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Quantitative fluorescence imaging of point-like sources in small animals
Physics in Medicine & Biology
Abstract
A planar imaging approach is described for the in vivo quantitative reconstruction of fluorescent point sources in small animals. The method uses the diffusion approximation as a forward model of light propagation from a point source in a homogeneous tissue to find source depth and strength. The tissue optical properties obtained from video reflectometry measurements were used to compensate for the effects of tissue heterogeneity. The method was evaluated on images of fluorescent sources implanted 2-8.5 mm deep in the thigh and abdomen of rats post mortem. In more than 70% of the total number of implants the source depth was retrieved with an error of less than 1 mm. The largest absolute error was 1.9 mm. In retrieving source strength, the errors ranged from 0.4% to 89% generally increasing with increased source depth.
... Fluorescent imaging was used as a means to determine the fluorophore concentration in each mouse lung lobe (Yi et al, 2010). The use of the probe, AlPCS, permitted measurements of the excitation and emission wavelengths in the near-infrared region where biological tissue auto-fluorescence is negligible and tissue attenuation is minimal (Chen et al, 2006, Comsa et al, 2008, Mohajerani et al, 2009, . In addition, the relationship between the intensity within a pixel and the number of fluorescent molecules within the rectangular prism was established by empirically accounting for absorption of light by tissue. ...
... Here, it must be appreciated that in addition to loss of light by the above mechanisms, there is also distortion. That is, images obtained with the Maestro system do not appear sharply focused because tissue affects the path of light by reflection, refraction and scattering (Kovar et al, 2007, Comsa et al, 2008. This in turns causes photons to be accumulated in a CCD that is not directly above the rectangular prism of the tissue. ...
... The far right curve is the uncorrected data, which appears as a Gaussian broadened curve. This is consistent with the work from Comsa et al (2008), which indicated that light distortion can be modeled a random process. ...
Better methods are needed to quantify the distribution of drug among the airways of the lungs of small animals to facilitate the development of agents that can target specific airways. Mice were exposed to aerosols of aluminum phthalocyanine tetrasulfonic acid (AlPCS) that ranged in concentration and size (0.2-2.8 μm). The trachea and lobes were removed and placed between glass slides, and fluorescent images were obtained at two different compression thicknesses. The intensity, normalized by the area, exposure time, and thickness, was then plotted as a function of compression thickness, from which the concentration and attenuation coefficient were estimated for each lobe and then for each pixel of the image. The latter was then used to generate an image reflective of the concentration. The lobe volume, concentration, and tissue attenuation of AlPCS was consistent among the lobes. The deposition fraction increased with decreasing particle size. The network of lines in the concentration image indicated that connective tissue has a lower concentration. The central airways were clearly evident in the images of mice exposed to the very small and large aerosols. This approach provides a rapid, economical means to obtain high resolution images of mouse lungs from which detailed analysis of the distribution of deposited aerosol particles can be obtained.
... Fluorescence imaging is an attractive approach to assess the deposition of particles in the lung, since measurements can be carried out in live animals (5)(6)(7)(8). In principle, there is a relationship between the intensity of light within a pixel, representing a two-dimensional projection of the biological sample, and the number of fluorescent molecules within the rectangular prism (7,9,10). ...
... Fluorescence imaging is an attractive approach to assess the deposition of particles in the lung, since measurements can be carried out in live animals (5)(6)(7)(8). In principle, there is a relationship between the intensity of light within a pixel, representing a two-dimensional projection of the biological sample, and the number of fluorescent molecules within the rectangular prism (7,9,10). However, both the incident excitation and the emitted fluorescent light are scattered, reflected, and absorbed by tissue, which affects the relationship between light captured by the detector and number of molecules. ...
... Light absorption and autofluorescence are problems that can be corrected in a relatively straightforward manner (7,10). Light absorption is typically treated in an empirically predictable manner with the assumption that the attenuation is proportional to an exponential function of thickness. ...
Lung samples were prepared to investigate the perturbing effects of light absorption for quantifying the fluorescence signal of aluminum phthalocyanine tetrasulfonic acid (AlPCS). Standard solutions of known concentration and depth were imaged with different exposure times and analyzed. The intensity was found to be a linear function of concentration, depth, exposure time, and area. Mice were exposed to an aerosol of AlPCS with a mass median aerodynamic diameter of 390 nm and geometric standard deviation of 1.8. Images of intact lung lobes and lung homogenates were obtained and then analyzed to allow quantifying the concentration of AlPCS among the lung lobes and trachea. For the distribution of aerosols, the results indicate that the concentration was uniform among the different lobes. Combining the quantitative analysis of the concentration with image analysis of the area/thickness, the mass deposited in each lobe was readily determined. This approach provides a quantitative means to determine the selectivity of drug delivery to mouse lower respiratory tract.
... Distributions of fluorescent probes in biological tissues are, however, commonly confined to specific organs or regions where the molecular target is located, taking the form of inclusions that can be assumed point-like because of their small spatial dimensions [32,33]. In addition, fluorescent sources can be considered as isotropic light emitters [34]. ...
... These facts can be used to advantage to devise simpler and faster algorithms for localizing fluorescence. Localizing fluorescent inclusions is a problem of high interest in biomedical optics with possible uses in small animal molecular imaging for disease development studies and drug discovery [32,33,35], in prostate cancer imaging [36], in breast tumor detection and brain imaging [37,38]. In this work, time-domain data [so-called time point-spread functions (TPSFs)] are exploited in a geometrical approach for localizing point-like fluorescence inclusions using early photon arrival times. ...
We introduce a novel approach for localizing a plurality of discrete point-like fluorescent inclusions embedded in a thick turbid medium using time-domain measurements. The approach uses early photon information contained in measured time-of-flight distributions originating from fluorescence emission. Fluorescence time point-spread functions (FTPSFs) are acquired with ultrafast time-correlated single photon counting after short pulse laser excitation. Early photon arrival times are extracted from the FTPSFs obtained from several source-detector positions. Each source-detector measurement allows defining a geometrical locus where an inclusion is to be found. These loci take the form of ovals in 2D or ovoids in 3D. From these loci a map can be built, with the maxima thereof corresponding to positions of inclusions. This geometrical approach is supported by Monte Carlo simulations performed for biological tissue-like media with embedded fluorescent inclusions. To validate the approach, several experiments are conducted with a homogeneous phantom mimicking tissue optical properties. In the experiments, inclusions filled with indocyanine green are embedded in the phantom and the fluorescence response to a short pulse of excitation laser is recorded. With our approach, several inclusions can be localized with low millimeter positional error. Our results support the approach as an accurate, efficient, and fast method for localizing fluorescent inclusions embedded in highly turbid media mimicking biological tissues. Further Monte Carlo simulations on a realistic mouse model show the feasibility of the technique for small animal imaging.
... Biological tissue, which has the capability to auto-fluoresce, gives rise to heterogeneous background signal (Yi et al., 2010). This problem can be solved by subtracting auto-fluorescence with blank tissue or current instrumentation that emits light at long wavelengths in the near-infrared region of the spectrum (Adams et al., 2007; Comsa et al., 2008). Fluorescent dyes such as aluminum (III) phthalocyanine chloride tetrasulfonic acid, can be administered in aerosol form and then image can be taken by in vivo imaging system to visualize particle deposition in different lobes of the lungs followed by quantification of particles using image analyzer. ...
... Fluorescent dyes such as aluminum (III) phthalocyanine chloride tetrasulfonic acid, can be administered in aerosol form and then image can be taken by in vivo imaging system to visualize particle deposition in different lobes of the lungs followed by quantification of particles using image analyzer. By this method, it is possible to reduce tissue attenuation by using molecular probes that absorb and emit light in the near infrared region (Adams et al., 2007; Comsa et al., 2008; Kovar et al., 2007). ...
