-containing liposomes showed that the local spatial confinement of PS (dictated by the PS chemistry) into different domains within the liposome directly controls the temperature-response. Membrane-bound photosensitizers were less sensitive to temperature effects as illustrated by the decay dynamics observed in solu, that is, they developed a unique decay behavior that correlated with the phase transition of the membrane. The fluorescent lifetime of PS-encapsulated liposomes in deoxygenated environments, relevant to oxygen independent type I phototoxicity, was also probed in the frequency-domain revealing that liposome-confined PS display very different trends than those observed in solu.

Dual-spatial integration for longitudinal localization of inclusions in turbid media.

We introduce a technique called dual-spatial integration (DSI) that is used to isolate and enhance inclusions that differ only by their longitudinal placement within a scattering medium. DSI uses three different source-detector configurations to section a scattering medium into three longitudinal zones. This sectioning permits the extraction of structures close to surfaces and the enhancement of those structures located in the central part of the medium. Both the simulation and the experimental results indicate that DSI has potential interest for applications in biomedical imaging such as optical mammography.

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.