No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Fluorescence molecular tomography resolves protease activity
Abstract
Systematic efforts are under way to develop novel technologies that would allow molecular sensing in intact organisms in vivo. Using near-infrared fluorescent molecular beacons and inversion techniques that take into account the diffuse nature of photon propagation in tissue, we were able to obtain three-dimensional in vivo images of a protease in orthopic gliomas. We demonstrate that enzyme-activatable fluorochromes can be detected with high positional accuracy in deep tissues, that molecular specificities of different beacons towards enzymes can be resolved and that tomography of beacon activation is linearly related to enzyme concentration. The tomographic imaging method offers a range of new capabilities for studying biological function; for example, identifying molecular-expression patterns by multispectral imaging or continuously monitoring the efficacy of therapeutic drugs.
... Fluorescence diffuse optical tomography (FDOT) or fluorescence molecular tomography (FMT) is one of the imaging techniques for fluorescence in strongly scattering material like biological tissues. This imaging technique is potentially powerful in medical and biological applications because some fluorescencetagged regions, which are specific to diseases and biological activities, can be identified in a non-invasive way [16,18,20]. However, many usual imaging techniques cannot be applied to this problem because the spatial distribution of fluorescence is significantly blurred by strong scattering, and the identification of the distribution needs to solve an inverse problem with a light propagation model for such media. ...
... This is the same as the case b = 0, i.e., β = 0 as in [7, Corollary 2.3]. However, a new feature here is that there is a factor ρ c3 ρ c3 + bτ 2 (20) in (11), which is an effect coming from the boundary. ...
... By (58), we can compute that the smallest peak time t min peak = 458.2 ps for given x c = (7,17,20) and L = 8. For the noisy data, the orders of jitter contained in the peak time (57) are at least 0.4582 ps, 4.582 ps and 22.91 ps for 0.1%, 1% and 5% relative jitter levels, respectively. ...
... FMT is capable of visualizing fluorescence distribution in 3D in tissue by using a raster scanning method and a model-based reconstruction algorithm [13]. More specifically, a free-space near-infrared laser beam scans the surface of the object in a point-by-point raster scan pattern. ...
... Both the lateral and axial resolution of FMT may be deteriorated for fluorophores distributed in deep brain regions, which is related to the reconstruction algorithm applied. 13. Opportunities and advances in FMT technique: due to its noninvasive nature, 3D macroscopic fluorescence is a highly valuable and attractive method, especially for brain imaging of proteinopathies such as tau deposits and alpha-synuclein inclusions [43], as well as neuroinflammation assisted with contrast agents [44]. ...
Alzheimer’s disease is pathologically featured by the accumulation of amyloid-beta (Aβ) plaque and neurofibrillary tangles. Compared to small animal positron emission tomography, optical imaging features nonionizing radiation, low cost, and logistic convenience. Optical detection of Aβ deposits is typically implemented by 2D macroscopic imaging and various microscopic techniques assisted with Aβ-targeted contrast agents. Here, we introduce fluorescence molecular tomography (FMT), a macroscopic 3D fluorescence imaging technique, convenient for in vivo longitudinal monitoring of the animal brain without the involvement of cranial window opening operation. This chapter aims to provide the protocols for FMT in vivo imaging of Aβ deposits in the brain of rodent model of Alzheimer’s disease. The materials, stepwise method, notes, limitations of FMT, and emerging opportunities for FMT techniques are presented.
... In contrast to the aforementioned optical imaging methods, fluorescence molecular tomography (FMT) is a truly non-invasive alternative that covers a large FOV comparable to traditional fluorescence reflectance imaging (FRI) and resolves the 3D fluorophore distribution in tissue [29]. More specifically, FMT belongs to a subclass of diffuse optical imaging modalities that adopt near-infrared light for exciting the fluorescence probe and utilize a model-based reconstruction algorithm to recover the 3D distribution of the fluorophore [30]. ...
