Fast ICCD-based temperature modulated fluorescence tomography.

Fluorescence tomography (FT) has become a powerful preclinical imaging modality with a great potential for several clinical applications. Although it has superior sensitivity and utilizes low-cost instrumentation, the highly scattering nature of bio-tissue makes FT in thick samples challenging, resulting in poor resolution and low quantitative accuracy. To overcome the limitations of FT, we previously introduced a novel method, termed temperature modulated fluorescence tomography (TMFT), which is based on two key elements: (1) temperature-sensitive fluorescent agents (ThermoDots) and (2) high-intensity focused ultrasound (HIFU). The fluorescence emission of ThermoDots increases up to hundredfold with only several degree temperature elevation. The exceptional and reversible response of these ThermoDots enables their modulation, which effectively allows their localization using the HIFU. Their localization is then used as functional a priori during the FT image reconstruction process to resolve their distribution with higher spatial resolution. The last version of the TMFT system was based on a cooled CCD camera utilizing a step-and-shoot mode, which necessitated long total imaging time only for a small selected region of interest (ROI). In this paper, we present the latest version of our TMFT technology, which uses a much faster continuous HIFU scanning mode based on an intensified CCD (ICCD) camera. This new, to the best of our knowledge, version can capture the whole field-of-view (FOV) of 50×30m m 2 at once and reduces the total imaging time down to 30 min, while preserving the same high resolution (∼1.3m m) and superior quantitative accuracy (<7% error) as the previous versions. Therefore, this new method is an important step toward utilization of TMFT for preclinical imaging.

Feasibility study of high spatial resolution multimodality fluorescence tomography in ex vivo biological tissue.

Previously, we demonstrated that temperature-modulated fluorescence tomography (TM-FT) could provide fluorescence images with high quantitative accuracy and the spatial resolution of focused ultrasound. TM-FT is based on scanning the focused ultrasound across the medium to activate temperature-reversible fluorescent nanoprobes (ThermoDots). This technique can resolve small fluorescent targets located several centimeters deep in turbid media with millimeter resolution. Our past studies with this multimodality technique used agar phantoms, which could not represent the true heterogeneous nature of the acoustic and optical properties of biological tissue. In this work, we report the results of the first TM-FT study performed on ex vivo chicken breast tissue. In order to improve the spatial resolution of this technique, diffuse optical tomography is also used to better estimate the optical property maps of the tissue, which is utilized as functional a priori for the TM-FT reconstruction algorithm. These ex vivo results show that TM-FT can accurately recover the concentration and position of a 1.5 mm×5 mm inclusion filled with ThermoDots. Since the inclusion is embedded 2 cm deep in the chicken breast sample, these results demonstrate the great potential of TM-FT for future in vivo small animal imaging.

Experimental evaluation of the resolution and quantitative accuracy of temperature-modulated fluorescence tomography.

Previously, we reported on the spatial resolution and quantitative accuracy of temperature-modulated fluorescence tomography (TM-FT) using simulation studies. TM-FT is a novel fully integrated multimodality imaging technique that combines fluorescence diffuse optical tomography (FT) with focused ultrasound. Utilizing unique thermo-reversible fluorescent nanocapsules (ThermoDots), TM-FT provides high-resolution cross-sectional fluorescence images in thick tissue (up to 6 cm). Focused ultrasound and temperature-sensitive ThermoDots are combined to provide accurate localization of these fluorescent probes and functional a priori information to constrain the conventional FT reconstruction algorithm. Our previous simulation studies evaluated the performance of TM-FT using synthetic phantoms with multiple fluorescence targets of various sizes located at different depths. In this follow-up work, we perform experimental studies to evaluate the performance of this hybrid imaging system, in particular, the effect of size, depth, and concentration of the fluorescence target. While FT alone is unable to accurately locate and resolve the fluorophore target in many cases, TM-FT is able to resolve the size and concentration of the ThermoDots within a thick turbid medium with high accuracy for all cases. The maximum error in the recovered ThermoDots concentration and target sizes with TM-FT are 12% and 25%, respectively.

Differentiation of tumor vasculature heterogeneity levels in small animals based on total hemoglobin concentration using magnetic resonance-guided diffuse optical tomography in vivo.

Insight into the vasculature of the tumor in small animals has the potential to impact many areas of cancer research. The heterogeneity of the vasculature of a tumor is directly related to tumor stage and disease progression. In this small scale animal study, we investigated the feasibility of differentiating tumors with different levels of vasculature heterogeneity in vivo using a previously developed hybrid magnetic resonance imaging (MRI) and diffuse optical tomography (DOT) system for small animal imaging. Cross-sectional total hemoglobin concentration maps of 10 Fisher rats bearing R3230 breast tumors are reconstructed using multi-wavelength DOT measurements both with and without magnetic resonance (MR) structural a priori information. Simultaneously acquired MR structural images are used to guide and constrain the DOT reconstruction, while dynamic contrast-enhanced MR functional images are used as the gold standard to classify the vasculature of the tumor into two types: high versus low heterogeneity. These preliminary results show that the stand-alone DOT is unable to differentiate tumors with low and high vascular heterogeneity without structural a priori information provided by a high resolution imaging modality. The mean total hemoglobin concentrations comparing the vasculature of the tumors with low and high heterogeneity are significant (p-value 0.02) only when MR structural a priori information is utilized.

Implementation of a new scanning method for high-resolution fluorescence tomography using thermo-sensitive fluorescent agents.

