Scott B. Raymond

Design, Synthesis, and Testing of Difluoroboron-Derivatized Curcumins as Near-Infrared Probes for in Vivo Detection of Amyloid-β Deposits

Amyloid-beta (Abeta) deposits have been identified as key players in the progression of Alzheimers disease (AD). Recent evidence indicates that the deposits probably precede and induce the neuronal atrophy. Therefore, methods that enable monitoring the pathology before clinical symptoms are observed would be beneficial for early AD detection. Here, we report the design, synthesis, and testing of a curcumin-derivatized near-infrared (NIR) probe, CRANAD-2. Upon interacting with Abeta aggregates, CRANAD-2 undergoes a range of changes, which include a 70-fold fluorescence intensity increase, a 90 nm blue shift (from 805 to 715 nm), and a large increase in quantum yield. Moreover, this probe also shows a high affinity for Abeta aggregates (K(d) = 38.0 nM), a reasonable log P value (log P = 3), considerable stability in serum, and a weak interaction with albumin. After intravenous injection of this probe, 19-month-old Tg2576 mice exhibited significantly higher relative signal than that of the control mice over the same period of time. In summary, CRANAD-2 meets all the requirements for a NIR contrast agent for the detection of Abeta plaques both in vitro and in vivo. Our data point toward the feasibility of monitoring the progress of the disease by NIR imaging with CRANAD-2. In addition, we believe that our probe could be potentially used as a tool for drug screening.

Ultrasound Enhanced Delivery of Molecular Imaging and Therapeutic Agents in Alzheimer's Disease Mouse Models

Alzheimers disease is a neurodegenerative disorder typified by the accumulation of a small protein, beta-amyloid, which aggregates and is the primary component of amyloid plaques. Many new therapeutic and diagnostic agents for reducing amyloid plaques have limited efficacy in vivo because of poor transport across the blood-brain barrier. Here we demonstrate that low-intensity focused ultrasound with a microbubble contrast agent may be used to transiently disrupt the blood-brain barrier, allowing non-invasive, localized delivery of imaging fluorophores and immunotherapeutics directly to amyloid plaques. We administered intravenous Trypan blue, an amyloid staining red fluorophore, and anti-amyloid antibodies, concurrently with focused ultrasound therapy in plaque-bearing, transgenic mouse models of Alzheimers disease with amyloid pathology. MRI guidance permitted selective treatment and monitoring of plaque-heavy anatomical regions, such as the hippocampus. Treated brain regions exhibited 16.5±5.4-fold increase in Trypan blue fluorescence and 2.7±1.2-fold increase in anti-amyloid antibodies that localized to amyloid plaques. Ultrasound-enhanced delivery was consistently reproduced in two different transgenic strains (APPswe:PSEN1dE9, PDAPP), across a large age range (9–26 months), with and without MR guidance, and with little or no tissue damage. Ultrasound-mediated, transient blood-brain barrier disruption allows the delivery of both therapeutic and molecular imaging agents in Alzheimers mouse models, which should aid pre-clinical drug screening and imaging probe development. Furthermore, this technique may be used to deliver a wide variety of small and large molecules to the brain for imaging and therapy in other neurodegenerative diseases.

A Time Domain Fluorescence Tomography System for Small Animal Imaging

We describe the application of a time domain diffuse fluorescence tomography system for whole body small animal imaging. The key features of the system are the use of point excitation in free space using ultrashort laser pulses and noncontact detection using a gated, intensified charge-coupled device (CCD) camera. Mouse shaped epoxy phantoms, with embedded fluorescent inclusions, were used to verify the performance of a recently developed asymptotic lifetime-based tomography algorithm. The asymptotic algorithm is based on a multiexponential analysis of the decay portion of the data. The multiexponential model is shown to enable the use of a global analysis approach for a robust recovery of the lifetime components present within the imaging medium. The surface boundaries of the imaging volume were acquired using a photogrammetric camera integrated with the imaging system, and implemented in a Monte-Carlo model of photon propagation in tissue. The tomography results show that the asymptotic approach is able to separate axially located fluorescent inclusions centered at depths of 4 and 10 mm from the surface of the mouse phantom. The fluorescent inclusions had distinct lifetimes of 0.5 and 0.95 ns. The inclusions were nearly overlapping along the measurement axis and shown to be not resolvable using continuous wave (CW) methods. These results suggest the practical feasibility and advantages of a time domain approach for whole body small animal fluorescence molecular imaging, particularly with the use of lifetime as a contrast mechanism.

