5-Aminolevelunic acid-induced protoporphyrin IX (PpIX) fluorescence-guided resection (FGR) enables maximum safe resection of glioma by providing real-time tumor contrast. However, the subjective visual assessment and the variable intrinsic optical attenuation of tissue limit this technique to reliably delineating only high-grade tumors that display strong fluorescence. We have previously shown, using a fiber-optic probe, that quantitative assessment using noninvasive point spectroscopic measurements of the absolute PpIX concentration in tissue further improves the accuracy of FGR, extending it to surgically curable low-grade glioma. More recently, we have shown that implementing spatial frequency domain imaging with a fluorescent-light transport model enables recovery of two-dimensional images of [PpIX], alleviating the need for time-consuming point sampling of the brain surface. We present first results of this technique modified for in vivo imaging on an RG2 rat brain tumor model. Despite the moderate errors in retrieving the absorption and reduced scattering coefficients in the subdiffusive regime of 14% and 19%, respectively, the recovered [PpIX] maps agree within 10% of the point [PpIX] values measured by the fiber-optic probe, validating its potential as an extension or an alternative to point sampling during glioma resection.
An explicit dosimetry model has been developed to calculate the apparent reacted 1O2 concentration ([1O2]rx) in an in-vivo model. In the model, a macroscopic quantity, g, is introduced to account for oxygen perfusion to the medium during PDT. In this study, the SOED model is extended for PDT treatment in phantom conditions where vasculature is not present; the oxygen perfusion is achieved through the air-phantom interface instead. The solution of the SOED model is obtained by solving the coupled photochemical rate equations incorporating oxygen perfusion through the air-liquid interface. Experiments were performed for two photosensitizers (PS), Rose Bengal (RB) and Photofrin, in solution, using SOED and SOLD measurements to determine both the instantaneous [1O2] as well as cumulative [1O2]rx concentrations, where [1O2=(1/τ▵)•∫[1O2]dt. The PS concentrations varied between 10 and 100 mM for RB and ~200 mM for Photofrin. The resulting magnitudes of [1O2] were compared between SOED and SOLD.
Intraoperative fluorescence guidance enables maximum safe resection of, for example, glioblastomas by providing
surgeons with real-time tumor optical contrast. Specifically, 5-aminolevulinic acid (ALA)-induced protoporphyrin IX
(PpIX) fluorescence guided resection can improve surgical outcomes by better defining tumor margins and identifying
satellite tumor foci. However, visual assessment of PpIX fluorescence is subjective and limited by the distorting effects
of light attenuation (absorption and scattering) by tissue and background tissue autofluorescence. We have previously
shown, using a point fluorescence-reflectance fiberoptic probe, that non-invasive measurement of the absolute PpIX
concentration, [PpIX], further improves sensitivity and specificity, leading to the demonstration that the technique can
also detect low-grade gliomas as well as otherwise undetectable residual tumor foci in high-grade disease. Here, we
extend this approach to wide-field quantitative fluorescence imaging (qFI) by implementing spatial frequency domain
imaging (SFDI) to recover the tissue optical absorption and transport scattering coefficients across the field of view. We
report on the performance of this approach to determine the intrinsic fluorescence intensity in tissue-simulating
phantoms in both the fully diffusive (i.e. scatter-dominated) and sub-diffusive (low transport albedo) regimes, for which
higher spatial frequencies are used. The performance of qFI is compared to a Born- normalization correction scheme, as
well as to the values obtained using the fiberoptic probe on homogeneous tissue phantoms containing PpIX.
In recent years numerous studies have shown the potential advantages of molecular imaging in vitro and in vivo using contrast agents based on surface enhanced Raman scattering (SERS), however the low throughput of traditional point-scanned imaging methodologies have limited their use in biological imaging. In this work we demonstrate that direct widefield Raman imaging based on a tunable filter is capable of quantitative multiplex SERS imaging in vivo, and that this imaging is possible with acquisition times which are orders of magnitude lower than achievable with comparable point-scanned methodologies. The system, designed for small animal imaging, has a linear response from (0.01 to 100 pM), acquires typical in vivo images in <10 s , and with suitable SERS reporter molecules is capable of multiplex imaging without compensation for spectral overlap. To demonstrate the utility of widefield Raman imaging in biological applications, we show quantitative imaging of four simultaneous SERS reporter molecules in vivo with resulting probe quantification that is in excellent agreement with known quantities (R 2 >0.98 ).