... Imaging Aβ plaque distribution in the mouse brain using FMT is a challenging problem, as we have to consider complex anatomical structures such as the scalp and skull during reconstruction. The problem is further complicated by the fact that we do not consider focal brain lesions, e.g., in a glioma model [29], but rather a plaque distribution that is diffuse across cortical and, to some extent, subcortical structures. Thus, a low target-to-background ratio for the distribution of fluorescent dye across large brain areas is expected. ...
Abnormal cerebral accumulation of amyloid-beta peptide (Aβ) is a major hallmark of Alzheimer’s disease. Non-invasive monitoring of Aβ deposits enables assessing the disease burden in patients and animal models mimicking aspects of the human disease as well as evaluating the efficacy of Aβ-modulating therapies. Previous in vivo assessments of plaque load have been predominantly based on macroscopic fluorescence reflectance imaging (FRI) and confocal or two-photon microscopy using Aβ-specific imaging agents. However, the former method lacks depth resolution, whereas the latter is restricted by the limited field of view preventing a full coverage of the large brain region. Here, we utilized a fluorescence molecular tomography (FMT)-magnetic resonance imaging (MRI) pipeline with the curcumin derivative fluorescent probe CRANAD-2 to achieve full 3D brain coverage for detecting Aβ accumulation in the arcAβ mouse model of cerebral amyloidosis. A homebuilt FMT system was used for data acquisition, whereas a customized software platform enabled the integration of MRI-derived anatomical information as prior information for FMT image reconstruction. The results obtained from the FMT-MRI study were compared to those from conventional planar FRI recorded under similar physiological conditions, yielding comparable time courses of the fluorescence intensity following intravenous injection of CRANAD-2 in a region-of-interest comprising the brain. In conclusion, we have demonstrated the feasibility of visualizing Aβ deposition in 3D using a multimodal FMT-MRI strategy. This hybrid imaging method provides complementary anatomical, physiological and molecular information, thereby enabling the detailed characterization of the disease status in arcAβ mouse models, which can also facilitate monitoring the efficacy of putative treatments targeting Aβ.
... The development of Aβ-specific fluorescent probes such as AOI987 (Hintersteiner et al., 2005), CRANAD-2/-3 (Ran et al., 2009;Zhang et al., 2015), and luminescent conjugated oligothiophenes (Calvo-Rodriguez et al., 2019;Ni et al., 2020a;Shirani et al., 2015) using methoxy-X04, BTA-1 and PIB (Bacskai et al., 2003;Hefendehl et al., 2011;Klunk et al., 2002;Meyer-Luehmann et al., 2008) enabled the application of various optical imaging methods in preclinical studies. Optical detection of Aβ deposits by using ex vivo optical projection tomography (Nguyen et al., 2019), light-sheet microscopy (Ni et al., 2020b) and various in vivo fluorescence microscopy methods, such as multiphoton microscopy, enabled monitoring of Aβ at fluorescence reflectance imaging (FRI) and resolves the fluorophore distribution in tissue in 3D (Ntziachristos et al., 2002). More specifically, FMT belongs to a subclass of diffuse optical imaging modalities that adopt near-infrared light for exciting the fluorescence probe and utilize a model-based reconstruction algorithm to recover the 3D distribution of the fluorophore (Arridge and Schotland, 2009). ...
... Imaging Aβ plaque distribution in the mouse brain using FMT is a challenging problem, as we have to consider complex anatomical structures such as the scalp and skull during reconstruction. The problem is further complicated by the fact that we do not consider focal brain lesions, e.g., in a glioma model (Ntziachristos et al., 2002), but rather a plaque distribution that is diffuse across cortical and, to some extent, subcortical structures. Thus, a low target-to-background ratio for the distribution of fluorescent dye across large brain areas is expected. ...
