Pascal Gallant

Determination of reference values for optical properties of liquid phantoms based on Intralipid and India ink

A multi-center study has been set up to accurately characterize the optical properties of diffusive liquid phantoms based on Intralipid and India ink at near-infrared (NIR) wavelengths. Nine research laboratories from six countries adopting different measurement techniques, instrumental set-ups, and data analysis methods determined at their best the optical properties and relative uncertainties of diffusive dilutions prepared with common samples of the two compounds. By exploiting a suitable statistical model, comprehensive reference values at three NIR wavelengths for the intrinsic absorption coefficient of India ink and the intrinsic reduced scattering coefficient of Intralipid-20% were determined with an uncertainty of about 2% or better, depending on the wavelength considered, and 1%, respectively. Even if in this study we focused on particular batches of India ink and Intralipid, the reference values determined here represent a solid and useful starting point for preparing diffusive liquid phantoms with accurately defined optical properties. Furthermore, due to the ready availability, low cost, long-term stability and batch-to-batch reproducibility of these compounds, they provide a unique fundamental tool for the calibration and performance assessment of diffuse optical spectroscopy instrumentation intended to be used in laboratory or clinical environment. Finally, the collaborative work presented here demonstrates that the accuracy level attained in this work for optical properties of diffusive phantoms is reliable.

Sensitivity characterization of a time-domain fluorescence imager: eXplore Optix

A key issue in the practical application of fluorescence imaging is the presence of a background signal detected during data acquisition when no target fluorescent material is present. Regardless of the technology employed, background signals cannot be completely eliminated, which limits the detection sensitivity of fluorescence imaging systems, especially for in vivo applications. We present a methodology to characterize the sensitivity of fluorescence imaging devices by taking the background effect into account through the fluorescent signal-to-background ratio (SBR). In an initial application of the methodology, tissuelike liquid phantoms with Cy5.5 fluorescent inclusions were investigated experimentally over a wide range of varying parameters, such as tissue absorption coefficient, scattering coefficient, fluorophore concentration, and inclusion location. By defining detectable and quantifiable SBR thresholds, empirical relations are established, and the sensitivity performance of Advanced Research Technologiess eXplore Optix using Cy5.5 is characterized.

A quantitative time-domain optical imager for small animals in vivo fluorescence studies

ART as developed a time-domain optical molecular imager that recovers size, position and concentration of fluorescent inclusions embedded in turbid media within 15-30% accuracy. Fluorescent lifetime also gives the capability to discriminate different fluorescent sources.

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.

System IRF impact on fluorescence lifetime fitting in turbid medium

Fluorescence lifetime imaging is independent of signal intensity and is thus efficient and robust. Additionally, lifetime can be used to differentiate fluorophores and sense fluorophore micro-environment change. A time-resolved optical system is usually used to measure fluorescent decay kinetics, and then one fits the decay to get lifetime. Since the system impulse response function (IRF) is finite, it impacts lifetime fitting. Deconvolution of the IRF can diminish its impact. In thick tissues, light diffusion due to scattering is also convolved with the fluorescence decay. One can recover the decay using an inversion algorithm. However, processing data in this way is computationally intensive and therefore not practical for real time imaging. We present here results of our studies on the IRF impact to fluorescence lifetime fitting in a turbid medium over a wide range of parameters, using a unique time-domain imaging system. Fluorophores were submerged inside a turbid medium that models tissue. Analytical analysis and computation show that when the lifetime is 1.5 times larger than the FWHM of system IRF, reasonable fluorescence lifetimes can be obtained by fitting the decay tail without taking into account IRF. For small source-fluorophore-detector separation, the effect of optical diffusion on the lifetime fitting is also negligible. This gives a guidance of system precision limit for fluorescence lifetime imaging by fast tail fitting. Experimental data using a fs laser with a streak camera and a pulsed diode laser with PMT-TCSPC for ICG, Cy5.5, and ATTO 680 support the theoretical results.

Inter-Laboratory Comparison of Optical Properties Performed on Intralipid and India Ink

Intrinsic reduced scattering coefficient of Intralipid and intrinsic absorption coefficient of Indian ink at NIR wavelengths are accurately assessed in a multi-center study involving different techniques, instrumental set-ups, and analysis methods.

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.

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.

Improved photoacoustic dosimetry for retinal laser surgery

Lasers are employed for numerous medical interventions by exploiting ablative, disruptive or thermal effects. In ocular procedures, lasers have been used for decades to treat diseases such as diabetic retinopathy, macular edema and aged related macular degeneration via photocoagulation of retinal tissues. Although laser photocoagulation is well established in today’s practice, efforts to improve clinical outcomes by reducing the collateral damage from thermal diffusion is leading to novel treatments using shorter (μs) laser pulses (e.g. selective retinal therapy) which result in physical rather than thermal damage. However, for these new techniques to be widely utilized, a method is required to ensure safe but sufficient dosage has been applied, since no visible effects can be seen by ophthalmoscopy directly post treatment. Photoacoustic feedback presents an attractive solution, as the signal is dependent directly on absorbed dosage. Here, we present a method that takes advantage of temporal pulse formatting technology to minimize variation in absorbed dose in ophthalmic laser treatment and provide intelligent dosimetry feedback based on photoacoustic (PA) response. This method tailors the pulse to match the frequency response of the sample and/or detection chain. Depending on the system, this may include the absorbing particle size, the laser beam diameter, the laser pulse duration, tissue acoustic properties and the acoustic detector frequency response. A significant improvement (<7x) of photoacoustic signal-to-noise ratio over equivalent traditional pulse formats have been achieved, while spectral analysis of the detected signal provides indications of cavitation events and other sample properties.

New fiber-based approaches for optical biopsy (Conference Presentation)

Optical biopsy of tissue using fiber optic probes has proven to be a powerful tool for non-invasive and minimally invasive diagnostics. However, there are still many challenges to improving diagnostic value and commercial translation of these techniques. Many fiber-based methods are limited by background noise, which impairs sensitivity and specificity. Aspects of quality control, such as adequacy of the target of interest sampled and validation of optical measurements with histopathology can be problematic. Complexity, cost, and disposability or sterilizability are roadblocks to widespread clinical use. Here, we present new approaches to using fibers for optical biopsy aimed at solving these problems. Specifically, the new concepts are designed with the goals of being simple and disposable, to improve control of light delivery and collection from the sample, and to inherently enable better quality control of the biopsy process. A concept-of-operation aimed at nearly zero impact to the work flow of the biopsy and standard pathology procedures will be outlined. Several concepts for fiber implementations will be presented. A trade-off analysis of the concepts used to select a first implementation for testing will be presented. Preliminary experimental validation in phantoms and tissue samples will be presented for the selected configuration.