In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

Schematic demonstrating protease-mediated activation of the nanosensor, Prosense/VM110.

source: ©2011 Journal of Visualized Experiments

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.

Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries. The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.

Fig. 1 Schematic of 2D NIRF Catheter To extend the clinical potential of a 1D NIRF sensing approach, we constructed a novel 2-D NIRF-catheter for intravascular imaging. The custom-built catheter consists of an optical fiber (125 micron diameter housed in polyethylene tubing: 2.9F) that illuminates using a 750 nm laser excitation source. Laser light is emitted at a 90 degree angle relative to fiber axis. The system utilizes two automated motors (rotational and translational) to enable concomitant 360 degree imaging and longitudinal pullback to obtain true 2D imaging. Images used with permission from reference 11.

Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR ‘window’ can substantially improve the potential for in vivo imaging.

Inflammatory cysteine proteases have been well studied using activatable NIRF probes, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata. In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline. Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.

In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach, is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation. The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.
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M. A. Calfon, A. Rosenthal, G. Mallas, A. Mauskapf, R. N. Nudelman, V. Ntziachristos, F. A. Jaffer, „In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis,” J. Vis. Exp., Issue 54 (2011).

Two-Dimensional Intravascular Near-Infrared Fluorescence Molecular Imaging of Inflammation in Atherosclerosis and Stent-Induced Vascular Injury

Representative In Vivo Molecular and Anatomical Imaging of Inflamed Atheromata

source: ©2011 Journal of the American College of Cardiology

Objectives: This study sought to develop a 2-dimensional (2D) intravascular near-infrared fluorescence (NIRF) imaging strategy for investigation of arterial inflammation in coronary-sized vessels.

Background: Molecular imaging of arterial inflammation could provide new insights into the pathogenesis of acute myocardial infarction stemming from coronary atheromata and implanted stents. Presently, few high-resolution approaches can image inflammation in coronary-sized arteries in vivo.

Methods: A new 2.9-F rotational, automated pullback 2D imaging catheter was engineered and optimized for 360° viewing intravascular NIRF imaging. In conjunction with the cysteine protease-activatable imaging reporter Prosense VM110 (VisEn Medical, Woburn, Massachusetts), intra-arterial 2D NIRF imaging was performed in rabbit aortas with atherosclerosis (n =10) or implanted coronary bare-metal stents (n = 10, 3.5-mm diameter, day 7 post-implantation). Intravascular ultrasound provided coregistered anatomical images of arteries. After sacrifice, specimens underwent ex vivo NIRF imaging, fluorescence microscopy, and histological and immunohistochemical analyses.

Fig. 1 Schematic of the Constructed 2D NIRF Imaging System. The tip of the fiber contains a right angle coated prism that reflects the guide’s laser light into the artery wall and couples the subsequent fluorescent light back into the fiber. The fluorescent light is then directed to a dichroic beam splitter that selectively reflects it into a photomultiplier tube. The beam passes additional filters to minimize the parasitic signals of laser photons and autofluorescence. The inset shows the spectra of the 3 filters (I, II, III) used in the system. 2D = 2-dimensional; NIRF = near-infrared fluorescence.

Results: Imaging of coronary artery–scaled phantoms demonstrated 8-sector angular resolution and submillimeter axial resolution, nanomolar sensitivity to NIR fluorochromes, and modest NIRF light attenuation through blood. High-resolution NIRF images of vessel wall inflammation with signal-to-noise ratios >10 were obtained in real-time through blood, without flushing or occlusion. In atherosclerosis, 2D NIRF, intravascular ultrasound–NIRF fusion, microscopy, and immunoblotting studies provided insight into the spatial distribution of plaque protease activity. In stent-implanted vessels, real-time imaging illuminated an edge-based pattern of stent-induced arterial inflammation.