Abnormal cerebral accumulation of amyloid-beta peptide (Aβ) is a major hallmark of Alzheimer's disease. Non-invasive monitoring of Aβ deposits enables assessing the disease burden in patients and animal models mimicking aspects of the human disease as well as evaluating the efficacy of Aβ-modulating therapies. Previous in vivo assessments of plaque load in mouse models of cerebral amyloidosis have been predominantly based on two-dimensional diffuse fluorescence reflectance imaging (2D-FRI) and two-photon microscopy (2PM) using Aβ-specific imaging agents. However, 2D-FRI lacks depth resolution, whereas 2PM is restricted by the limited field of view preventing coverage of large brain regions. Here, we utilized a magnetic resonance imaging (MRI) and fluorescence molecular tomography (FMT) pipeline with the curcumin derivative fluorescent probe CRANAD-2 to achieve full 3D brain coverage for detecting Aβ accumulation in the arcAβ mouse model of cerebral amyloidosis. A homebuilt FMT system was used for data acquisition in combination with a customized software platform enabling the integration of anatomical information derived from MRI as prior information for FMT image reconstruction. The results obtained from the FMT-MRI study were compared to data obtained from conventional 2D-FRI recorded under similar physiological conditions. The two methods yielded comparable time courses of the fluorescence intensity following intravenous injection of CRANAD-2 in a region of interest comprising the mouse brain. The depth resolution inherent to FMT allowed separation of signal contributions from the scalp. CC-BY-NC
... CCD technology has been widely adopted in a large number of NIR fluorescence imaging systems. Some of the most prominent NIR fluorescence imaging systems include fluorescence molecular tomography [30] as well as NIR fluorescence-guided surgery imaging systems such as the Storz D-Light and SPY imaging systems [31]. The FCCD is designed with the polysilicon gate located at the front, the photodiodes at the back, and the wiring in between. ...
To translate near-infrared (NIR) and shortwave infrared (SWIR) fluorescence imaging into the clinic, the paired imaging device needs to detect trace doses of fluorescent imaging agents. Except for the filtration scheme and excitation light source, the image sensor used will finally determine the detection limitations of NIR and SWIR fluorescence imaging systems. In this review, we investigate the current state-of-the-art image sensors used in NIR and SWIR fluorescence imaging systems and discuss the advantages and limitations of their characteristics, such as readout architecture and noise factors. Finally, the imaging performance of these image sensors is evaluated and compared.
... This structure makes QDs overcome the surface deficiency and increases the quantum yield [5,6]. Also, some of the natural organic atoms could be applied by making them absorb on the QDs' surface and proceeded as covering specialists [7][8][9]. What needs to be concerned is that some of the QDs are based on single elements, for example, the carbon dots (C-Dots), which are regarded as a new choice for semiconductor materials, are exhibiting their potential for their applications in plenty of fields [10][11][12]. ...
Quantum dots (QDs) are semiconductor-based nanocrystals. These nanoparticles have exhibited their unique optical and electronic properties. With these characteristics, QDs attracted scientists interest in biomedical areas such as bioimaging, drug delivery and biosensors apart from other applications like photocatalysis, light-emitting, and solar cells in recent years, and plenty of QDs and QD-based materials have exhibited their unique properties in their applications of biomedical areas. Nevertheless, the potential toxicity of QDs becomes the limitation of QDs application in biomedical areas. The toxicity of QDs might come from: the toxicity core material of QDs; the toxicity substances from the surface of QDs; the free radicals or reactive species released from QDs; the biological environment induced QDs toxicity. In this article, we review the properties of QDs, the applications of QDs in different biomedical fields, how QDs cause toxicity, and how to reduce or prevent QDs potential toxicity. In the future, we expect the improvement and further application of QDs in biomedical areas.
... It is achieved by using reconstruction algorithms to invert the localization and quantitative distribution of targeted objects within living body based on light intensity distribution measured on the surface of biological tissues (Ntziachristos et al 2005, Ale et al 2012. As a non-invasive whole-body imaging modality, 3D OI exhibits the advantages of high sensitivity and specificity, and can be applied to preclinical studies such as early detection of cancer, drug manufacturing and efficacy assessment (Ntziachristos et al 2002, O'Neill et al 2010. Depending on the optical signal generation mechanism, 3D OI is mainly divided into different modalities such as bioluminescence tomography, fluorescence tomography, Cerenkov luminescence tomography, x-ray excited luminescent tomography (Pratx et al 2010, Darne et al 2014, Wang and Huang 2021, Zhang et al 2021a. ...
