Quantitative fluorescence imaging of point-like sources in small animals

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.     

Accuracy of quantitative reconstructions in SPECT/CT imaging

The goal of this study was to determine the quantitative accuracy of our OSEM-APDI reconstruction method based on SPECT/CT imaging for Tc-99m, In-111, I-123, and I-131 isotopes. Phantom studies were performed on a SPECT/low-dose multislice CT system (Infinia-Hawkeye-4 slice, GE Healthcare) using clinical acquisition protocols. Two radioactive sources were centrally and peripherally placed inside an anthropometric Thorax phantom filled with non-radioactive water. Corrections for attenuation, scatter, collimator blurring and collimator septal penetration were applied and their contribution to the overall accuracy of the reconstruction was evaluated. Reconstruction with the most comprehensive set of corrections resulted in activity estimation with error levels of 3-5% for all the isotopes.     

MR-based keyhole SPECT for small animal imaging

The rationale for multi-modality imaging is to integrate the strengths of different imaging technologies while reducing the shortcomings of an individual modality. The work presented here proposes a limited-field-of-view (LFOV) SPECT reconstruction technique that can be implemented on a multi-modality MR/SPECT system that can be used to obtain simultaneous MRI and SPECT images for small animal imaging. The reason for using a combined MR/SPECT system in this work is to eliminate any possible misregistration between the two sets of images when MR images are used as a priori information for SPECT. In nuclear imaging the target area is usually smaller than the entire object; thus, focusing the detector on the LFOV results in various advantages including the use of a smaller nuclear detector (less cost), smaller reconstruction region (faster reconstruction) and higher spatial resolution when used in conjunction with pinhole collimators with magnification. The MR/SPECT system can be used to choose a region of interest (ROI) for SPECT. A priori information obtained by the full field-of-view (FOV) MRI combined with the preliminary SPECT image can be used to reduce the dimensions of the SPECT reconstruction by limiting the computation to the smaller FOV while reducing artifacts resulting from the truncated data. Since the technique is based on SPECT imaging within the LFOV it will be called the keyhole SPECT (K-SPECT) method. At first MRI images of the entire object using a larger FOV are obtained to determine the location of the ROI covering the target organ. Once the ROI is determined, the animal is moved inside the radiofrequency (rf) coil to bring the target area inside the LFOV and then simultaneous MRI and SPECT are performed. The spatial resolution of the SPECT image is improved by employing a pinhole collimator with magnification >1 by having carefully calculated acceptance angles for each pinhole to avoid multiplexing. In our design all the pinholes are focused to the center of the LFOV. K-SPECT reconstruction is accomplished by generating an adaptive weighting matrix using a priori information obtained by simultaneously acquired MR images and the radioactivity distribution obtained from the ROI region of the SPECT image that is reconstructed without any a priori input. Preliminary results using simulations with numerical phantoms show that the image resolution of the SPECT image within the LFOV is improved while minimizing artifacts arising from parts of the object outside the LFOV due to the chosen magnification and the new reconstruction technique. The root-mean-square-error (RMSE) in the out-of-field artifacts was reduced by 60% for spherical phantoms using the K-SPECT reconstruction technique and by 48.5-52.6% for the heart in the case with the MOBY phantom. The K-SPECT reconstruction technique significantly improved the spatial resolution and quantification while reducing artifacts from the contributions outside the LFOV as well as reducing the dimension of the reconstruction matrix.     

Recent advances in imaging the lungs of intact small animals

A new generation of imaging devices now make it possible to generate both structural and functional images for the study of lung biology in small animals, including common laboratory mouse and rat models. "Micro" X-ray computed tomography and positron emission tomography scanners, highly sensitive cooled charge coupled device cameras for bioluminescence and fluorescence imaging, high magnetic field magnetic resonance imaging scanners, and recent advances in ultrasound system technology can be used to study such diverse processes as ventilation, perfusion, pulmonary hypertension, lung inflammation, and gene transfer, among others. Images from more than one modality can also be fused, allowing structure-function and function-function relationships to be studied on a regional basis. These new instruments, part of an emerging suite of techniques collectively known as "molecular imaging," provide an enormous potential for elucidating lung biology in intact animal models and systems.     