Delivery of therapeutic agents via the pulmonary route has gained significant attention over the past few decades because this route of administration offers multiple advantages over traditional routes that include localized action, non-invasive nature and favorable lung-to- plasma ratio. However, assessment of post administration behavior of inhaled pharmaceuticals-such as deposition of particles over the respiratory airways, interaction with the respiratory fluid and movement across the air-blood barrier-is challenging because the lung is a very complex organs that is composed of airways with thousands of bifurcations with variable diameters. Thus, much effort has been put forward to develop models that mimic human lungs and allow evaluation of various pharmaceutical and physiological factors that influence the deposition and absorption profiles of inhaled formulations. In this review, we sought to discuss in vitro, in vivo and ex vivo models that have been extensively used to study the behaviors of airborne particles in the lungs and determine the absorption of drugs after pulmonary administration. We have provided a summary of lung cast models, cascade impactors, noninvasive imaging, intact animals, cell culture and isolated perfused lung models as tools to evaluate the distribution and absorption of inhaled particles. We have also outlined the limitations of currently used models and proposed future studies to enhance the reproducibility of these models.
... Many important cellular events, such as cell proliferation, apoptosis, and other complicated molecular events, involve molecular behavior characterization and multimolecular interaction occurring on a nanoscale, and how to achieve dynamic imaging of cellular events in a single cell level is becoming increasingly required in cell and molecular biology sciences [1][2][3][4][5][6][7]. The transferrin receptor (TfR), is located in the cell membrane surface and plays an important role for transporting ferric ion and is an accessible portal for the cancer drug delivery due to the expression level of TfR on cancer cell being high, relative to the normal cell [8,9]. ...
A metal nanoparticles-based surface-enhanced Raman scattering (SERS) technique has been developed for biosensing and bioimaging due to its advantages in ultra-narrow line width for multiplexing, ultra-high sensitivity and excellent photostability. However, the “hotspots” effect between nanoparticles usually leads to unstable and nonuniform Raman enhancement, and this will greatly reduce the quality of SERS imaging. In this study, we employ the bridge gaps-enhanced Raman tags (BGERTs) to perform SERS imaging, in which BGERTs can not only reduce the influence of the “hotspots” effect between nanoparticles on Raman signal intensity but provide a great Raman enhancement when the Gold (Au) shell is thick enough. Based on BGERTs and its conjugation with the thiol-terminated polyethylene glycol (PEG) and transferrin, we construct a targeted Transferrin (TF)-PEG-BGERTs SERS nanoprobe and achieve the dynamic imaging of transferrin receptor (TfR) molecules on a single live cell, in which the role of transferrin-conjugated PEG-BGERT is for targeting TfR molecules located in cellular membrane surface. Importantly, this BGERTs-based SERS imaging could potentially provide a useful tool for studying the precise mechanism during the receptor-mediated nanoparticles endocytosis or cell proliferation, apoptosis, and other complicated molecular events.
... This work and others [16] demonstrate that modeling the fluorophore as a point-like source rather than as its true cylindrical shape does not undermine the diffusefluorescence model. The modulation patterns were projected parallel to the inclusion, so that the demodulated images fully reconstructed it without boundary-induced artifacts. ...
Mapping the optical absorption and scattering properties of tissues using spatial frequency-domain imaging (SFDI) enhances quantitative fluorescence imaging of protoporphyrin IX (PpIX) in gliomas in the preclinical setting. The feasibility of using SFDI in the operating room was investigated here. A benchtop SFDI system was modified to mount directly to a commercial operating microscope. A digital light processing module imposed a selectable spatial light pattern from a broad-band xenon arc lamp to illuminate the surgical field. White light excitation and a liquid crystal-tunable filter allowed the diffuse reflectance images to be recorded at discrete wavelengths from 450 to 720 nm on a sCMOS camera. The performance was first tested in tissue-simulating phantoms, and data were then acquired intraoperatively during brain tumor resection surgery. The optical absorption and transport scattering coefficients could be estimated with average errors of 3.2% and 4.5% for the benchtop and clinical systems, respectively, with spatial resolution of better than 0.7 mm. These findings suggest that SFDI can be implemented in a clinically relevant configuration to achieve accurate mapping of the optical properties in the surgical field that can then be applied to achieve quantitative imaging of the fluorophore.
... This work and others [16] demonstrate that modeling the fluorophore as a point-like source rather than as its true cylindrical shape does not undermine the diffusefluorescence model. The modulation patterns were projected parallel to the inclusion, so that the demodulated images fully reconstructed it without boundary-induced artifacts. ...
The rate of complete resection of glioma has improved with the introduction of 5‐aminolevulinic acid‐induced protoporphyrin IX (ALA‐PpIX) fluorescence image guidance. Surgical outcomes are further enhanced when the fluorescence signal is decoupled from the intrinsic tissue optical absorption and scattering obtained from diffuse reflectance measurements, yielding the absolute PpIX concentration, [PpIX]. Spatial frequency domain imaging (SFDI) was used previously to measure [PpIX] in near‐surface tumors under blue fluorescence excitation. Here, we extend this to subsurface [PpIX] fluorescence under red‐light excitation. The decay rate of the modulation amplitude of the fluorescence signal was used to calculate the PpIX depth, which was then applied in a forward diffusion model to estimate [PpIX] at depth. For brain‐like optical properties in phantoms with PpIX fluorescent inclusions, the depth can be recovered up to depths of 9.5 mm ± 0.4 mm, with [PpIX] ranging from 5 to 15 μg/ml within an average deviation of 15% from the true [PpIX] value.
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... The autofluorescence background can be measured using untreated tissue and subtracted from the experimental background or the use of near-infrared wavelengths can be used to overcome this issue (Adams K.E. et al., 2007;Comsa D.C. et al., 2008;Kovar J.L. et al., 2007;Murata M. et al., 2014). Fluorescent dyes are aerosolized, deliv-ered to the lungs via inhalation, intracheal, or intranasal routes, and imaged using an in vivo imaging system (IVIS). ...
This article reviews the pulmonary route of administration, aerosol delivery devices, characterization of pulmonary drug delivery systems, and discusses the rationale for inhaled delivery of siRNA. Diseases with known protein malfunctions may be mitigated through the use of siRNA therapeutics. The inhalation route of administration provides local delivery of siRNA therapeutics for the treatment of various pulmonary diseases, however barriers to pulmonary delivery and intracellular delivery of siRNA exists. siRNA loaded nanocarriers can be used to overcome the barriers associated with the pulmonary route, such as anatomical barriers, mucociliary clearance, and alveolar macrophage clearance. Apart from naked siRNA aerosol delivery, previously studied siRNA carrier systems comprise of lipidic, polymeric, peptide, or inorganic origin. Such siRNA delivery systems formulated as aerosols can be successfully delivered via an inhaler or nebulizer to the pulmonary region. Preclinical animal investigations of inhaled siRNA therapeutics rely on intratracheal and intranasal siRNA and siRNA nanocarrier delivery. Aerosolized siRNA delivery systems may be characterized using in vitro techniques, such as dissolution test, inertial cascade impaction, delivered dose uniformity assay, laser diffraction, and laser Doppler velocimetry. The ex vivo techniques used to characterize pulmonary administered formulations include the isolated perfused lung model. In vivo techniques like gamma scintigraphy, 3D SPECT, PET, MRI, fluorescence imaging and pharmacokinetic/pharmacodynamics analysis may be used for evaluation of aerosolized siRNA delivery systems. The use of inhalable siRNA delivery systems encounters barriers to their delivery, however overcoming the barriers while formulating a safe and effective delivery system will offer unique advances to the field of inhaled medicine.
