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.

Monitoring Distribution of the Therapeutic Agent Dimethyl Sulfoxide via Solvatochromic Shift of Albumin-Bound Indocyanine Green.

We recently developed a novel hyperspectral excitation-resolved near-infrared fluorescence imaging system (HER-NIRF) based on a continuous-wave wavelength-swept laser. In this study, this technique is applied to measure the distribution of the therapeutic agent dimethyl sulfoxide (DMSO) by utilizing solvatochromic shift in the spectral profile of albumin-bound Indocyanine green (ICG). Using wide-field imaging in turbid media, complex dynamics of albumin-bound ICG are measured in mixtures of dimethyl sulfoxide (DMSO) and water. Phantom experiments are conducted to evaluate the performance of the HER-NIRF system. The results show that the distribution of DMSO can be visualized in the wide-field reflection geometry. One of the main purposes of the DMSO is to act as a carrier for other drugs, enhancing their effects by facilitating skin penetration. Understanding the solubility and permeability of drugs in vivo is very important in drug discovery and development. Hence, this HER-NIRF technique has great potential to advance the utilization of the therapeutic agent DMSO by mapping its distribution via the solvatochromic shift of ICG. By customizing the operational wavelength range, this system can be applied to any other fluorophores in the near-infrared region and utilized for a wide variety of drug delivery studies.

Multiwavelength photo-magnetic imaging algorithm improved for direct chromophore concentration recovery using spectral constraints.

Multiwavelength photo-magnetic imaging (PMI) is a novel combination of diffuse optics and magnetic resonance imaging, to the best of our knowledge, that yields tissue chromophore concentration maps with high resolution and quantitative accuracy. Here, we present the first experimental results, to the best of our knowledge, obtained using a spectrally constrained PMI image reconstruction method, where chromophore concentration maps are directly recovered, unlike the conventional two-step approach that requires an intermediate step of reconstructing wavelength-dependent absorption coefficient maps. The imposition of the prior spectral information into the PMI inverse problem improves the reconstructed image quality and allows recovery of highly quantitative concentration maps, which are crucial for effective cancer detection and characterization. The obtained results demonstrate the higher performance of the direct reconstruction method. Indeed, the reconstructed concentration maps are not only of higher quality but also obtained approximately 2 times faster than the conventional method.

Multi-Wavelength Photo-Magnetic Imaging System for Photothermal Therapy Guidance.

BACKGROUND AND OBJECTIVES: In photothermal therapy, cancerous tissue is treated by the heat generated from absorbed light energy. For effective photothermal therapy, the parameters affecting the induced temperature should be determined before the treatment by modeling the increase in temperature via numerical simulations. However, accurate simulations can only be achieved when utilizing the accurate optical, thermal, and physiological properties of the treated tissue. Here, we propose a multi-wavelength photo-magnetic imaging (PMI) technique that provides quantitative and spatially resolved tissue optical absorption maps at any wavelength within the near-infrared (NIR) window to assist accurate photothermal therapy planning. STUDY DESIGN/MATERIALS AND METHODS: The study was conducted using our recently developed multi-wavelength PMI system, which operates at four laser wavelengths (760, 808, 860, and 980 nm). An agar tissue-simulating phantom containing water, lipid, and ink was illuminated using these wavelengths, and the slight internal laser-induced temperature rise was measured using magnetic resonance thermometry (MRT). The phantom optical absorption was recovered at the used wavelengths using our dedicated PMI image reconstruction algorithm. These absorption maps were then used to resolve the concentration of the tissue chromophores, and thus deduce its optical absorption spectrum in the NIR region based on the Beer-Lambert law. RESULTS: The optical absorption of the phantom was successfully recovered at the used four wavelengths with an average error of ~1.9%. The recovered absorption coefficient was then used to simulate temperature variations inside the phantom. A comparison between the modeled temperature maps and the MRT measured ones showed that these maps are in a good agreement with an average pseudo R2 statistic of 0.992. These absorption values were used to successfully recover the concentration of the used chromophores. Finally, these concentrations are used to accurately calculate the total absorption spectrum of the phantom in the NIR spectral window with an average error as low as ~2.3%. CONCLUSIONS: Multi-wavelength PMI demonstrated a great ability to assess the distribution of tissue chromophores, thus providing its total absorption at any wavelength within the NIR spectral range. Therefore, applications of photothermal therapy applied at NIR wavelengths can benefit from the absorption spectrum recovered by PMI to determine important parameters such as laser power as well as the laser exposure time needed to attain a specific increase in temperature prior to treatment. Lasers Surg. Med. 00:00-00, 2020. © 2020 Wiley Periodicals LLC.