Objective: The reconstruction of three-dimensional optical imaging that can quantitatively acquire the target distribution from surface measurements is a serious ill-posed problem. The objective of this work is to develop a highly robust reconstruction framework to solve the existing problems. Approach: This paper proposes a physical model constrained neural networks-based reconstruction framework. In the framework, the neural networks are to generate a target distribution from surface measurements, while the physical model is used to calculate the surface light distribution based on this target distribution. The mean square error between the calculated surface light distribution and the surface measurements is then used as a loss function to optimize the neural network. To further reduce the dependence on a priori information, a moveable region is randomly selected and then traverses the entire solution interval. We reconstruct the target distribution in this moveable region and the results are used as the basis for its next movement. Main Results: The performance of the proposed framework is evaluated with a series of simulations and in vivo experiment, including accuracy robustness of different target distributions, noise immunity, depth robustness, and spatial resolution. The results collectively demonstrate that the framework can reconstruct targets with a high accuracy, stability and versatility. Significance: The proposed framework has high accuracy and robustness, as well as good generalizability. Compared with traditional regularization-based reconstruction methods, it eliminates the need to manually delineate feasible regions and adjust regularization parameters. Compared with emerging deep learning assisted methods, it does not require any training dataset, thus saving a lot of time and resources and solving the problem of poor generalization and robustness of deep learning methods. Thus, the framework opens up a new perspective for the reconstruction of three-dimension optical imaging.
... On the aspect of hardware, early implementations of DOT, FMT, and BLT employ optical fibers that contact directly to the tissue for light emission and detection [10]- [13]. More recently, noncontact optical tomography systems that utilize free-space light illumination and fiber-free detection, such as charge-coupled device (CCD) and complementary metal-oxide semiconductor (CMOS) cameras, have gained large popularity due to better flexibility in both system design and experimental operation, considering the fact that the fiber-based solutions necessitate an optically coupling medium where the object is immersed [14]. Furthermore, detecting emitted light with CCD/CMOS cameras in noncontact systems enables highresolution image acquisition up to megapixels and provides more detector-source pairs which can potentially improve the reconstruction results [15]- [17], thus is more advantageous than the fiber-based contact systems where the number and locations of attached fibers are considerably restricted. ...
italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Objective:
Macroscopic optical tomography is a non-invasive method that can visualize the 3D distribution of intrinsic optical properties or exogenous fluorophores, making it highly attractive for small animal imaging. However, reconstructing the images requires prior knowledge of surface information. To address this, existing systems often use additional hardware components or integrate multimodal information, which is expensive and introduces new issues such as image registration. Our goal is to develop a multifunctional optical tomography system that can extract surface information using a concise hardware design.
Methods:
Our proposed system uses a single programmable scanner to implement both surface extraction and optical tomography functions. A unified pinhole model is used to describe both the illumination and detection procedures for capturing 3D point cloud. Line-shaped scanning is adopted to improve both spatial resolution and speed of surface extraction. Finally, we integrate the extracted surface information into the optical tomographic reconstruction to more accurately map the fluorescence distribution.
Result:
Comprehensive phantom experiments with different levels of complexity were designed to evaluate the performance of surface extraction and fluorescence tomography. We also imaged the axillary lymph nodes in living mice after injection of fluorophore, demonstrating the proposed system facilitates more reliable fluorescence tomography.
Conclusion:
We have successfully developed a versatile optical tomography system by leveraging concise hardware design and unified pinhole modeling. Phantom validation demonstrates that our system provides high-precision surface information with a maximum error of 0.1 mm, while the surface-guided FMT reconstruction is more reliable than the blind reconstruction using simplified surface geometry, elevating several quantitative metrics including RMSE, CNR, and Dice.
Significance:
Our work explores the feasibility of obtaining additional surface information using existing components of standalone optical tomography. This makes the optical tomographic technique more accurate and more accessible to biomedical researchers.
... Fluorescence molecular tomography (FMT) has been widely used in preclinical small animal imaging studies to assess the progression of diseases such as cancer, aiding in the development of new treatments and therapies [1][2][3][4]. ...