Conclusions: A new 2D intravascular NIRF imaging strategy provides high-resolution in vivo spatial mapping of arterial inflammation in coronary-sized arteries and reveals increased inflammation-regulated cysteine protease activity in atheromata and stent-induced arterial injury. 
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F. A. Jaffer, M. A. Calfon, A. Rosenthal, G. Mallas, R. N. Razansky, A. Mauskapf, R. Weissleder, P. Libby, V. Ntziachristos, “Two-Dimensional Intravascular Near-Infrared Fluorescence Molecular Imaging of Inflammation in Atherosclerosis and Stent-Induced Vascular Injury,” J. Am. Coll. Cardiol., Vol. 57, pp. 2516-2526 (2011).

Model-Based Optoacoustic inversions with incomplete projection data

The phantom used in the numerical study representing the map of local laser energy deposition

source: © 2011 Medical Physics

Optoacoustic imaging is an emerging noninvasive imaging modality that can resolve optical contrast through several millimeters to centimeters of tissue with diffraction‐limited resolution of ultrasound. Yet, quantified reconstruction of tissue absorption maps requires optoacoustic signals to be collected from as many locations around the object as possible. In many tomographic imaging scenarios, however, only limited‐view or partial projection data are available, which has been shown to generate image artifacts and overall loss of quantification accuracy.

In this article, the recently introduced interpolated‐matrix‐model optoacoustic inversion method is tested in different limited‐view scenarios and compared to the standard backprojection algorithm. Both direct (TGSVD) and iterative (PLSQR) regularization methods are proposed to improve the accuracy of image reconstructions with their performance tested on simulated and experimental data.

Fig.1 (a) Reconstructable (bright solid line) and “invisible” (bright dashed line) boundaries of a round object with a square insertion partially lying in the “visibility region” (shaded area) and the “invisibility domain” for a detector moving along the dark solid arc. Dashed boundaries blur away since they do not fulfill detection criterion, i.e., they do not have a normal passing at least through one detector position. (b) Backprojection reconstruction of the phantom shown in (a). Boundaries that do not fulfill the detection criterion blur away. (c) The condition number of the matrix urn:x-wiley:00942405:mp6916:equation:mp6916-math-0138 as a function of the detection arc. The higher the condition number, the more error‐prone the inversion of urn:x-wiley:00942405:mp6916:equation:mp6916-math-0139 becomes. (d) IMMI reconstruction of the phantom. IMMI reconstructions show stripe artifacts in the invisibility domain.

While for full‐view tomographic data the model‐based inversion has been generally shown to attain higher reconstruction accuracy compared to backprojection algorithms, the incomplete tomographic datasets lead to ill‐conditioned forward matrices and, consequently, to error‐prone inversions, with strong artifacts following a distinct ripple‐type pattern. The proposed regularization techniques are shown to stabilize the inversion and eliminate the artifacts.

Overall, it has been determined that the regularized interpolated‐matrix‐model‐based optoacoustic inversions show higher accuracy than reconstructions with the standard backprojection algorithm. Finally, the combination of model‐based inversion with PLSQR or TGSVD regularization methods can lead to an accurate reconstruction of limited‐projection angle optoacoustic data and practical systems for optoacoustic imaging in many realistic cases where the full‐view dataset is unavailable. 
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A. Buehler, A. Rosenthal , T. Jetzfellner , A. Dima , D. Razansky, and V. Ntziachristos, „Model-Based Optoacoustic inversions with incomplete projection data,” Med. Phys., Vol. 38, pp.1694-1704 (2011)

Optoacoustic methods for frequency calibration of ultrasonic sensors

A schematic description of the different geometries used to measure the frequency response of the acoustic detector