Ex vivo validation of photo-magnetic imaging.

We recently introduced a new high-resolution diffuse optical imaging technique termed photo-magnetic imaging (PMI), which utilizes magnetic resonance thermometry (MRT) to monitor the 3D temperature distribution induced in a medium illuminated with a near-infrared light. The spatiotemporal temperature distribution due to light absorption can be accurately estimated using a combined photon propagation and heat diffusion model. High-resolution optical absorption images are then obtained by iteratively minimizing the error between the measured and modeled temperature distributions. We have previously demonstrated the feasibility of PMI with experimental studies using tissue simulating agarose phantoms. In this Letter, we present the preliminary ex vivo PMI results obtained with a chicken breast sample. Similarly to the results obtained on phantoms, the reconstructed images reveal that PMI can quantitatively resolve an inclusion with a 3 mm diameter embedded deep in a biological tissue sample with only 10% error. These encouraging results demonstrate the high performance of PMI in ex vivo biological tissue and its potential for in vivo 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.

Breast density quantification using structured-light-based diffuse optical tomography simulations.

We present the feasibility of structured-light-based diffuse optical tomography (DOT) to quantify the breast density with an extensive simulation study. This study is performed on multiple numerical breast phantoms built from magnetic resonance imaging (MRI) images. These phantoms represent realistic tissue morphologies and are given typical breast optical properties. First, synthetic data are simulated at five wavelengths using our structured-light-based DOT forward problem. Afterwards, the inverse problem is solved to obtain the absorption images and subsequently the chromophore concentration maps. Parameters, such as segmented volumes and mean concentrations, are extracted from these maps and used in a regression model to estimate the percent breast densities. These estimations are correlated with the true values from MRI, r=0.97, showing that our new technique is promising in measuring breast density.

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.

Effect of Shot Noise on Simultaneous Sensing in Frequency Division Multiplexed Diffuse Optical Tomographic Imaging Process.

Diffuse optical tomography (DOT) has been studied for use in the detection of breast cancer, cerebral oxygenation, and cognitive brain signals. As optical imaging studies have increased significantly, acquiring imaging data in real time has become increasingly important. We have developed frequency-division multiplexing (FDM) DOT systems to analyze their performance with respect to acquisition time and imaging quality, in comparison with the conventional time-division multiplexing (TDM) DOT. A large tomographic area of a cylindrical phantom 60 mm in diameter could be successfully reconstructed using both TDM DOT and FDM DOT systems. In our experiment with 6 source-detector (S-D) pairs, the TDM DOT and FDM DOT systems required 6.18 and 1 s, respectively, to obtain a single tomographic data set. While the absorption coefficient of the reconstruction image was underestimated in the case of the FDM DOT, we experimentally confirmed that the abnormal region can be clearly distinguished from the background phantom using both methods.

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.

Comprehensive analytical model for CW laser induced heat in turbid media.