Multiphoton imaging of ultrasound/Optison mediated cerebrovascular effects in vivo

Ultrasound (US) enhanced with microbubble contrast agents may transiently disrupt the blood–brain barrier (BBB) with minimal damage, providing a technique for noninvasive, localized drug-delivery deep within the brain. The mechanism and temporal profile of disruption are not understood, owing to the limitations of imaging modalities used previously. In this study, we monitored US-induced BBB disruption with multiphoton microscopy, providing high-resolution temporal and spatial information about the permeabilization mechanism and immediate effects of US exposure. Anesthetized C57 mice were prepared with a craniotomy and injected intravenously with fluorescent dyes to permit visualization of the vasculature and BBB integrity. The animals were imaged through a cranial window while exposed to low-intensity US (f =1.029 MHz, power = 0.2 W) with a coincident intravenous injection of Optison (a microbubble contrast agent). We observed arteriolar vasoconstriction on US exposure that disrupted blood flow and lasted up to 5 mins; BBB disruption occurred via two characteristically distinct processes—perivascular fluorescence gradually increased (over minutes) along the length of the affected vessel without apparent rupture of the vessel wall or rapidly (seconds) increased in select, focal regions. These data corroborated previous studies suggesting increased endothelial transcytosis and breached tight junctions and demonstrated vasoconstriction, which might alter BBB permeability by modifying cerebral blood flow.

Time resolved fluorescence tomography of turbid media based on lifetime contrast

A general linear model for time domain (TD) fluorescence tomography is presented that allows a lifetime-based analysis of the entire temporal fluorescence response from a turbid medium. Simulations are used to show that TD fluorescence tomography is optimally performed using two complementary approaches: A direct TD analysis of a few time points near the peak of the temporal response, which provides superior resolution; and an asymptotic multi-exponential analysis based tomography of the decay portion of the temporal response, which provides accurate localization of yield distributions for various lifetime components present in the imaging medium. These results indicate the potential of TD technology for biomedical imaging with lifetime sensitive targeted probes, and provide useful guidelines for an optimal approach to fluorescence tomography with TD data.

Smart optical probes for near-infrared fluorescence imaging of Alzheimer's disease pathology.

PurposeNear-infrared fluorescent probes for amyloid-beta (Aβ) are an exciting option for molecular imaging in Alzheimer’s disease research and may translate to clinical diagnostics. However, Aβ-targeted optical probes often suffer from poor specificity and slow clearance from the brain. We are designing smart optical probes that emit characteristic fluorescence signal only when bound to Aβ.MethodsWe synthesized a family of dyes and tested Aβ-binding sensitivity with fluorescence spectroscopy and tissue-staining.ResultsSelect compounds exhibited Aβ-dependent changes in fluorescence quantum yield, lifetime, and emission spectra that may be imaged microscopically or in vivo using new lifetime and spectral fluorescence imaging techniques.ConclusionSmart optical probes that turn on when bound to Aβ will improve amyloid detection and may enable quantitative molecular imaging in vivo.

Comparison of frequency-domain and time-domain fluorescence lifetime tomography.

We compare frequency-and time-domain formulations of deep-tissue fluorescence imaging of turbid media. Simulations are used to show that time-domain fluorescence tomography, implemented via the asymptotic lifetime-based approach, offers a significantly better separability of multiple lifetime targets than a frequency-domain approach. We also demonstrate experimentally, using complex-shaped phantoms, the advantages of the asymptotic time-domain approach over a Fourier-based approach for analyzing time-domain fluorescence data.

Feasibility of in vivo imaging of fluorescent proteins using lifetime contrast

We show that fluorescence lifetime is a powerful contrast mechanism that can enhance the whole-body imaging of fluorescent proteins (FPs), in the presence of background tissue autofluorescence (AF). The nonexponential AF decay is characterized from time-domain (TD) measurements on multiple nude mice and separated from the FP fluorescence using a linear fit to a priori basis functions. We illustrate this approach using an orthotopic mouse tumor model of breast adenocarcinoma. We also report that four commonly used FPs show distinct lifetimes, indicating their suitability for in vivo lifetime multiplexing. These results suggest the potential for exploiting fluorescence lifetime for imaging FPs for a variety of whole-body small-animal imaging applications.

Focused ultrasound thermal surgery, imaging, and elastometry using the same phased array: feasibility study

A multifunctional array capable of imaging, surgery, and real-time surgery monitoring would significantly improve current focused ultrasound procedures. We tested a 24-element array to determine the feasibility of building a full, 128-element array capable of all three functions. The test array results suggested that a 128-element array would have sufficient power output and imaging capabilities to perform B-mode imaging, focused ultrasound surgery, and localized harmonic motion imaging for monitoring surgery.

Imaging Brain Collaterals: Quantification, Scoring, and Potential Significance.

Abstract Leptomeningeal collaterals provide the primary source of perfusion to ischemic brain tissue following the onset of acute ischemic stroke and are becoming an important imaging biomarker for stroke therapy triage. Collateral circulation is predictive of infarct growth, end infarct volume, and response to endovascular therapy. The strength of the collateral circulation varies among patients and is partially dependent on genetic and modifiable risk factors. Collateral circulation may be assessed by standard angiographic techniques, including digital subtraction angiography, computed tomography and magnetic resonance (MR) angiography, as well as a growing array of advanced MR techniques including arterial spin labeling and dynamic MR angiography. Simple scoring systems are used to estimate the relative strength of the collateral circulation for a given patient, although there are some discrepancies in the predictive value of these systems. In this review, we discuss methods and techniques for determining the robustness of the collateral circulation and the role of the collateral circulation in acute ischemic stroke assessment and triage.