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... Most of the current approaches assume a homogeneous background with known absorption and scattering parameters. On the other hand, normalized Born ratios [172], video reflectometry measurements [25] and DOT guided FMT [150,184] are proposed to deal with the heterogeneity. ...
Diffuse optical tomography (DOT) and fluorescence molecular tomography (FMT) are two attractive imaging techniques for in vivo physiological and psychological research. They have distinct advantages such as non-invasiveness, non-ionizing radiation, high sensitivity and longitudinal monitoring. This paper reviews the key components of DOT and FMT. Light propagation model, mathematical reconstruction algorithm, imaging instrumentation and medical applications are included. Future challenges and perspective on optical tomography are discussed.
... One current goal of optical tomography is the reconstruction of source strength, its absolute position (Comsa et al 2008), and, in the case of fluorescence tomography using timedependent methods, lifetime. As PET standard uptake value (SUV) or percentage injected dose gm −1 tissue is used to ascertain change in disease markers in preclinical therapeutic studies, a comparable quantitative figure of merit that can reproducibly reflect changes in disease marker availability is needed for optical tomography, whether for bioluminescent and fluorescent protein gene reporters, or targeted exogenous fluorescent agents. ...
Emerging fluorescence and bioluminescence tomography approaches have several common, yet several distinct features from established emission tomographies of PET and SPECT. Although both nuclear and optical imaging modalities involve counting of photons, nuclear imaging techniques collect the emitted high energy (100-511 keV) photons after radioactive decay of radionuclides while optical techniques count low-energy (1.5-4.1 eV) photons that are scattered and absorbed by tissues requiring models of light transport for quantitative image reconstruction. Fluorescence imaging has been recently translated into clinic demonstrating high sensitivity, modest tissue penetration depth, and fast, millisecond image acquisition times. As a consequence, the promise of quantitative optical tomography as a complement of small animal PET and SPECT remains high. In this review, we summarize the different instrumentation, methodological approaches and schema for inverse image reconstructions for optical tomography, including luminescence and fluorescence modalities, and comment on limitations and key technological advances needed for further discovery research and translation.
... 1). The determination of the location and intensity of a luminescent point source in such a diffusive medium is a standard problem in molecular imaging [16, 17]. ...
In this communication, the intrinsic precision in the localization of a fluorescent source in a turbid medium is analyzed for various fluorescence diffuse optical tomography (fDOT) setups in reflection geometry via a rigorous statistical methodology, the Cramer-Rao bound. Firstly, a spatially and temporally resolved imaging technique (TD) is considered and the strong impact of the fluorescence life-time is revealed. Then, these performances are compared with other standard setups, namely a spatially resolved in a continuous wave setup (CW) and a single detector in a time resolved setup (ITD).
... Fluorescence imaging has become an important investigational tool for preclinical studies. In particular, fluorescence diffuse optical tomography (FDOT) offers the capability of noninvasive 3-D imaging, which is critical for in vivo experiments123456. In a traditional setup for FDOT experiments, optical fibers are typically used to couple light to and from the animal, while it is enclosed in a compressing chamber filled with optical matching fluid. ...
Image reconstruction is one of the main challenges for fluorescence tomography. For in vivo experiments on small animals, in particular, the inhomogeneous optical properties and irregular surface of the animal make free-space image reconstruction challenging because of the difficulties in accurately modeling the forward problem and the finite dynamic range of the photodetector. These two factors are fundamentally limited by the currently available forward models and photonic technologies. Nonetheless, both limitations can be significantly eased using a signal processing approach. We have recently constructed a free-space panoramic fluorescence diffuse optical tomography system to take advantage of co-registered microCT data acquired from the same animal. In this article, we present a data processing strategy that adaptively selects the optical sampling points in the raw 2-D fluorescent CCD images. Specifically, the general sampling area and sampling density are initially specified to create a set of potential sampling points sufficient to cover the region of interest. Based on 3-D anatomical information from the microCT and the fluorescent CCD images, data points are excluded from the set when they are located in an area where either the forward model is known to be problematic (e.g., large wrinkles on the skin) or where the signal is unreliable (e.g., saturated or low signal-to-noise ratio). Parallel Monte Carlo software was implemented to compute the sensitivity function for image reconstruction. Animal experiments were conducted on a mouse cadaver with an artificial fluorescent inclusion. Compared to our previous results using a finite element method, the newly developed parallel Monte Carlo software and the adaptive sampling strategy produced favorable reconstruction results.
... Let us consider a fluorescent point-like volume located at rf and embedded in a 9mm thick homogeneous diffusive slab (see Fig. 1). The determination of the location and intensity of a luminescent point source in such a diffusive medium is a standard problem in molecular imaging [16, 17]. ...
Using a Cramer-Rao analysis, we study the theoretical performances of a time and spatially resolved fDOT imaging system for jointly estimating the position and the concentration of a point-wide fluorescent volume in a diffusive sample. We show that the fluorescence lifetime is a critical parameter for the precision of the technique. A time resolved fDOT system that does not use spatial information is also considered. In certain cases, a simple steady-state configuration may be as efficient as this time resolved fDOT system.
... This happens when dealing with highly vascularized tissues (e.g. heart, liver, etc., see reported values in [23]). Another circumstance comes about in optical imaging of NIR-activable fluorescent probes, which requires calculating the profile of the optical field in the tissue at the excitation wavelength. ...
We introduce a system of coupled time-dependent parabolic simplified spherical harmonic equations to model the propagation of both excitation and fluorescence light in biological tissues. We resort to a finite element approach to obtain the time-dependent profile of the excitation and the fluorescence light fields in the medium. We present results for cases involving two geometries in three-dimensions: a homogeneous cylinder with an embedded fluorescent inclusion and a realistically-shaped rodent with an embedded inclusion alike an organ filled with a fluorescent probe. For the cylindrical geometry, we show the differences in the time-dependent fluorescence response for a point-like, a spherical, and a spherically Gaussian distributed fluorescent inclusion. From our results, we conclude that the model is able to describe the time-dependent excitation and fluorescent light transfer in small geometries with high absorption coefficients and in nondiffusive domains, as may be found in small animal diffuse optical tomography (DOT) and fluorescence DOT imaging.
... Le choix de ce modèle simple repose sur plusieurs arguments. Non seulement il s'agit d'une situation canonique dans le sens où il est relativement facile d'obtenir des formulations analytiques des grandeurs qui nous intéressent, mais il a été aussi montré que la source ponctuelle était un modèle valide pour certaines applications biomédicales, en particulier sur l'os (Comsa et al. 2008), ou pour modéliser des objets relativement gros comme des tumeurs marquées de près d'un centimètre de diamètre (Milstein et al. 2005). ...
Molecular imaging is a major modality in the field of preclinical research. Among the existing methods, techniques based on optical detection of visible or near infrared radiation are the most recent and are mainly represented by luminescence optical tomography techniques. These methods allow for 3D characterization of a biological medium by reconstructing maps of concentration or localisation of luminescent beacons sensitive to biological and chemical processes at the molecular or cellular scale. Luminescence optical tomography is based on a model of light propagation in tissues, a protocol for acquiring surface signal and a numerical inversion procedure used to reconstruct the parameters of interest. This thesis is structured around these three axes and provides an answer to each problem. The main objective of this study is to introduce and present the tools to evaluate the theoretical performances of optical tomography methods. One of its major outcomes is the realisation of experimental tomographic reconstructions from images acquired by an optical imager designed for 2D planar imaging and developed by the company Quidd. In a first step we develop the theory of transport in scattering medium to establish the concept on which our work will rely. We present two different propagation models as well as resolution methods and theoretical difficulties associated with them. In a second part we introduce the statistical tools used to characterise tomographic systems. We define and apply a procedure to simple situations in luminescence optical tomography. The last part of this work presents the development of an inversion procedure. After introducing the theoretical frameworkwe validate the procedure fromnumerical data before successfully applying it to experimentalmeasurements.