In this work, we present a new analytical approach to model continuous wave laser induced temperature in highly homogeneous turbid media. First, the diffusion equation is used to model light transport and a comprehensive solution is derived analytically by obtaining a special Greens' function. Next, the time-dependent bio-heat equation is used to describe the induced heat increase and propagation within the medium. The bio-heat equation is solved analytically utilizing the separation of variables technique. Our theoretical model is successfully validated using numerical simulations and experimental studies with agarose phantoms and ex-vivo chicken breast samples. The encouraging results show that our method can be implemented as a simulation tool to determine important laser parameters that govern the magnitude of temperature rise within homogenous biological tissue or organs.

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.

Gold nanoparticle-mediated photothermal therapy guidance with multi-wavelength photomagnetic imaging.

Difficulty in heating tumors with high spatial selectivity while protecting surrounding healthy tissues from thermal harm is a challenge for cancer photothermal treatment (PTT). To mitigate this problem, PTT mediated by photothermal agents (PTAs) has been established as a potential therapeutic technique to boost selectivity and reduce damage to surrounding healthy tissues. Various gold nanoparticles (AuNP) have been effectively utilized as PTAs, mainly using strategies to target cancerous tissue and increase selective thermal damage. Meanwhile, imaging can be used in tandem to monitor the AuNP distribution and guide the PTT. Mainly, the parameters impacting the induced temperature can be determined using simulation tools before treatment for effective PTT. However, accurate simulations can only be performed if the amount of AuNPs accumulated in the tumor is known. This study introduces Photo-Magnetic Imaging (PMI), which can appropriately recover the AuNP concentration to guide the PTT. Using multi-wavelength measurements, PMI can provide AuNP concentration based on their distinct absorption spectra. Tissue-simulating phantom studies are conducted to demonstrate the potential of PMI in recovering AuNP concentration for PTT planning. The recovered AuNP concentration is used to model the temperature increase accurately in a small inclusion representing tumor using a multiphysics solver that takes into account the light propagation and heat diffusion in turbid media.

Self-Guided Algorithm for Fast Image Reconstruction in Photo-Magnetic Imaging: Artificial Intelligence-Assisted Approach.

Previously, we introduced photomagnetic imaging (PMI) that synergistically utilizes laser light to slightly elevate the tissue temperature and magnetic resonance thermometry (MRT) to measure the induced temperature. The MRT temperature maps are then converted into absorption maps using a dedicated PMI image reconstruction algorithm. In the MRT maps, the presence of abnormalities such as tumors would create a notable high contrast due to their higher hemoglobin levels. In this study, we present a new artificial intelligence-based image reconstruction algorithm that improves the accuracy and spatial resolution of the recovered absorption maps while reducing the recovery time. Technically, a supervised machine learning approach was used to detect and delineate the boundary of tumors directly from the MRT maps based on their temperature contrast to the background. This information was further utilized as a soft functional a priori in the standard PMI algorithm to enhance the absorption recovery. Our new method was evaluated on a tissue-like phantom with two inclusions representing tumors. The reconstructed absorption map showed that the well-trained neural network not only increased the PMI spatial resolution but also improved the accuracy of the recovered absorption to as low as a 2% percentage error, reduced the artifacts by 15%, and accelerated the image reconstruction process approximately 9-fold.

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.

Development of a theranostic preclinical fluorescence molecular tomography/cone beam CT-guided irradiator platform.

Image-guided small animal radiation research platforms allow more precise radiation treatment. Commercially available small animal X-ray irradiators are often equipped with a CT/cone-beam CT (CBCT) component for target guidance. Besides having poor soft-tissue contrast, CBCT unfortunately cannot provide molecular information due to its low sensitivity. Hence, there are extensive efforts to incorporate a molecular imaging component besides CBCT on these radiation therapy platforms. As an extension of these efforts, here we present a theranostic fluorescence tomography/CBCT-guided irradiator platform that provides both anatomical and molecular guidance, which can overcome the limitations of stand-alone CBCT. The performance of our hybrid system is validated using both tissue-like phantoms and mice ex vivo. Both studies show that fluorescence tomography can provide much more accurate quantitative results when CBCT-derived structural information is used to constrain the inverse problem. The error in the recovered fluorescence absorbance reduces nearly 10-fold for all cases, from approximately 60% down to 6%. This is very significant since high quantitative accuracy in molecular information is crucial to the correct assessment of the changes in tumor microenvironment related to radiation therapy.

