Scott C. Davis

Pre-clinical whole-body fluorescence imaging: Review of instruments, methods and applications

Fluorescence sampling of cellular function is widely used in all aspects of biology, allowing the visualization of cellular and sub-cellular biological processes with spatial resolutions in the range from nanometers up to centimeters. Imaging of fluorescence in vivo has become the most commonly used radiological tool in all pre-clinical work. In the last decade, full-body pre-clinical imaging systems have emerged with a wide range of utilities and niche application areas. The range of fluorescent probes that can be excited in the visible to near-infrared part of the electromagnetic spectrum continues to expand, with the most value for in vivo use being beyond the 630 nm wavelength, because the absorption of light sharply decreases. Whole-body in vivo fluorescence imaging has not yet reached a state of maturity that allows its routine use in the scope of large-scale pre-clinical studies. This is in part due to an incomplete understanding of what the actual fundamental capabilities and limitations of this imaging modality are. However, progress is continuously being made in research laboratories pushing the limits of the approach to consistently improve its performance in terms of spatial resolution, sensitivity and quantification. This paper reviews this imaging technology with a particular emphasis on its potential uses and limitations, the required instrumentation, and the possible imaging geometries and applications. A detailed account of the main commercially available systems is provided as well as some perspective relating to the future of the technology development. Although the vast majority of applications of in vivo small animal imaging are based on epi-illumination planar imaging, the future success of the method relies heavily on the design of novel imaging systems based on state-of-the-art optical technology used in conjunction with high spatial resolution structural modalities such as MRI, CT or ultrasound.

Spectrally resolved bioluminescence optical tomography.

Spectrally resolved bioluminescence optical tomography is an approach to recover images of luciferase activity within a volume using multiwavelength emission data from internal bioluminescence sources. The underlying problem of uniqueness associated with nonspectrally resolved intensity-based bioluminescence tomography is highlighted. Reconstructed images of bioluminescence are presented by using as input both simulated and real multiwavelength data from a tissue-simulating phantom. The location of the internal bioluminescence is obtained with 1 mm accuracy. Further, the amplitude of the reconstructed source is proportional to the actual bioluminescence intensity.

Magnetic resonance–coupled fluorescence tomography scanner for molecular imaging of tissue

A multichannel spectrally resolved optical tomography system to image molecular targets in small animals from within a clinical MRI is described. Long source/detector fibers operate in contact mode and couple light from the tissue surface in the magnet bore to 16 spectrometers, each containing two optical gratings optimized for the near infrared wavelength range. High sensitivity, cooled charge coupled devices connected to each spectrograph provide detection of the spectrally resolved signal, with exposure times that are automated for acquisition at each fiber. The design allows spectral fitting of the remission light, thereby separating the fluorescence signal from the nonspecific background, which improves the accuracy and sensitivity when imaging low fluorophore concentrations. Images of fluorescence yield are recovered using a nonlinear reconstruction approach based on the diffusion approximation of photon propagation in tissue. The tissue morphology derived from the MR images serves as an imaging template to guide the optical reconstruction algorithm. Sensitivity studies show that recovered values of indocyanine green fluorescence yield are linear to concentrations of 1 nM in a 70 mm diameter homogeneous phantom, and detection is feasible to near 10 pM. Phantom data also demonstrate imaging capabilities of imperfect fluorophore uptake in tissue volumes of clinically relevant sizes. A unique rodent MR coil provides optical fiber access for simultaneous optical and MR data acquisition of small animals. A pilot murine study using an orthotopic glioma tumor model demonstrates optical-MRI imaging of an epidermal growth factor receptor targeted fluorescent probe in vivo.

Image analysis methods for diffuse optical tomography.