Radiative transfer theory (RTT) is a valuable theoretical framework for describing the propagation of optical radiation in turbid media (Ishimaru, 1978; Wang and Wu, 2007). RTT has succeeded in fields such as astronomy and astrophysics (Chandrasekhar, 1960), remote sensing of the earth surface and atmosphere (Melnikova et al., 2012), heat transfer (Howell et al., 2010; Modest, 2003; Atalay, 2006), and, particularly, in biomedical optics (Wang and Wu, 2007; Hielscher et al., 2011; Klose, 2010a). The fundamental equation in RTT is the radiative transfer equation (RTE) (Wang and Wu, 2007). The RTE is the most accurate model for describing light propagation in biological tissue, with no approximation regarding the angular, spatial or temporal dependences (Hielscher et al., 2011). The RTE is an integrodifferential equation that depends on a set of optical parameters (index of refraction, absorption, scattering and scattering function) that describe the medium (Ishimaru, 1978). The validity limits of the RTE rest on the model conceived to describe light propagation, and should be established for each physical situation (Martí López et al., 2003). Analytical solutions of the RTE are only known for simple geometries (Ishimaru, 1978; Liemert and Kienle, 2011b).
We introduce a novel approach for localizing a plurality of discrete fluorescent inclusions embedded in a thick scattering medium. It exploits time-domain experimental data and intersections of ellipses where inclusions are likely to be found.
Fluorescence molecular tomography (FMT) supports monitoring molecular events non-invasively in vivo over a span of long time, and meets the demands of monitoring a process of life. Priori information can be applied to speed the complex and time-consuming reconstruction process and to enhance the quality of the reconstructed images for FMT. A method was proposed in the present paper to estimate rapidly the depth of fluorescence source. The estimation process was accomplished with an optimization algorithm, particle swarm optimization (PSO). Firstly the fluorescence intensity ratio Rf of two positions on the boundary of tissue was derived under extrapolated boundary condition and a diffusion model for the propagation of near-infrared photons in biological tissue. Then a PSO algorithm was applied to minimize the difference between the theoretical ratio R Tf and the measured ratio R Mf. The depth of fluorescence source was estimated after the rapid PSO optimization process. Two phantoms indicated that the proposed method can estimate the depth of single fluorescence source rapidly and easily without the time-consuming mesh generation and reconstruction process.
An ultrasound coupled handheld-probe-based optical fluorescence
molecular tomography (FMT) system has been in development for the
purpose of quantifying the production of Protoporphyrin IX (PPIX) in
aminolevulinic acid treated (ALA), Basal Cell Carcinoma (BCC) in vivo.
The design couples fiber-based spectral sampling of PPIX fluorescence
emission with a high frequency ultrasound imaging system, allowing
regionally localized fluorescence intensities to be quantified [1]. The
optical data are obtained by sequential excitation of the tissue with a
633nm laser, at four source locations and five parallel detections at
each of the five interspersed detection locations. This method of
acquisition permits fluorescence detection for both superficial and deep
locations in ultrasound field. The optical boundary data, tissue layers
segmented from ultrasound image and diffusion theory are used to
estimate the fluorescence in tissue layers. To improve the recovery of
the fluorescence signal of PPIX, eliminating tissue autofluorescence is
of great importance. Here the approach was to utilize measurements which
straddled the steep Qband excitation peak of PPIX, via the integration
of an additional laser source, exciting at 637 nm; a wavelength with a 2
fold lower PPIX excitation value than 633nm.The auto-fluorescence
spectrum acquired from the 637 nm laser is then used to spectrally
decouple the fluorescence data and produce an accurate fluorescence
emission signal, because the two wavelengths have very similar
auto-fluorescence but substantially different PPIX excitation levels.
The accuracy of this method, using a single source detector pair setup,
is verified through animal tumor model experiments, and the result is
compared to different methods of fluorescence signal recovery.
We present a diffuse optical tomography (DOT) algorithm for imaging the absorption distribution in a biological tissue using time-domain optical measurements. The time-dependent parabolic simplified spherical polynomials approximation of the radiative transfer equation (the TD-pSPN model) serves as the forward model. The DOT algorithm is implemented using a nested analysis and design (NAND) method developed for solving constrained optimization problems. Numerical experiments are provided for small geometry media to mimic small animal imaging. In these experiments, the optical absorption coefficient value is varied within typical values found in the near infrared range for biological tissues, including high absorption values. The results show good spatial and quantitative reconstructions and support our TD-pSPN-based DOT algorithm as an accurate approach to image absorption in biological media.
We investigate the problem of retrieving the optical properties (absorption and scattering) of biological tissue from a set of optical measurements. A diffuse optical tomography (DOT) algorithm that incorporates constrained optimization methods is implemented. To improve image quality, the DOT algorithm exploits full time-domain data. The time-dependent parabolic simplified spherical harmonics equations (TD-pSPN) are used as the forward model. Time-dependent adjoint variables are resorted to in the calculation of the gradient of the objective function. Several numerical experiments for small geometric media with embedded inclusions that mimic small animal imaging are performed. In the experiments, optical coefficient values are varied in the range of realistic values for the near-infrared spectrum, including high absorption values. Single and multiparameter reconstructions are performed with the diffusion equation and higher orders of the TD-pSPN equations. The results suggest the DOT algorithm based on the TD-pSPN model outperforms the DE, and accurately reconstructs optical parameter distributions of biological media both spatially and quantitatively.
Fluorescence diffuse optical tomography (FDOT) can provide important information in biomedical studies. In this ill-posed problem, suppression of background tissue autofluorescence is of utmost importance. We report a method for autofluorescence-insensitive FDOT using nonlinear upconverting nanoparticles (NaYF<sub>4</sub>:Yb<sup>3+</sup>/Tm<sup>3+</sup>) in a tissue phantom under excitation intensities well below tissue-damage thresholds. Even with the intrinsic autofluorescence from the phantom only, the reconstruction of the nanoparticles is of much better quality than the reconstruction of a Stokes-shifting dye. In addition, the nonlinear power dependence leads to more confined reconstructions and may increase the resolution in FDOT.
Different colors of visible light penetrate to varying depths in tissue due to the wavelength dependence of tissue optical absorption and elastic scattering. We exploit this to map the contour of the closest surface of a buried fluorescent object. This uses a novel algorithm based on the diffusion theory description of light propagation in tissue at each excitation wavelength to derive metrics that define the depth of the top surface of the object. The algorithm was validated using a tissue-simulating phantom. It was then demonstrated in vivo by subsurface brain tumor topography in a rodent model, using the fluorescence signal from protoporphyrin IX that is preferentially synthesized within malignant cells following systemic application of aminolevulinic acid. Comparisons to histomorphometry in the brain post mortem show the spatial accuracy of the technique. This method has potential for fluorescence image-guided tumor surgery, as well as other biomedical and nonbiological applications in subsurface sensing.
We present a simplified spherical harmonics approximation for the time-domain radiative transfer equation including the source-divergence effect. This leads to a set of coupled partial differential equations (PDEs) of the parabolic type that model diffuse light propagation in biological-tissue-like media. We introduce a finite element approach for solving these PDEs, thereby obtaining the time-dependent spatial profile of the fluence. We compare the results with the diffusion equation and Monte Carlo simulations. The fluence obtained via our model is shown to reproduce well the Monte Carlo results in all cases and improves on the solution of the diffusion equation in homogeneous diffusive-defying media. Our solution also shows more sensitivity than the diffusion equation to changes in the absorption coefficient of small inclusions.