Model-based compensation for quantitative 123I brain SPECT imaging

Previously we have developed a model-based method that can accurately estimate downscatter contamination from high-energy photons in 123I imaging. In this work we combined the model-based method with iterative reconstruction-based compensations for other image-degrading factors such as attenuation, scatter, the collimator-detector response function (CDRF) and partial volume effects to form a comprehensive method for performing quantitative 123I SPECT image reconstruction. In the model-based downscatter estimation method, photon scatter inside the object was modelled using the effective source scatter estimation (ESSE) technique, including contributions from all the photon emissions. The CDRFs, including the penetration and scatter components due to the high-energy 123I photons, were estimated using Monte Carlo (MC) simulations of point sources in air at various distances from the face of the collimator. The downscatter contamination was then compensated for during the iterative reconstruction by adding the estimated results to the projection steps. The model-based downscatter compensation (MBDC) was evaluated using MC simulated and experimentally acquired projection data. From the MC simulation, we found about 39% of the total counts in the energy window of 123I were attributed to the downscatter contamination, which reduced image contrast and caused a 1.5% to 10% overestimation of activities in various brain structures. Model-based estimates of the downscatter contamination were in good agreement with the simulated data. Compensation using MBDC removed the contamination and improved the image contrast and quantitative accuracy to that of the images obtained from 159 keV photons. The errors in absolute quantitation were reduced to within +/-3.5%. The striatal specific binding potential calculated based on the activity ratio to the background was also improved after MBDC. The errors were reduced from -4.5% to -10.93% without compensation to -0.55% to 4.87% after compensation. The model-based method provided accurate downscatter estimation and, when combined with iterative reconstruction-based compensations, accurate quantitation was obtained with minimal loss of precision.     

Design and simulation of a high-resolution stationary SPECT system for small animals

Exciting new SPECT systems can be created by combining pinhole imaging with compact high-resolution gamma cameras. These new systems are able to solve the problem of the limited sensitivity-resolution trade-off that hampers contemporary small animal SPECT. The design presented here (U-SPECT-III) uses a set of detectors placed in a polygonal configuration and a cylindrical collimator that contains 135 pinholes arranged in nine rings. Each ring contains 15 gold pinhole apertures that focus on the centre of the cylinder. A non-overlapping projection is acquired via each pinhole. Consequently, when a mouse brain is placed in the central field-of-view, each voxel in the cerebrum can be observed via 130 to 135 different pinholes simultaneously. A method for high-resolution scintillation detection is described that eliminates the depth-of-interaction problem encountered with pinhole cameras, and is expected to provide intrinsic detector resolutions better than 150 microm. By means of simulations U-SPECT-III is compared to a simulated dual pinhole SPECT (DP-SPECT) system with a pixelated array consisting of 2.0 x 2.0 mm NaI crystals. Analytic calculations indicate that the proposed U-SPECT-III system yields an almost four times higher linear and about sixty times higher volumetric system resolution than DP-SPECT, when the systems are compared at matching system sensitivity. In addition, it should be possible to achieve a 15 up to 30 times higher sensitivity with U-SPECT-III when the systems are compared at equal resolution. Simulated images of a digital mouse-brain phantom show much more detail with U-SPECT-III than with DP-SPECT. In a resolution phantom, 0.3 mm diameter cold rods are clearly visible with U-SPECT-III, whereas with DP-SPECT the smallest visible rods are about 0.6-0.8 mm. Furthermore, with U-SPECT-III, the image deformations outside the central plane of reconstruction that hamper conventional pinhole SPECT are strongly suppressed. Simulation results indicate that future pinhole SPECT systems are likely to bring about significant improvements in radio-molecular imaging of small animals.     

Multiplane spectroscopic whole-body photoacoustic imaging of small animals in vivo

We have successfully developed a multiscale acoustic-resolution photoacoustic tomography system in a single imaging platform. By switching between ultrasound transducers (center frequencies 5 and 40 MHz) and optical condensers, we have photoacoustically imaged microvasculatures of small animals in vivo at different scales. Further, we have extended the field of view of our imaging system to entire bodies of small animals. At different imaging planes, we have noninvasively imaged the major blood vessels (e.g., descending aorta, intercostal vessels, cephalic vessels, brachial vessels, femoral vessels, popliteal vessels, lateral marginal vessels, cranial mesenteric vessels, mammalian vessels, carotid artery, jugular vein, subclavian vessels, iliac vessels, and caudal vessels) as well as intact internal organs (e.g., spleen, liver, kidney, intestine, cecum, and spinal cord) of the animals in vivo. The spectroscopic whole-body photoacoustic imaging clearly reveals the spectral responses of the internal structures. Similar to other existing preclinical whole-body imaging systems, this whole-body photoacoustic tomography can be a useful tool for small-animal research.     

Bioluminescence imaging of point sources implanted in small animals post mortem: evaluation of a method for estimating source strength and depth

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.     