Three major analytical tools in imaging science are summarized and demonstrated relative to optical imaging in vivo. Standard resolution testing is optimal when infinite contrast is used and hardware evaluation is the goal. However, deep tissue imaging of absorption or fluorescent contrast agents in vivo often presents a different problem, which requires contrast-detail analysis. This analysis shows that the minimum detectable sizes are in the range of 1/10 the outer diameter, whereas minimum detectable contrast values are in the range of 10 to 20% relative to the continuous background values. This is estimated for objects being in the center of the domain being imaged, and as the heterogeneous region becomes closer to the surface, the lower limit on size and contrast can become arbitrarily low and more dictated by hardware specifications. Finally, if human observer detection of abnormalities in the images is the goal, as is standard in most radiological practice, receiver operating characteristic (ROC) curve and location receiver operating characteristic curve (LROC) are used. Each of these three major areas of image interpretation and analysis are reviewed in the context of medical imaging as well as how they are used to quantify the performance of diffuse optical imaging of tissue.

Subsurface diffuse optical tomography can localize absorber and fluorescent objects but recovered image sensitivity is nonlinear with depth.

Subsurface tomography with diffuse light has been investigated with a noncontact approach to characterize the performance of absorption and fluorescence imaging. Using both simulations and experiments, the reconstruction of local subsurface heterogeneity is demonstrated, but the recovery of target size and fluorophore concentration is not linear when changes in depth occur, whereas the mean position of the object for experimental fluorescent and absorber targets is accurate to within 0.5 and 1.45 mm when located within the first 10 mm below the surface. Improvements in the linearity of the response with depth appear to remain challenging and may ultimately limit the approach to detection rather than characterization applications. However, increases in tissue curvature and/or the addition of prior information are expected to improve the linearity of the response. The potential for this type of imaging technique to serve as a surgical guide is highlighted.

Fast segmentation and high-quality three-dimensional volume mesh creation from medical images for diffuse optical tomography

Abstract. Multimodal approaches that combine near-infrared (NIR) and conventional imaging modalities have been shown to improve optical parameter estimation dramatically and thus represent a prevailing trend in NIR imaging. These approaches typically involve applying anatomical templates from magnetic resonance imaging/computed tomography/ultrasound images to guide the recovery of optical parameters. However, merging these data sets using current technology requires multiple software packages, substantial expertise, significant time-commitment, and often results in unacceptably poor mesh quality for optical image reconstruction, a reality that represents a significant roadblock for translational research of multimodal NIR imaging. This work addresses these challenges directly by introducing automated digital imaging and communications in medicine image stack segmentation and a new one-click three-dimensional mesh generator optimized for multimodal NIR imaging, and combining these capabilities into a single software package (available for free download) with a streamlined workflow. Image processing time and mesh quality benchmarks were examined for four common multimodal NIR use-cases (breast, brain, pancreas, and small animal) and were compared to a commercial image processing package. Applying these tools resulted in a fivefold decrease in image processing time and 62% improvement in minimum mesh quality, in the absence of extra mesh postprocessing. These capabilities represent a significant step toward enabling translational multimodal NIR research for both expert and nonexpert users in an open-source platform.

Cerenkov emission induced by external beam radiation stimulates molecular fluorescence

PURPOSE Cerenkov emission is induced when a charged particle moves faster than the speed of light in a given medium. Both x-ray photons and electrons produce optical Cerenkov photons in everyday radiation therapy of tissue; yet, this phenomenon has never been fully documented. This study quantifies the emissions and also demonstrates that the Cerenkov emission can excite a fluorophore, protoporphyrin IX (PpIX), embedded in biological phantoms. METHODS In this study, Cerenkov emission induced by radiation from a clinical linear accelerator is investigated. Biological mimicking phantoms were irradiated with x-ray photons, with energies of 6 or 18 MV, or electrons at energies 6, 9, 12, 15, or 18 MeV. The Cerenkov emission and the induced molecular fluorescence were detected by a camera or a spectrometer equipped with a fiber optic cable. RESULTS It is shown that both x-ray photons and electrons, at MeV energies, produce optical Cerenkov photons in tissue mimicking media. Furthermore, we demonstrate that the Cerenkov emission can excite a fluorophore, protoporphyrin IX (PpIX), embedded in biological phantoms. CONCLUSIONS The results here indicate that molecular fluorescence monitoring during external beam radiotherapy is possible.