Fluorescence diffuse optical tomography (FDOT) is a biomedical imaging modality that can be used for localization and quantification of fluorescent molecules inside turbid media. In this ill-posed problem, the reconstruction quality is directly determined by the amount and quality of the information obtained from the boundary measurements. Regularly, more information can be obtained by increasing the number of excitation positions in an FDOT system. However, the maximum number of excitation positions is limited by the finite size of the excitation beam. In the present work, we demonstrate a method in FDOT to exploit the unique nonlinear power dependence of upconverting nanoparticles to further increase the amount of information in a raster-scanning setup by including excitation with two beams simultaneously. We show that the additional information can be used to obtain more accurate reconstructions.
We present a 3-D image reconstruction method for free-space fluorescence tomography of mice using hybrid anatomical prior information. Specifically, we use an optically reconstructed surface of the experimental animal and a digital mouse atlas to approximate the anatomy of the animal as structural priors to assist image reconstruction. Experiments are carried out on a cadaver of a nude mouse with a fluorescent inclusion (2.4-mm-diam cylinder) implanted in the chest cavity. Tomographic fluorescence images are reconstructed using an iterative algorithm based on a finite element method. Coregistration of the fluorescence reconstruction and micro-CT (computed tomography) data acquired afterward show good localization accuracy (localization error 1.2+/-0.6 mm). Using the optically reconstructed surface, but without the atlas anatomy, image reconstruction fails to show the fluorescent inclusion correctly. The method demonstrates the utility of anatomical priors in support of free-space fluorescence tomography.
The absorption, scattering, and anisotropy coefficients of the fat emulsion Intralipid-10% have been measured at 457.9, 514.5, 632.8, and 1064 nm. The size and shape distributions of the scattering particles in Intralipid-10% were determined by transmission electron microscopy. Mie theory calculations performed by using the particle size distribution yielded values for the scattering and anisotropy coefficients from 400 to 1100 nm. The agreement with experimental values is better than 6%.
In vertebrates, the development and integrity of the skeleton requires hydroxyapatite (HA) deposition by osteoblasts. HA deposition is also a marker of, or a participant in, processes as diverse as cancer and atherosclerosis. At present, sites of osteoblastic activity can only be imaged in vivo using gamma-emitting radioisotopes. The scan times required are long, and the resultant radioscintigraphic images suffer from relatively low resolution. We have synthesized a near-infrared (NIR) fluorescent bisphosphonate derivative that exhibits rapid and specific binding to HA in vitro and in vivo. We demonstrate NIR light-based detection of osteoblastic activity in the living animal, and discuss how this technology can be used to study skeletal development, osteoblastic metastasis, coronary atherosclerosis, and other human diseases.
We have performed modeling of fluorescence signals from inclusions inside turbid media to investigate the influence of a limited fluorescence contrast and how accurately the depth can be determined by using the spectral information. The depth was determined by forming a ratio of simulated fluorescence intensities at two wavelengths. The results show that it is important to consider the background autofluorescence in determining the depth of a fluorescent inclusion. It is also necessary to know the optical properties of the tissue to obtain the depth. A 20% error in absorption or scattering coefficients yields an error in the determined depth of approximately 2-3 mm (relative error of 10-15%) in a 20 mm thick tissue slab.
Using the method of images, we examine the three boundary conditions commonly applied to the surface of a semi-infinite turbid medium. We find that the image-charge configurations of the partial-current and extrapolated-boundary conditions have the same dipole and quadrupole moments and that the two corresponding solutions to the diffusion equation are approximately equal. In the application of diffusion theory to frequency-domain photon-migration (FDPM) data, these two approaches yield values for the scattering and absorption coefficients that are equal to within 3%. Moreover, the two boundary conditions can be combined to yield a remarkably simple, accurate, and computationally fast method for extracting values for optical parameters from FDPM data. FDPM data were taken both at the surface and deep inside tissue phantoms, and the difference in data between the two geometries is striking. If one analyzes the surface data without accounting for the boundary, values deduced for the optical coefficients are in error by 50% or more. As expected, when aluminum foil was placed on the surface of a tissue phantom, phase and modulation data were closer to the results for an infinite-medium geometry. Raising the reflectivity of a tissue surface can, in principle, eliminate the effect of the boundary. However, we find that phase and modulation data are highly sensitive to the reflectivity in the range of 80-100%, and a minimum value of 98% is needed to mimic an infinite-medium geometry reliably. We conclude that noninvasive measurements of optically thick tissue require a rigorous treatment of the tissue boundary, and we suggest a unified partial-current--extrapolated boundary approach.
A nonlinear, Bayesian optimization scheme is presented for reconstructing fluorescent yield and lifetime, the absorption coefficient, and the diffusion coefficient in turbid media, such as biological tissue. The method utilizes measurements at both the excitation and the emission wavelengths to reconstruct all unknown parameters. The effectiveness of the reconstruction algorithm is demonstrated by simulation and by application to experimental data from a tissue phantom containing the fluorescent agent Indocyanine Green.
Accurate and rapid detection of tumors is of great importance for interrogating the molecular basis of cancer pathogenesis, preventing the onset of complications, and implementing a tailored therapeutic regimen. In this era of molecular medicine, molecular probes that respond to, or target molecular processes are indispensable. Although numerous imaging modalities have been developed for visualizing pathologic conditions, the high sensitivity and relatively innocuous low energy radiation of optical imaging method makes it attractive for molecular imaging. While many human diseases have been studied successfully by using intrinsic optical properties of normal and pathologic tissues, molecular imaging of the expression of aberrant genes, proteins, and other pathophysiologic processes would be enhanced by the use of highly specific exogenous molecular beacons. This review focuses on the development of receptor-specific molecular probes for optical imaging of tumors. Particularly, bioconjugates of probes that absorb and fluoresce in the near infrared wavelengths between 750 and 900 nm will be reviewed.
The ability to image and quantitate fluorescently labeled markers in vivo has generally been limited by autofluorescence of the tissue. Skin, in particular, has a strong autofluorescence signal, particularly when excited in the blue or green wavelengths. Fluorescence labels with emission wavelengths in the near-infrared are more amenable to deep-tissue imaging, because both scattering and autofluorescence are reduced as wavelengths are increased, but even in these spectral regions, autofluorescence can still limit sensitivity. Multispectral imaging (MSI), however, can remove the signal degradation caused by autofluorescence while adding enhanced multiplexing capabilities. While the availability of spectral "libraries" makes multispectral analysis routine for well-characterized samples, new software tools have been developed that greatly simplify the application of MSI to novel specimens.
Inevitable discrepancies between the mouse tissue optical properties assumed by an experimenter and the actual physiological values may affect the tomographic localization of bioluminescent sources. In a previous work, the simplifying assumption of optically homogeneous tissues led to inaccurate localization of deep sources. Improved results may be obtained if a mouse anatomical map is provided by a high-resolution imaging modality and optical properties are assigned to segmented tissues. In this work, the feasibility of this approach was explored by simulating the effect of different magnitude optical property errors on the image formation process of a combined optical-PET system. Some comparisons were made with corresponding simulations using higher spatial resolution data that are typically attainable by CCD cameras. In addition, simulation results provided insights on some of the experimental conditions that could lead to poor localization of bioluminescent sources. They also provided a rough guide on how accurately tissue optical properties need to be known in order to achieve correct localization of point sources with increasing tissue depth under low background noise conditions.