Attenuation correction for small animal SPECT imaging using x-ray CT data

Photon attenuation in small animal nuclear medicine scans can be significant when using isotopes that emit lower energy photons such as iodine-125. We have developed a method to use microCT data to perform attenuation corrected small animal single-photon emission computed tomography (SPECT). A microCT calibration phantom was first imaged, and the resulting calibration curve was used to convert microCT image values to linear attenuation coefficient values that were then used in an iterative SPECT reconstruction algorithm. This method was applied to reconstruct a SPECT image of a uniform phantom filled with 125I-NaI. Without attenuation correction, the image suffered a 30% decrease in intensity in the center of the image, which was removed with the addition of attenuation correction. This reduced the relative standard deviation in the region of interest from 10% to 6%.     

A prototype instrument for single pinhole small animal adaptive SPECT imaging

The authors have designed and constructed a small-animal adaptive SPECT imaging system as a prototype for quantifying the potential benefit of adaptive SPECT imaging over the traditional fixed geometry approach. The optical design of the system is based on filling the detector with the region of interest for each viewing angle, maximizing the sensitivity, and optimizing the resolution in the projection images. Additional feedback rules for determining the optimal geometry of the system can be easily added to the existing control software. Preliminary data have been taken of a phantom with a small, hot, offset lesion in a flat background in both adaptive and fixed geometry modes. Comparison of the predicted system behavior with the actual system behavior is presented, along with recommendations for system improvements.     

Stereotactic imaging for radiotherapy: accuracy of CT,MRI, PET and SPECT

CT, MRI, PET and SPECT provide complementary information for treatment planning in stereotactic radiotherapy. Stereotactic correlation of these images requires commissioning tests to confirm the localization accuracy of each modality. A phantom was developed to measure the accuracy of stereotactic localization for CT, MRI, PET and SPECT in the head and neck region. To this end. the stereotactically measured coordinates of structures within the phantom were compared with their mechanically defined coordinates. For MRI, PET and SPECT, measurements were performed using two different devices. For MRI, T1- and T2-weighted imaging sequences were applied. For each measurement, the mean radial deviation in space between the stereotactically measured and mechanically defined position of target points was determined. For CT, the mean radial deviation was 0.4 +/- 0.2 mm. For MRI, the mean deviations ranged between 0.7 +/- 0.2 mm and 1.4 +/- 0.5 mm, depending on the MRI device and the imaging sequence. For PET, mean deviations of 1.1 +/- 0.5 mm and 2.4 +/- 0.3 mm were obtained. The mean deviations for SPECT were 1.6 +/- 0.5 mm and 2.0 +/- 0.6 mm. The phantom is well suited to determine the accuracy of stereotactic localization with CT, MRI, PET and SPECT in the head and neck region. The obtained accuracy is well below the physical resolution for CT, PET and SPECT, and of comparable magnitude for MRI. Since the localization accuracy may be device dependent, results obtained at one device cannot be generalized to others.     

Functional imaging of cerebrovascular activities in small animals using high-resolution photoacoustic tomography

Photoacoustic imaging (PAI) is a noninvasive, nonionizing modality based on the differences in light absorption of various biological tissues. PAI utilizes the endogenous contrast characteristics of traditional optical imaging, while benefiting from high spatial resolution of the ultrasound imaging. A PAI system was developed to reconstruct the two-dimensional cross section image and to visualize the cerebrovascular activities of mouse in vivo. The spatial resolution of the PAI system was determined to be 0.110 mm by a two-point-source phantom with the Rayleigh criterion. The potential applications of the system were clearly demonstrated by successfully mapping a traumatic lesion in the mouse brain cerebral cortex, by its ability to monitor physiological changes in the brain due to carotid ligation and drug stimulation, and two-dimensional sliced images of a traumatic mouse brain at different depths were also provided. Our experimental results indicate that PAI has the potential for studying of traumatic brain injury and physiological functions of the brain.     

Noninvasive photoacoustic imaging of the thoracic cavity and the kidney in small and large animals

The internal organs of rats and rabbits were clearly imaged noninvasively using a deeply penetrating reflection-mode photoacoustic imaging system. This imaging system had previously been found to provide an imaging depth limit of approximately 38 mm. In the thoracic cavity, major blood vessels connecting to the heart were imaged, and the right atrium was imaged as deeply as approximately 8 mm. In the abdominal cavities, the kidney and vena cava inferior were also imaged in situ. The vena cava inferior approximately 13.7 mm deep was imaged. The kidney of a New Zealand white rabbit was also imaged. This study shows the deep internal organ imaging capability of the system in animals. This technology can potentially be used to study tumors in internal organs, and be adapted to clinical diagnosis.     