Spectrally resolved bioluminescence tomography using the reciprocity approach

Spectrally resolved bioluminescence optical tomography is an approach to recover images of, for example, Luciferase activity within a volume using multiwavelength emission data from internal bioluminescence sources. The underlying problem of uniqueness associated with nonspectrally resolved intensity-based bioluminescence tomography is demonstrated and it is shown that using a non-negative constraint inverse algorithm, an accurate solution for the source distribution can be calculated from the measured data. Reconstructed images of bioluminescence are presented using both simulated complex and heterogeneous small animal models as well as real multiwavelength data from a tissue-simulating phantom. The location of the internal bioluminescence source using experimental data is obtained with 0.5 mm accuracy and it is shown that small (2.5 mm diameter) sources of up to 12.5 mm deep, within a complex mouse model, can be resolved accurately using a single view data collection strategy. Finally, using the reciprocity approach for image reconstruction, a dramatic improvement in computational time is shown without loss to image accuracy with both experimental and simulated data, potentially reducing computing time from 402 to 3.75 h.

Projection imaging of photon beams by the Čerenkov effect

PURPOSE A novel technique for beam profiling of megavoltage photon beams was investigated for the first time by capturing images of the induced Čerenkov emission in water, as a potential surrogate for the imparted dose in irradiated media. METHODS A high-sensitivity, intensified CCD camera (ICCD) was configured to acquire 2D projection images of Čerenkov emission from a 4 × 4 cm(2) 6 MV linear accelerator (LINAC) x-ray photon beam operating at a dose rate of 400 MU∕min incident on a water tank with transparent walls. The ICCD acquisition was gated to the LINAC sync pulse to reduce background light artifacts, and the measurement quality was investigated by evaluating the signal to noise ratio and measurement repeatability as a function of delivered dose. Monte Carlo simulations were used to derive a calibration factor for differences between the optical images and deposited dose arising from the anisotropic angular dependence of Čerenkov emission. Finally, Čerenkov-based beam profiles were compared to a percent depth dose (PDD) and lateral dose profile at a depth of d(max) from a reference dose distribution generated from the clinical Varian ECLIPSE treatment planning system (TPS). RESULTS The signal to noise ratio was found to be 20 at a delivered dose of 66.6 cGy, and proportional to the square root of the delivered dose as expected from Poisson photon counting statistics. A 2.1% mean standard deviation and 5.6% maximum variation in successive measurements were observed, and the Monte Carlo derived calibration factor resulted in Čerenkov emission images which were directly correlated to deposited dose, with some spatial issues. The dose difference between the TPS and PDD predicted by Čerenkov measurements was within 20% in the buildup region with a distance to agreement (DTA) of 1.5-2 mm and ±3% at depths beyond d(max). In the lateral profile, the dose difference at the beam penumbra was within ±13% with a DTA of 0-2 mm, ±5% in the central beam region, and 2%-3% in the beam umbra. CONCLUSIONS The results from this initial study demonstrate the first documented use of Čerenkov emission imaging to profile x-ray photon LINAC beams in water. The proposed modality has several potential advantages over alternative methods, and upon future refinement may prove to be a robust and novel dosimetry method.

Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo

The up-regulation of cell surface receptors has become a central focus in personalized cancer treatment; however, because of the complex nature of contrast agent pharmacokinetics in tumor tissue, methods to quantify receptor binding in vivo remain elusive. Here, we present a dual-tracer optical technique for noninvasive estimation of specific receptor binding in cancer. A multispectral MRI-coupled fluorescence molecular tomography system was used to image the uptake kinetics of two fluorescent tracers injected simultaneously, one tracer targeted to the receptor of interest and the other tracer a nontargeted reference. These dynamic tracer data were then fit to a dual-tracer compartmental model to estimate the density of receptors available for binding in the tissue. Applying this approach to mice with deep-seated gliomas that overexpress the EGF receptor produced an estimate of available receptor density of 2.3 ± 0.5 nM (n = 5), consistent with values estimated in comparative invasive imaging and ex vivo studies.