As small-animal fluorescence imaging becomes increasingly accessible to a broad spectrum of users, many lab animal researchers are just beginning to be exposed to its challenges. One setback to fluorescence imaging is background autofluorescence generated in animal tissue and in ingested food. The authors bring this issue into focus, and show how autofluorescence can be reduced in nude mice through selection of appropriate excitation wavelength and mouse diet.
A relatively new strategy to longitudinally monitor tumor load in intact animals and the effects of therapy is noninvasive bioluminescence imaging (BLI). The validity of BLIf or quantitative assessment of tumor load in small animals is critically evaluated in the present review. Cancer cells are grafted in mice or rats after transfection with a luciferase gene--usually that of a firefly. To determine tumor load, animals receive the substrate agent luciferin intraperitoneally, which luciferase converts into oxyluciferin in an ATP-dependent manner Light emitted by oxyluciferin in viable cancer cells is captured noninvasively with a highly sensitive charge-coupled device (CCD) camera. Validation studies indicate that BLI is useful to determine tumor load in the course of time, with each animal serving as its own reference. BLI is rapid, easy to perform, and sensitive. It can detect tumor load shortly after inoculation, even when relatively few cancer cells (2500-10,000) are used. BLI is less suited for the determination of absolute tumor mass in an animal because of quenching of bioluminescence by tissue components and the exact location of tumors because its spatial resolution is limited. Nevertheless, BLI is a powerful tool for high-throughput longitudinal monitoring of tumor load in small animals and allows the implementation of more advanced orthotopic tumor models in therapy intervention studies with almost the same simplicity as when measuring traditional ectopic subcutaneous models in combination with calipers.
Diabetics would benefit greatly from a device capable of providing continuous noninvasive monitoring of their blood glucose levels. The optical scattering coefficient of tissue depends on the concentration of glucose in the extracellular fluid. A feasibility study was performed to evaluate the sensitivity of the tissue reduced scattering coefficient in response to step changes in the blood glucose levels of diabetic volunteers. Estimates of the scattering coefficient were based on measurements of the diffuse reflectance on the skin at distances of 1-10 mm from a point source. A correlation was observed between step changes in blood glucose concentration and tissue reduced scattering coefficient in 30 out of 41 subjects measured.
We examined the effect of temperature change on the diffuse reflectance of the skin. The optical probe consists of several optical fibers located at the center of a thermal electric device, which controls the temperature at the surface of the skin in contact. Measured light reflectance profile between 0.4-1.9 mm was fitted to a mathematical model obtained by Monte Carlo simulation, and absorption and scattering coefficients were estimated. The reduced scattering coefficient of the forearms consistently showed a positive relationship with temperature between 22 and 42 degree(s)C. This dependency was reversible without apparent delay. The same effect was observed on ex vivo pigskin. It is possible to explain the positive instantaneous dependency of scattering on temperature by the change of the refractive index of intercellular fluid. The scattering coefficient of the subcutaneous fat of pigskin showed a negative dependence on temperature. This negative dependency of scattering can be attributed to a phase change as a function of temperature. The absorption coefficient in vivo also increased with temperature from 22 to 42 degree(s)C. But the change was not immediately reversible after temperature reached 40 degree(s)C. This relationship was similar to the nonlinear increase in blood perfusion observed in laser Doppler measurements.
We present the first tomographic reconstruction algorithm for optical molecular imaging that is based on the equation of radiative transfer. The reconstruction code recovers the spatial distribution of fluorescent sources in highly scattering biological tissue. An objective function, which describes the discrepancy of measured near-infrared light with predicted numerical data on the tissue surface, is iteratively minimized to find a solution of the inverse source problem. At each iteration step the predicted data are calculated by a forward model for light propagation based on the equation of radiative transfer. The unknown source distribution is updated along a search direction that is provided by an adjoint differentiation technique.
Extract: Cancer frequently spreads to bone, a process termed bone metastasis. Up to 70% of patients with breast cancer or prostate cancer, and 15 to 30% of patients with lung, colon, bladder or kidney cancer develop bone metastasis. Once tumors go to bone, such as in patients with breast cancer or prostate cancer, they are incurable, and only 20% of patients with breast cancer are still alive five years after they are found to have bone metastasis. It is estimated that about 350,000 people die with bone metastasis each year in the United States. Bone metastasis causes severe bone pain and can result in fractures without any injury, as well as other life-threatening conditions. There are two major types of bone metastasis, one in which bone destruction is the predominant feature and the other one in which new bone formation is predominant. Bone metastasis where bone destruction is the predominant feature is known as osteolytic, and that in which new bone formation is the primary feature is called osteoblastic. This classification for metastasis is really two extremes of a continuum because many patients can have both osteolytic and osteoblastic or mixtures of both in their bone metastasis. In fact, patients with prostate cancer who usually have bone metastasis that shows increased new bone formation also have increased bone destruction in the same lesions.
A model based upon steady-state diffusion theory which describes the radial dependence of diffuse reflectance of light from tissues is developed. This model incorporates a photon dipole source in order to satisfy the tissue boundary conditions and is suitable for either refractive index matched or mismatched surfaces. The predictions of the model were compared with Monte Carlo simulations as well as experimental measurements made with tissue simulating phantoms. The model describes the reflectance data accurately to radial distances as small as 0.5 mm when compared to Monte Carlo simulations and agrees with experimental measurements to distances as small as 1 mm. A nonlinear least-squares fitting procedure has been used to determine the tissue optical properties from the radial reflectance data in both phantoms and tissues in vivo. The optical properties derived for the phantoms are within 5%-10% of those determined by other established techniques. The in vivo values are also consistent with those reported by other investigators.
To build and test an optical imaging system that is sensitive to near-infrared fluorescent molecular probes activated by specific enzymes in tumor tissues in mice.
The imaging system consisted of a source that delivered 610-650-nm excitation light within a lighttight chamber, a 700-nm longpass filter for selecting near-infrared fluorescence emission photons from tissues, and a charge-coupled device (CCD) for recording images. The molecular probe was a biocompatible autoquenched near-infrared fluorescent compound that was activated by tumor-associated proteases for cathepsins B and H. Imaging experiments were performed 0-72 hours after intravenous injection of the probe in nude mice that bore human breast carcinoma (BT-20).
The imaging system had a maximal spatial resolution of 60 microns, with a field of view of 14 cm2. The detection threshold of the nonquenched near-infrared fluorescent dye was subpicomolar in the imaging phantom experiments. In tissue, 250 pmol of fluorochrome was easily detected during the 10-second image acquisition. After intravenous injection of the probe into the tumor-bearing animals, tumors as small as 1 mm became detectable because of tumor-associated enzymatic activation of the quenched compound.
Tumor proteases can be used as molecular targets, allowing visualization of millimeter-sized tumors. The development of this technology, probe design, and optical imaging systems hold promise for molecular imaging, cancer detection, and evaluation of treatment.
To advance our understanding of biological processes as they occur in living animals, imaging strategies have been developed and refined that reveal cellular and molecular features of biology and disease in real time. One rapid and accessible technology for in vivo analysis employs internal biological sources of light emitted from luminescent enzymes, luciferases, to label genes and cells. Combining this reporter system with the new generation of charge coupled device (CCD) cameras that detect the light transmitted through the animal's tissues has opened the door to sensitive in vivo measurements of mammalian gene expression in living animals. Here, we review the development and application of this imaging strategy, in vivo bioluminescence imaging (BLI), together with in vivo fluorescence imaging methods, which has enabled the real-time study of immune cell trafficking, of various genetic regulatory elements in transgenic mice, and of in vivo gene transfer. BLI has been combined with fluorescence methods that together offer access to in vivo measurements that were not previously available. Such studies will greatly facilitate the functional analysis of a wide range of genes for their roles in health and disease.