Simultaneous fluorescence and positron emission tomography for in vivo imaging of small animals

Simultaneous positron emission tomography (PET) and fluorescence tomography (FT) for in vivo imaging of small animals is proposed by a dual-modality system. This system combines a charge-coupled device-based near-infrared fluorescence imaging with a planar detector pair-based PET. With [(18)F]-2-fluoro-2-deoxy-d-glucose radioactive tracer and the protease activated fluorescence probe, on the one hand, the simultaneous metabolic activity and protease activity in tumor region are revealed by the PET and FT, respectively. On the other hand, the protease activity both on the surface layer and the deep tissue of the tumor is provided by the fluorescence reflection imaging and FT, respectively.     

Upconversion fluorescence imaging of cells and small animals using lanthanide doped nanocrystals

Upconversion fluorescence imaging technique with excitation in the near-infrared (NIR) region has been used for imaging of biological cells and tissues. This has several advantages, including absence of photo-damage to living organisms, very low auto-fluorescence, high detection sensitivity, and high light penetration depth in biological tissues. In this report we demonstrate the use of a new upconversion fluorophore, lanthanide doped nanocrystals, for imaging of cells and some deep tissues in animal. Polyethyleneimine (PEI) coated NaYF(4):Yb,Er nanoparticles were synthesized, which produce very strong upconversion fluorescence when excited at 980 nm by a NIR laser. The nanoparticles were shown to be stable in physiologic buffered saline (PBS), non-toxic to bone marrow stem cells, and resistant to photo-bleaching. The nanoparticles delivered into some cell lines or injected intradermally and intramuscularly into some tissues either near the body surface or deep in the body of rats showed visible fluorescence, when exposed to a 980 nm NIR laser. To the best of our knowledge, this represents the first demonstration of use of upconversion fluorophores for cellular and tissue imaging.     

Three-dimensional optical tomographic brain imaging in small animals, part 1: hypercapnia

In this study, we explore the potential of diffuse optical tomography for brain oximetry. While several groups have already reported on the sensitivity of optical measurements to changes in oxyhemoglobin, deoxyhemoglobin, and blood volume, these studies were often limited to single source-detector geometries or topographic maps, where signals obtained from within the brain are projected onto 2-D surface maps. In this two-part study, we report on our efforts toward developing a volumetric optical imaging system that allows one to spatially resolve 3-D hemodynamic effects in rat brains. In part 1, we describe the instrumentation, optical probe design, and the model-based iterative image reconstruction algorithm employed in this work. Consideration of how a priori anatomical knowledge can be incorporated in the reconstruction process is presented. This system is then used to monitor global hemodynamic changes that occur in the brain under various degrees of hypercapnia. The physiologic cerebral response to hypercapnia is well known and therefore allows an initial performance assessment of the imaging system. As expected, we observe global changes in blood volume and oxygenation, which vary linearly as a function of the concentration of the inspired carbon dioxide. Furthermore, experiments are designed to determine the sensitivity of the reconstructions of only 1 mm to inaccurate probe positioning. We determine that shifts can significantly influence the reconstructions. In part 2 we focus on more local hemodynamic changes that occur during unilateral carotid occlusion performed at lower-than-normal systemic blood pressure. In this case, the occlusion leads to a predominantly monohemispherically localized effect, which is well described in the literature. Having explored the system with a well-characterized physiologic effect, we investigate and discuss the complex compensatory cerebrovascular hemodynamics that occur at normotensive blood pressure. Overall, these studies demonstrate the potential and limitations of our diffuse optical imager for visualizing global and focal hemodynamic phenomenon three dimensionally in the brains of small animals.     

Possible sources of error in the evaluation of psychopathology

Mental health professionals have been described as biased in the evaluation of mental illness, almost always in favor of illness, If a professional bias does exist, two factors could be responsible-situational expectancies and/or professional training. Undergraduate students (N = 32) and psychiatric residents (N = 16) listened to four recorded interviews. Two levels of mental illness expectancy were obtained by changing the context of the interviews. The Ss rated the interviewees on scales of psychopathology and recorded their impression of the interviewees. Multivariate and univariate analyses of variance and x² tests were used to evaluate the data. Evidence indicated that the bised perception of mental illness occurred for all Ss when mental illness expectancies were high. The data also indicated that residents are not more inclined than students to label people mentally ill.