source: © 2011 IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control

The frequency response of ultrasonic detectors is commonly calibrated by finding their sensitivity to incident plane waves at discrete frequencies. For certain applications, such as the emerging field of optoacoustic tomography, it is the response to point sources emitting broadband spectra that needs to be found instead. Although these two distinct sensitivity characteristics are interchangeable in the case of a flat detector and a point source at infinity, it is not the case for detectors with size considerably larger than the acoustic wavelength of interest or those having a focused aperture. Such geometries, which are common in optoacoustics, require direct calibration of the acoustic detector using a point source placed in the relevant position. In this paper, we report on novel cross-validating optoacoustic methods for measuring the frequency response of wideband acoustic sensors. The approach developed does not require pre-calibrated hydrophones and therefore can be readily adopted in any existing optoacoustic measurement configuration. The methods are successfully confirmed experimentally by measuring the frequency response of a common piezoelectric detector having a cylindrically focused shape.
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Fig.1 The different 2-D configurations analyzed in this paper for measuring the frequency response of acoustic detectors. The acoustic sources are equal to one in the gray areas and to zero outside them; the detectors are on the far right (point, flat, and curved). a and b denote the lateral and axial dimensions of the acoustic source, respectively; c denotes the distance from the source to the detector; d denotes the vertical length of the detector; and v denotes the speed of sound. (a) A source with a smooth boundary and similar dimensions on the axial and lateral axes and a point detector. The dashed curves represent the two extreme arcs over which the integral in (2) is nonzero. (b) A heuristic description of the integral in (2) and of (c) the spectrum of pÎŽ(r,t) for the geometry in Fig. 1(a). (d)–(f) A rectangular optoacoustic source with point, flat, and curved detectors, respectively. The curved detector is focused on to the middle of the proximal edge of the source. The dashed lines represent the longest distances from any point on the detectors to any point on the proximal edge of the sources.

A. Rosenthal, V. Ntziachristos, and D. Razansky,“Optoacoustic methods for frequency calibration of ultrasonic sensors,” IEEE Trans. Ultrason. Ferroelectr. Freq. Control, Vol. 58, pp. 316-326 (2011)

High-sensitivity compact ultrasonic detector based on a pi-phase-shifted fiber Bragg grating

Schematic description of the detection scheme. Including a Tunable CW laser and an Electric Pulser.

source: © 2011 Optical Society of America

A highly sensitive compact hydrophone, based on a pi-phase-shifted fiber Bragg grating, has been developed for the measurement of wideband ultrasonic fields. The grating exhibits a sharp resonance, whose centroid wavelength is pressure sensitive. The resonance is monitored by a continuous-wave (CW) laser to measure ultrasound-induced pressure variations within the grating. In contrast to standard fiber sensors, the high finesse of the resonance—which is the reason for the sensor’s high sensitivity—is not associated with a long propagation length. Light localization around the phase shift reduces the effective size of the sensor below that of the grating and is scaled inversely with the resonance spectral width. In our system, an effective sensor length of 270 Όm, pressure sensitivity of 440 Pa, and effective bandwidth of 10 MHz were achieved. This performance makes our design attractive for medical imaging applications, such as optoacoustic tomography, in which compact, sensitive, and wideband acoustic detectors are required.
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Schematic description of the detection scheme. Including a Tunable CW laser and an Electric Pulser.

Fig. 1 Schematic description of the detection scheme. A CW laser is used to monitor the reflection of an FBG.ï»ż

A. Rosenthal, D. Razansky, and V. Ntziachristos, “High-sensitivity compact ultrasonic detector based on a pi-phase-shifted fiber Bragg grating,” Opt. Let., Vol. 36, pp. 1833-1835 (2011).

Interpolated model-matrix optoacoustic tomography of the mouse brain

source: © 2011 Applied Physics Letters

Neuroscience investigations may significantly benefit from the availability of accurate imaging methods of brain parameters in small animals. In this letter, we investigate the imaging performance of the recently introduced interpolated model-matrix inversion (IMMI), in quantitative optoacoustic imaging of the mouse head. We compare the findings of the method against back-projection inversion methods that have more commonly been considered. We find that cross-sectional images of the mouse head accurately match anatomical structures seen on cryosliced head images serving as the gold standard. Moreover, superior imaging performance is found for IMMI compared to previously reported optoacoustic imaging of the mouse head.  [Read more…]

Fig 2.Several cross-sectional slices of the head region of a mouse back projection based reconstructions of (a) head region, (b) lower part of the head, (c) and (d) corresponding IMMI reconstructions, (e) and (f) corresponding IMMI high-pass filtered images, and (g) and (h) cryoslices. Anatomical structure: 1, 3, 4, 6—eye sockets 2, 5, 7, 8—blood vessels.