Noninvasive imaging should facilitate the analysis of changes in experimental tumors and metastases-expressing photoproteins and result in improved data consistency and experimental animal welfare. We analyzed quantitative aspects of noninvasive imaging of luciferase-labeled tumors by comparing the efficiency of noninvasive light detection with in vitro quantification of luciferase activity. An intensified charge coupled device video camera was used to noninvasively image luciferase-expressing human prostate tumors and metastases in nude mice, after ip inoculation of luciferin. Repeated imaging of anesthetized animals after intervening growth periods allowed monitoring of tumor and metastases development. Comparison of photon events recorded in tumor images with the number of relative light units from luminometric quantification of homogenates from the same tumors, revealed that the efficiency with which light escapes tumors is inversely related to tumor size and that intensified charge coupled device images alone are not sufficient for quantitative evaluation of tumor growth. However, a combined videometric and luminometric approach did allow quantification and was used to show the cytostatic effects of paclitaxel in three different human prostate tumors growing in nude mice.
We used the bioluminescent human prostate carcinoma cell line PC-3M-luc-C6 to non-invasively monitor in vivo growth and response of tumors and metastasis before, during and after treatments. Our goal was to determine the utility of a luciferase-based prostate cancer animal model to specifically assess tumor and metastatic recurrence in vivo following chemotherapy. Bioluminescent PC-3M-luc-C6 cells, constitutively expressing luciferase, were implanted into the prostate or under the skin of mice for primary tumor assessment. Cells were also injected into the left ventricle of the heart as an experimental metastasis model. Weekly serial in vivo images were taken of anesthetized mice that were untreated or treated with 5-fluorouracil or mitomycin C. Ex vivo imaging and/or histology was used to confirm and localize metastatic lesions in various tissues initially detected by images in vivo. Our in vivo data detected and quantified early inhibition of subcutaneous and orthotopic prostate tumors in mice as well as significant tumor regrowth post-treatment. Local and distal metastasis was observed within seven days following intracardiac injection of PC-3M-luc-C6 cells. Differential drug responses and metastatic tumor relapse patterns were distinguished over time by in vivo imaging depending on the metastatic site. The longitudinal evaluation of bioluminescent tumor and metastatic development within the same cohorts of animals permitted sensitive and quantitative assessment of both primary and metastatic prostate tumor response and recurrence in vivo.
Many morphological studies of the postmortem interval were carried out under conditions in which the tissue was incubated in vitro after extirpation. However, the extirpation affects cell viability. We examined the ultrastructural changes in the kidney, pancreas, liver, heart and skeletal muscle of male Wistar rats occurring postmortem in situ. In each organ, cell edema (cell swelling), appearance of amorphous dense deposits in the mitochondria, loss of glycogen granules, dilation of the endoplasmic reticulum, clumping and margination of nuclear chromatin, and/or condensation of nuclear chromatin were observed, but the duration of the period of ultrastructural change was organ specific. Most of the ultrastructural changes occurred earlier in kidney. In hepatocytes, the morphological degeneration occurred later than in the renal tubule epithelium and earlier than that in the myocardium. Of the five organs we examined, skeletal muscle showed the greatest delay in postmortem change. In the distal tubule epithelium and pancreatic acinar cells, two forms of nuclear change were seen: one resembled necrotic change and the other resembled apoptotic change. The effect of lysosomes and hydrolytic enzymes was not as great as previous findings.
Diffuse optical tomography is emerging as a viable new biomedical imaging modality. Using visible and near-infrared light this technique can probe the absorption and scattering properties of biological tissues. The main applications are currently in brain, breast, limb and joint imaging; however, optical tomographic imaging of small animals is attracting increasing attention. This interest is fuelled by recent advances in the transgenic manipulation of small animals that has led to many models of human disease. In addition, an ever increasing number of optically reactive biochemical markers has become available, which allow diseases to be detected at the molecular level long before macroscopic symptoms appear. The past three years have seen an array of novel technological developments that have led to the first optical tomographic studies of small animals in the areas of cerebral ischemia and cancer.
The metastasis of prostate cancer to bone is the most significant cause of morbidity and mortality in this disease. An estimated 28,900 men die annually secondary to prostate cancer bone metastasis. Current treatments increase survival for 2 months and only bisphosphonates offer any palliative benefit. This shortcoming is due in part to inadequate models in which to study the molecular biology of the disease and evaluate therapeutic regimens. We examined the breadth of models available that recapitulate the process of prostate cancer metastasis to bone.
A PubMed search was done for publications concerning prostate cancer metastasis to bone and the imaging of bone metastases. Only studies focusing on model systems of disease progression and imaging of the process were included. Additional studies were found by cross-reference searching.
Prostate cancer metastasis to bone is a lengthy, complex process characterized by multiple stages. This has made it difficult to find adequate laboratory models in which to recreate the disease process. Each available model has characteristics of particular phases of disease progression to bone. The most widely used models are transgenic mice, variations of SCID mice, and the traditional orthotopic and xenotransplantation models. Furthermore, investigators have started to adapt their models to incorporate imaging modalities for following the progression of prostate cancer to bone.
The development of models of prostate cancer metastasis to bone is an evolving discipline. A deeper understanding of the metastatic process has served to improve current models and it will continue to do so in the future.
Fluorescence molecular tomography (FMT) has emerged as a means of quantitatively imaging fluorescent molecular probes in three dimensions in living systems. To assess the accuracy of FMT in vivo, translucent plastic tubes containing a turbid solution with a known concentration of Cy 5.5 fluorescent dye are constructed and implanted subcutaneously in nude mice, simulating the presence of a tumor accumulating a fluorescent molecular reporter. Comparisons between measurements of fluorescent tubes made before and after implantation demonstrate that the accuracy of FMT reported for homogeneous phantoms extends to the in vivo situation. The sensitivity of FMT to background fluorescence is tested by imaging fluorescent tubes in mice injected with Cy 5.5-labeled Annexin V. For small tube fluorochrome concentrations, the presence of background fluorescence results in increases in the reconstructed concentration. This phenomenon is counteracted by applying a simple subtraction correction to the measured fluorescence data. The effects of varying tumor photon absorption are simulated by imaging fluorescent tubes with varying ink concentrations, and are found to be minor. These findings demonstrate the in vivo quantitative accuracy of fluorescence tomography, and encourage further development of this imaging modality as well as application of FMT in molecular imaging studies using fluorescent reporters.
Using an area-illumination and area-detection scheme, we acquire fluorescence frequency domain measurements from a tissue phantom with an embedded fluorescent target and obtain tomographic reconstructions of the interior fluorescence absorption map with an adaptive finite element based scheme. The tissue phantom consisted of a clear acrylic cubic box (512 ml) filled with 1% Liposyn solution, while the fluorescent targets were 5 mm diameter glass bulbs filled with 1 microM Indocyanine Green dye solution in 1% Liposyn. Frequency domain area illumination and detection employed a planar excitation source using an expanded intensity modulated (100 MHz) 785 nm diode laser light and a gain modulated image intensified charge coupled device camera, respectively. The excitation pattern was characterized by isolating the singly scattered component with cross polarizers and was input into a dual adaptive finite element-based scheme for three dimensional reconstructions of fluorescent targets embedded beneath the phantom surface. Adaptive mesh refinement techniques allowed efficient simulation of the incident excitation light and the reconstruction of fluorescent targets buried at the depths of 1 and 2 cm. The results demonstrate the first clinically relevant noncontact fluorescence tomography with adaptive finite element methods.
