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.
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.
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.
This project uses function-based detection via a fundamental understanding of the genetic markers of AR to distinguish
harmful organisms from innocuous ones. This approach circumvents complex analyses to unravel the taxonomic details
of 1399 pathogen species, enormously simplifying detection requirements. Laval Hospital's fast permeabilization
strategy enables AR revelation in <1hr. Packaging the AR protocols in liquid-processing cartridges and coupling these to
our in-house miniature fiber optic flow cell (FOFC) provides first responders with timely information on-site. INO's
FOFC platform consists of a specialty optical fiber through which a hole is transversally bored by laser micromachining.
The analyte solution is injected into the hole of the fiber and the particles are detected and counted. The advantage with
respect to classic free space FC is that alignment occurs in the fabrication process only and complex excitation and
collection optics are replaced by optical fibers. Moreover, we use a sheathless configuration which has the advantage of
increase the portability of the system, to reduce excess biohazard material and the need for weekly maintenance. In this
paper we present the principle of our FOFC along with a, demonstration of the basic capability of the platform for
detection of bacillus cereus spores using permeabilized staining.
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.
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, fluo, was compared for PSs free in aqueous solution and in a liposome-associated state at varied temperatures (25 to 78°C) and oxygen concentrations (0–190 µM). The analysis of 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 lipsomes. 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 fluo increase for all PSs in free solution, while the opposite, a fluo decrease, occurred in all liposomal PSs.
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.
Nonlinear optical imaging technologies offer some intriguing medical diagnostic applications. Examples include fast
imaging of elastin and collagen distributions in diseased tissues using two-photon fluorescence (TPF) and second
harmonic generation (SHG), respectively. The 3D sectioning capabilities and biochemical specificity that enable fast
imaging in highly scattering biological media lie at the heart of the appeal of these nonlinear approaches for medical
applications. One of these promising nonlinear techniques relies on the resonance enhancement of the third order
nonlinear susceptibility by a vibrational mode of a molecule. Coherent Anti-Stokes Raman Scattering (CARS) can
provide similar vibrational information as a spontaneous Raman spectrum. The technique has been shown to be orders
of magnitude more sensitive than spontaneous Raman, with video rate imaging demonstrated recently. In this work,
we investigate the potential use of broadband CARS spectroscopy and CARS imaging for biochemical analysis of
arterial tissue. Biochemical imaging data from broadband CARS is compared with spontaneous Raman
microspectroscopy. The broadband CARS system comprised of a single femtosecond-laser is presented in detail.
Issues related to data analysis, the advantages and current limitations of the CARS technique in biodiagnostics are
discussed.
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.
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