Conventional fluorescence tomography provides images of the distribution of fluorescent agents within highly scattering media, but suffers from poor spatial resolution. Previously, we introduced a new method termed "temperature-modulated fluorescence tomography" (TM-FT) that generates fluorescence images with high spatial resolution. TM-FT first uses focused ultrasound to locate the distribution of temperature-sensitive fluorescence probes. Afterward, this a priori information is utilized to improve the performance of the inverse solver for conventional fluorescence tomography and reveal quantitatively accurate fluorophore concentration maps. However, the disadvantage of this novel method is the long data acquisition time as the ultrasound beam was scanned in a step-and-shoot mode. In this Letter, we present a new, fast scanning method that reduces the imaging time 40 fold. By continuously scanning the ultrasound beam over a 50 mm by 25 mm field-of-view, high-resolution fluorescence images are obtained in less than 29 min, which is critical for in vivo small animal imaging.

Simulation-based evaluation of the resolution and quantitative accuracy of temperature-modulated fluorescence tomography.

Conventional fluorescence tomography (FT) can recover the distribution of fluorescent agents within a highly scattering medium. However, poor spatial resolution remains its foremost limitation. Previously, we introduced a new fluorescence imaging technique termed "temperature-modulated fluorescence tomography" (TM-FT), which provides high-resolution images of fluorophore distribution. TM-FT is a multimodality technique that combines fluorescence imaging with focused ultrasound to locate thermo-sensitive fluorescence probes using a priori spatial information to drastically improve the resolution of conventional FT. In this paper, we present an extensive simulation study to evaluate the performance of the TM-FT technique on complex phantoms with multiple fluorescent targets of various sizes located at different depths. In addition, the performance of the TM-FT is tested in the presence of background fluorescence. The results obtained using our new method are systematically compared with those obtained with the conventional FT. Overall, TM-FT provides higher resolution and superior quantitative accuracy, making it an ideal candidate for in vivo preclinical and clinical imaging. For example, a 4 mm diameter inclusion positioned in the middle of a synthetic slab geometry phantom (D:40 mm×W:100 mm) is recovered as an elongated object in the conventional FT (x=4.5 mm; y=10.4 mm), while TM-FT recovers it successfully in both directions (x=3.8 mm; y=4.6 mm). As a result, the quantitative accuracy of the TM-FT is superior because it recovers the concentration of the agent with a 22% error, which is in contrast with the 83% error of the conventional FT.

Development of a preclinical CCD-based temperature modulated fluorescence tomography platform.

In preclinical research, fluorescence molecular tomography (FMT) is the most sensitive imaging modality to interrogate whole-body and provide 3D distribution of fluorescent contract agents. Despite its superior sensitivity, its mediocre spatial-resolution has been the main barrier to its clinical translation. This limitation is mainly due to the high scattering of optical photons in biological tissue together with the limited boundary measurements that lead to an undetermined and ill-posed inverse problem. To overcome the limitations of FMT, we previously introduced a novel method termed, Temperature Modulated Fluorescence Tomography (TMFT). TMFT utilizes thermos-sensitive fluorescent agents (ThermoDots) as a key component and localizes them with high-intensity focused ultrasound (HIFU). Scanning the focused HIFU beam having a diameter Ø = 1.3 mm across the tissue while monitoring the variation in the measured fluorescence signals reveals the position of the ThermoDots with high spatial accuracy. We have formerly built a prototype TMFT system that uses optical fibers for detection. In this paper, we present an upgraded version using a CCD camera-based detection that enables non-contact imaging. In this version, the animal under investigation is placed on an ultrasound transparent membrane, which eliminates the need for its immersion in optical matching fluids that were required by the fiber-based system. This CCD-based system will pave the way for convenient and wide-spread use of TMFT in preclinical research. Its performance validation on phantom studies demonstrates that high spatial-resolution (∼1.3 mm) and quantitative accuracy in recovered fluorophore concentration (<3% error) can be achieved.

A thermo-sensitive fluorescent agent based method for excitation light leakage rejection for fluorescence molecular tomography.

Fluorescence molecular tomography (FMT) is widely used in preclinical oncology research. FMT is the only imaging technique able to provide 3D distribution of fluorescent probes within thick highly scattering media. However, its integration into clinical medicine has been hampered by its low spatial resolution caused by the undetermined and ill-posed nature of its reconstruction algorithm. Another major factor degrading the quality of FMT images is the large backscattered excitation light component leaking through the rejection filters and coinciding with the weak fluorescent signal arising from a low tissue fluorescence concentration. In this paper, we present a new method based on the use of a novel thermo-sensitive fluorescence probe. In fact, the excitation light leakage is accurately estimated from a set of measurements performed at different temperatures and then is corrected for in the tomographic data. The obtained results show a considerable improvement in both spatial resolution and quantitative accuracy of FMT images due to the proper correction of fluorescent signals.

Temperature-modulated fluorescence tomography based on both concentration and lifetime contrast.

It is challenging to image fluorescence objects with high spatial resolution in a highly scattering medium. Recently reported temperature-sensitive indocyanine green-loaded pluronic nanocapsules can potentially alleviate this problem. Here we demonstrate a frequency-domain temperature-modulated fluorescence tomography system that could acquire images at high intensity-focused ultrasound resolution with use of these nanocapsules. The system is experimentally verified with a phantom study, where a 3-mm fluorescence object embedded 2 cm deep in a turbid medium is successfully recovered based on both intensity and lifetime contrast.

Temperature-modulated fluorescence tomography in a turbid media.

High scattering in biological tissues makes fluorescence tomography inverse problem very challenging in thick medium. We describe an approach termed "temperature-modulated fluorescence tomography" that can acquire fluorescence images at focused ultrasound resolution. By utilizing recently emerged temperature sensitive fluorescence contrast agents, this technique provides fluorescence images with high resolution prior to any reconstruction process. We demonstrate that this technique is well suited to resolve small fluorescence targets located several centimeters deep in tissue.