There is a wealth of new fluorescent reporter technologies for tagging of many cellular and subcellular processes in vivo. This imposed contrast is now captured with an increasing number of available imaging methods that offer new ways to visualize and quantify fluorescent markers distributed in tissues. This is an evolving field of imaging sciences that has already achieved major advances but is also facing important challenges. It is nevertheless well poised to significantly impact the ways of biological research, drug discovery, and clinical practice in the years to come. Herein, the most pertinent technologies associated with in vivo noninvasive or minimally invasive fluorescence imaging of tissues are summarized. Focus is given to small-animal imaging. However, while a broad spectrum of fluorescence reporter technologies and imaging methods are outlined, as necessary for biomedical research, and clinical translation as well.
A simple approach for estimating the location and power of a bioluminescent point source inside tissue is reported. The strategy consists of using a diffuse reflectance image at the emission wavelength to determine the optical properties of the tissue. Following this, bioluminescence images are modelled using a single point source and the optical properties from the reflectance image, and the depth and power are iteratively adjusted to find the best agreement with the experimental image. The forward models for light propagation are based on the diffusion approximation, with appropriate boundary conditions. The method was tested using Monte Carlo simulations, Intralipid tissue-simulating phantoms and ex vivo chicken muscle. Monte Carlo data showed that depth could be recovered within 6% for depth 4-12 mm, and the corresponding relative source power within 12%. In Intralipid, the depth could be estimated within 8% for depth 4-12 mm, and the relative source power, within 20%. For ex vivo tissue samples, source depths of 4.5 and 10 mm and their relative powers were correctly identified.
A new method is described for obtaining a 3-D reconstruction of a bioluminescent light source distribution inside a living animal subject, from multispectral images of the surface light emission acquired on charge-coupled device (CCD) camera. The method uses the 3-D surface topography of the animal, which is obtained from a structured light illumination technique. The forward model of photon transport is based on the diffusion approximation in homogeneous tissue with a local planar boundary approximation for each mesh element, allowing rapid calculation of the forward Green's function kernel. Absorption and scattering properties of tissue are measured a priori as input to the algorithm. By using multispectral images, 3-D reconstructions of luminescent sources can be derived from images acquired from only a single view. As a demonstration, the reconstruction technique is applied to determine the location and brightness of a source embedded in a homogeneous phantom subject in the shape of a mouse. The technique is then evaluated with real mouse models in which calibrated sources are implanted at known locations within living tissue. Finally, reconstructions are demonstrated in a PC3M-luc (prostate tumor line) metastatic tumor model in nude mice.
The performance of a simple approach for the in vivo reconstruction of bioluminescent point sources in small animals was evaluated. The method uses the diffusion approximation as a forward model of light propagation from a point source in a homogeneous tissue to find the source depth and power. The optical properties of the tissue are estimated from reflectance images obtained at the same location on the animal. It was possible to localize point sources implanted in mice, 2-8 mm deep, to within 1 mm. The same performance was achieved for sources implanted in rat abdomens when the effects of tissue surface curvature were eliminated. The source power was reconstructed within a factor of 2 of the true power for the given range of depths, even though the apparent brightness of the source varied by several orders of magnitude. The study also showed that reconstructions using optical properties measured in situ were superior to those based on data in the literature.
Fluorescence molecular tomography (FMT) is a novel tomographic near-infrared (NIR) imaging modality that enables 3D quantitative determination of fluorochrome distribution in tissues of live small animals at any depth. This study demonstrates a noninvasive, quantitative method of monitoring engineered bone remodeling via FMT. Murine mesenchymal stem cells overexpressing the osteogenic gene BMP2 (mMSCs-BMP2) were implanted into the thigh muscle and into a radial nonunion bone defect model in C3H/HeN mice. Real-time imaging of bone formation was performed following systemic administration of the fluorescent bisphosphonate imaging agent OsteoSense, an hydroxyapatite-directed bone-imaging probe. The mice underwent imaging on days 7, 14, and 21 postimplantation. New bone formation at the implantation sites was quantified using micro-computed tomography (micro-CT) imaging. A higher fluorescent signal occurred at the site of the mMSC-BMP2 implants than that found in controls. Micro-CT imaging revealed a mass of mature bone formed in the implantation sites on day 21, a finding also confirmed by histology. These findings highlight the effectiveness of FMT as a functional platform for molecular imaging in the field of bone regeneration and tissue engineering.
We present a normalized Born expansion that facilitates fluorescence reconstructions in turbid, tissuelike media. The algorithm can be particularly useful for tissue investigations of fluorochrome distributionin vivo, since it does not require absolute photon-field measurements or measurements before contrast-agent administration. This unique advantage can be achieved only in fluorescence mode. We used this algorithm to three-dimensionally image and quantify an indocyanine fluorochrome phantom, using a novel fluorescence tomographic imager developed for animals.
A condition on nonuniqueness in optical tomography is stated. The main result applies to steady-state (dc) diffusion-based optical tomography, wherein we demonstrate that simultaneous unique recovery of diffusion and absorption coefficients cannot be achieved. A specific example of two images that give identical dc data is presented. If the refractive index is considered an unknown, then nonuniqueness also occurs in frequency-domain and time-domain optical tomography, if the underlying model of the diffusion approximation is employed.
Most instruments used to measure tissue optical properties noninvasively employ data-analysis algorithms that rely on the simplifying assumption that the tissue is semi-infinite and homogeneous. The influence of a layered tissue architecture on the determination of the scattering and absorption coefficients has been investigated in this study. Reflectance as a function of distance from a point source for a two-layered tissue architecture that simulates skin overlying fat was calculated by using a Monte Carlocode. These data were analyzed by using a diffusion theory modelfor a homogeneous semi-infinite medium to calculate the scatter and absorption coefficients. Depending on the algorithm and the radial distance, the estimated tissue optical properties were different from those of either layer, and under some circumstances, physically impossible. In addition, the sensitivity and cross talk of the estimated optical properties to changes in input optical properties were calculated for different layered geometries. For typical optical properties of skin, the sensitivity to changes in optical properties is highly dependent on the layered architecture, the measurement distance, and the fitting algorithm. Furthermore, a change in the input absorption coefficient may result in an apparent change in the measured scatter coefficient, and a change in the in put scatter coefficient may result in an apparent change in the measured absorption coefficient.
A combined magnetic resonance and near-infrared (MRI-NIR) imaging modality can potentially yield high resolution maps of optical properties from noninvasive simultaneous measurement. The main disadvantage of near-infrared (NIR) tomography lies in the low spatial resolution resulting from the highly scattering nature of tissue for these wavelengths. MRI has achieved high resolution, but suffers from low specificity. In this study, NIR image reconstruction algorithms that incorporate a priori structural information provided by MRI are investigated in an attempt to optimize recovery of a simulated optical property distribution. The effect of high levels of tissue heterogeneity are evaluated to determine the limitations of incorporating prior information into a realistic set of patient breast images. We assume absorption coefficient (μ<sub>a</sub>) variations near ±40%, and transport scattering coefficient (μ<sub>s</sub><sup></sup>/) variations near ±20%, in a coronal breast MRI geometry. Changes in tissue pathology due to tumor growth can be observed with NIR tompgraphy, and so the goal here is to determine how best to quantify these tumor-based contrast regions within the presence of high tissue heterogeneity. By applying knowledge of tissue's layered structure in reconstruction through various constraints in the iterative algorithm, quantitative recovery of the tumor optical properties improves from 69% to 74%, and localization improves as well. However, only when the true heterogeneity of the tissue distribution was included was accurate quantification of the tumor region possible. Using a good initial guess of μ<sub>a</sub> and μ<sub>s</sub><sup></sup>/, derived from the regional structure of the model, quantification of the region reaches 99% of the true value, and spatial resolution retains a similar value to the original MRI image.
Ntziachristos V and Weissleder R 2001 Experimental three-dimensional fluorescence reconstruction of diffuse media using a normalized Born approximation
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- Farrell T J