Thomas Jetzfellnera, Amir Rosenthal, K.-H. Englmeier, Alexander Dima, Miguel Ángel Araque Caballero, Daniel Razansky, and Vasilis Ntziachristos, “Interpolated model-matrix optoacoustic tomography of the mouse brain,” Appl. Phys. Lett. 98, 163701 (2011)

Model‐based optoacoustic inversion with arbitrary‐shape detectors

source: © 2011 American Association of Physicists in Medicine

Optoacoustic imaging enables mapping the optical absorption of biological tissue using optical excitation and acoustic detection. Although most image‐reconstruction algorithms are based on the assumption of a detector with an isotropic sensitivity, the geometry of the detector often leads to a response with spatially dependent magnitude and bandwidth. This effect may lead to attenuation or distortion in the recorded signal and, consequently, in the reconstructed image.

Herein, an accurate numerical method for simulating the spatially dependent response of an arbitrary‐shape acoustic transducer is presented. The method is based on an analytical solution obtained for a two‐dimensional line detector. The calculated response is incorporated in the forward model matrix of an optoacoustic imaging setup using temporal convolution, and image reconstruction is performed by inverting the matrix relation.  [Read more…]

Fig. 8 Experimental reconstructions of a point optoacoustic source detected by a flat detector with a width of 1.3 cm obtained using (a) the back‐projection algorithm (b) IMMI modeled with a point detector (c) IMMI modeled with a 1.3‐mm flat detector using spatial convolution and (d) IMMI modeled with a 1.3‐mm flat detector using temporal convolution. The point source was obtained by applying plane‐selective illuminating on a black hair embedded in a clear agar phantom, as shown in Fig. 6(a). Although both the spatial‐ and temporal‐convolution methods managed enhancing the reconstruction resolution, the temporal‐convolution method yielded a more accurate reconstruction with less background texture.

The method was numerically and experimentally demonstrated in two dimensions for both flat and focused transducers and compared to the spatial‐convolution method. In forward simulations, the developed method did not suffer from the numerical errors exhibited by the spatial‐convolution method. In reconstruction simulations and experiments, the use of both temporal‐convolution and spatial‐convolution methods lead to an enhancement in resolution compared to a reconstruction with a point detector model. However, because of its higher modeling accuracy, the temporal‐convolution method achieved a noise figure approximated three times lower than the spatial‐convolution method.

The demonstrated performance of the spatial‐convolution method shows it is a powerful tool for reducing reconstruction artifacts originating from the detector finite size and improving the quality of optoacoustic reconstructions. Furthermore, the method may be used for assessing new system designs. Specifically, detectors with nonstandard shapes may be investigated.

Amir Rosenthal Vasilis Ntziachristos Daniel Razansky, “Model‐based optoacoustic inversion with arbitrary‐shape detectors,” Medical Physics Volume38, Issue7,July 2011,Pages 4285-4295

Multispectral optoacoustic tomography by means of normalized spectral ratio

source: © 2011 Optics Letters

Quantification of biomarkers using multispectral optoacoustic tomography can be challenging due to photon fluence variations with depth and spatially heterogeneous tissue optical properties. Herein we introduce a spectral ratio approach that accounts for photon fluence variations. The performance and imaging improvement achieved with the proposed method is showcased both numerically and experimentally in phantoms and mice.  [Read more…]

Fig. 3 (a) Optoacoustic image of a mouse with ICG filled tubes at 800 nm . (b) Sketch of the mouse and the implanted tubes. (c) Spectral difference. (d) Spectral ratio. (e) Profiles along the dashed lines of (b) for spectral ratio and spectral difference. (f) Superimposed image of (d) on (a) after application of a threshold at đŒ=0.5 .

Thomas Jetzfellner, Amir Rosenthal, Andreas Buehler, Karl-Hans Englmeier, Daniel Razansky, and Vasilis Ntziachristos, “Multispectral optoacoustic tomography by means of normalized spectral ratio,” Opt. Lett. 36, 4176-4178 (2011)