Interstitial near-infrared laser thermal therapy (LITT) is currently undergoing clinical trials as an alternative to watchful waiting or radical surgery in patients with low-risk focal prostate cancer. Currently, we use magnetic resonance image (MRI)-based thermography to monitor treatment delivery and determine indirectly the completeness of the target tissue destruction while avoiding damage to adjacent normal tissues, particularly the rectal wall. However, incomplete tumor destruction has occurred in a significant fraction of patients due to premature termination of treatment, since the photocoagulation zone is not directly observed. Hence, we are developing transrectal diffuse optical tomography (TRDOT), in combination with transrectal 3D ultrasound (3D-TRUS), to address his limitation. This is based on the large changes in optical scattering expected upon tissue coagulation. Here, we present forward simulations of a growing coagulated lesion with optical scattering contrast, using an established finite element analysis software platform (NIRFAST). The simulations were validated in tissue-simulating phantoms, with measurements acquired by a state-of-the-art continuous wave (CW) TRDOT system and a recently assembled bench-top CW-DOT system, with specific source-detector configurations. Two image reconstruction schemes were investigated and evaluated, specifically for the accurate delineation of the posterior boundary of the coagulation zone as the critical parameter for treatment guidance in this clinical application.
We are developing a method of monitoring treatment progression of interstitial photothermal therapy of focal prostate cancer using transrectal diffuse optical tomography (TRDOT) combined with transrectal 3D ultrasound (3D-TRUS). Measurements of prostate tissue optical properties were made on ex vivo human prostate samples prior to and post coagulation. Interstitial photothermal treatments were delivered to the ex vivo samples and monitored using an interstitial probe near the treatment fiber. After treatment, bulk optical properties were measured on native and coagulated zones of tissue. Changes in optical properties across the boundary between native and coagulated tissues were spatially mapped using a small diffuse reflectance probe. The optical property estimates and spatial information obtained using each method was compared.
As molecular imaging moves towards lower detection limits, the elimination of endogenous background signals becomes imperative. We present a facile background-suppression technique that specifically segregates the signal from surface-enhanced Raman scattering (SERS)-active nanoparticles (NPs) from the tissue autofluorescence background in vivo. SERS NPs have extremely narrow spectral peaks that do not overlap significantly with endogenous Raman signals. This can be exploited, using specific narrow-band filters, to image picomolar (pM) concentrations of NPs against a broad tissue autofluorescence background in wide-field mode, with short integration times that compare favorably with point-by-point mapping typically used in SERS imaging. This advance will facilitate the potential applications of SERS NPs as contrast agents in wide-field multiplexed biomarker-targeted imaging in vivo.
We report on the design and testing of a prototype widefield surface enhanced Raman scattering (SERS) imaging system
based on a fiber optic bronchoscope using bandpass filters for Raman signal selection. The SERS contrast agents
employed consist of gold nanoparticles encoded with a Raman-active dye and made specific for lung adenocarcinoma
tissue through the use of an anti-epidermal growth factor receptor (EGFR) antibody. By exploiting the extremely narrow
SERS spectral peaks we demonstrate a facile method of background fluorescence rejection that can be implemented at
sub-video rates. The system has been tested on in-vivo tissues and performance metrics, including the maximum tissue
penetration and minimum detectable nanoparticle quantity have been determined in a standardized fashion.
Optical tissue phantoms are very important tools for the development of biomedical imaging applications. Optical
phantoms are often used as ground truth against which instruments results can be compared. It is therefore important that
the optical properties of reference phantoms be measured in a manner that is traceable to the international system of
units. SI traceability insures long term consistency of results and will therefore improve the effectiveness of diffuse
optics research effort more effective by reducing unwanted variability in the data produced and shared by the
community. The ultimate benefit of rigorous SI traceability is the reduction of variability in the data produced by novel
diagnostic devices, which will in turn increase the statistical power of clinical trials aiming at validating their clinical
usefulness. SI traceability, and therefore uncertainty analysis, is also relevant to traceability aspects mandated by FDA
regulations. SI traceability is achieved through a thorough analysis of the measurement principle and its potential error
sources. The uncertainty analysis should be ultimately validated by inter-laboratory comparison until a consensus is
attained on the best practices for measuring the optical properties of 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.
We present a video-rate optical microscope that allows simultaneous imaging of two-photon excited fluorescence
(TPEF), second harmonic generation (SHG) and reflectance. The ms time resolution of the system together with its
submicrometer spatial resolution make it an ideal tool for studying fast neuronal activity and signaling, to understand
how action potentials are decoded molecularly. Transient trans-membrane potentials are measured with SHG, while the
evoked calcium oscillations are monitored with TPEF. The ability of this system to monitor both signals simultaneously
in multiple sub-compartments of living neurons should open the way to study how the electrical activity of neurons is
encoded intracellularly.
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