TBX3 over-expression causes mammary gland hyperplasia and increases mammary stem-like cells in an inducible transgenic mouse model.

BACKGROUND: The T-box transcription factor TBX3 is necessary for early embryonic development and for the normal development of the mammary gland. Homozygous mutations, in mice, are embryonic lethal while heterozygous mutations result in perturbed mammary gland development. In humans, mutations that result in the haploinsufficiency of TBX3 causes Ulnar Mammary Syndrome (UMS) characterized by mammary gland hypoplasia as well as other congenital defects. In addition to its role in mammary gland development, various studies have also supported a role for Tbx3 in breast cancer development. TBX3 is over-expressed in various breast cancer cell lines as well as cancer tissue and has been found to contribute to breast cancer cell migration. Previous studies have suggested that TBX3 contributes to cancer development by its ability to bypass senescence by repressing the expression of p14(ARF)-tumor suppressor. Although many studies have shown that a dysregulation of TBX3 expression may contribute to cancer progression, no direct evidence shows TBX3 causes breast cancer. RESULTS: In this study, we created doxycycline inducible double transgenic mice (MMTV-rtTA;tet-myc-TBX3-IRES-Luciferase) to test whether TBX3 over-expression can induce tumor formation within the mammary gland. Although over-expression of TBX3, alone, did not induce tumor formation it did promote accelerated mammary gland development by increasing mammary epithelial cell proliferation. We also show that TBX3 directly binds to and represses NFκBIB, an inhibitor of the NF-κB pathway known to play a role in regulating cell proliferation. Lastly, we also show that the over-expression of TBX3 is associated with an increase in mammary stem-like cells. CONCLUSIONS: Overall, our data suggests that over-expression of TBX3 may contribute to breast cancer development by promoting accelerated mammary gland development through the inhibition of the NF-κB pathway and stimulation of both mammary epithelial cell and stem-like cell proliferation.

Quantitative fluorescence tomography using a combined tri-modality FT/DOT/XCT system.

In this work, a first-of-its-kind fully integrated tri-modality system that combines fluorescence, diffuse optical and x-ray tomography (FT/DOT/XCT) into the same setting is presented. The purpose of this system is to perform quantitative fluorescence tomography using multi-modality imaging approach. XCT anatomical information is used as structural priori while optical background heterogeneity information obtained by DOT measurements is used as functional priori. The performance of the hybrid system is evaluated using multi-modality phantoms. In particular, we show that a 2.4 mm diameter fluorescence inclusion located in a heterogeneous medium can be localized accurately with the functional a priori information, although the fluorophore concentration is recovered with 70% error. On the other hand, the fluorophore concentration can be accurately recovered within 8% error only when both DOT optical background functional and XCT structural a priori information are utilized to guide and constrain the FT reconstruction algorithm.

Fully linear reconstruction method for fluorescence yield and lifetime through inverse complex-source formulation: simulation studies.

In fluorescence imaging, both fluorescence yield and lifetime are of great importance. Traditionally, with the frequency-domain data, two parameters can be directly recovered through a nonlinear formulation. However, the reconstruction accuracy highly depends on initial guesses. To overcome this hurdle, we propose the linear scheme via an inverse complex-source formulation. Using the real and imaginary parts of the frequency-domain data, the proposed method is fully linear; it is not sensitive to initial guesses and is stable with high-level noise. Meanwhile, the algorithm is efficient, and the reconstruction takes one or a few iterations. In addition, the colocalization constraint due to the unique feature of fluorescence imaging is imposed to enhance algorithm performance. The algorithms are tested with simulated data.