The fast iterative shrinkage thresholding algorithm (FISTA)
has been shown to be an efficient method for solving least
squares with l1-norm regularization problems and has been
applied to optical molecular tomography. It adopts a linear
increase scheme to provide the Lipschitz constant, which
determines the step-size of the internal gradient. The
Lipschitz constant, however, will not change if the proximal
gradient condition is satisfied after the linear increase. Then
it restricts the convergence speed of FISTA further. In this
work, a non-linear search scheme, which contains the
gradient information, is proposed to obtain the suitable
Lipschitz constant. It can provide a variable step-size in each
iteration, which can accelerate the convergence of the
standard FISTA. We called is as VFISTA. Phantom and in
vivo experiments have been performed to show that VFISTA
can speed up the reconstruction process effectively for the
inverse problem of FMT compared to FISTA.
... The initial-boundary value problem (1) is used for light propagation in highly scattering media, such as biological tissue and has been applied for the quantitative analysis of optical properties of random media. [4][5][6][7][8] This kind of analysis includes diffuse optical spectroscopy (DOS) and diffuse optical tomography (DOT). Both DOS and DOT are used to identify the unknown absorption coefficient μ a (x) and which depends on both space and time. ...
Light propagation through diffusive media can be described by the diffusion equation in a space–time domain. Furthermore, fluorescence can be described by a system of coupled diffusion equations. This paper analyzes time-domain measurements. In particular, the temporal point-spread function is measured at the boundary of a diffusive medium. Moreover, the temporal profile of fluorescence is considered. In both cases, we refer to the maximum temporal position of measured light as the peak time. In this paper, we provide proofs of the existence and uniqueness of the peak time and give explicit expressions of the peak time. The relationship between the peak time and the object position in a medium is clarified.
... Fluorescence molecular tomography (FMT) is a promising imaging technology that noninvasively and dynamically offers a 3D visualization of the biological process in-vivo at the cellular and molecular levels. [1][2][3][4] Consequently, it greatly promotes its application in small animal research and preclinical diagnosis. 4,5 However, the reconstruction of FMT is severe ill-posed caused by the strong scattering of near-infrared photons propagation in biological tissues. ...
Significance:
Fluorescence molecular tomography (FMT) is a promising imaging modality, which has played a key role in disease progression and treatment response. However, the quality of FMT reconstruction is limited by the strong scattering and inadequate surface measurements, which makes it a highly ill-posed problem. Improving the quality of FMT reconstruction is crucial to meet the actual clinical application requirements.
Aim:
We propose an algorithm, neighbor-based adaptive sparsity orthogonal least square (NASOLS), to improve the quality of FMT reconstruction.
Approach:
The proposed NASOLS does not require sparsity prior information and is designed to efficiently establish a support set using a neighbor expansion strategy based on the orthogonal least squares algorithm. The performance of the algorithm was tested through numerical simulations, physical phantom experiments, and small animal experiments.
Results:
The results of the experiments demonstrated that the NASOLS significantly improves the reconstruction of images according to indicators, especially for double-target reconstruction.
Conclusion:
NASOLS can recover the fluorescence target with a good location error according to simulation experiments, phantom experiments and small mice experiments. This method is suitable for sparsity target reconstruction, and it would be applied to early detection of tumors.
... Optical molecular tomography (OMT) has considerable potential in various small animal modelbased studies, such as the early detection and diagnosis of tumors, analysis of pathological tumor characteristics, and efficacy evaluation of anticancer drug discovery [1,2]. The inverse inference of the 3D distribution of luminous sources in complex biological tissues based on acquired optical images has always been a topic of considerable research interest in OMT [3], including bioluminescence tomography (BLT) [4] and fluorescence molecular tomography imaging (FMT) [5]. ...
Optical molecular tomography (OMT) is an emerging imaging technique. To date, the poor universality of reconstruction algorithms based on deep learning for various imaged objects and optical probes limits the development and application of OMT. In this study, based on a new mapping representation, a multimodal and multitask reconstruction framework-3D deep optical learning (3DOL), was presented to overcome the limitations of OMT in universality by decomposing it into two tasks, optical field recovery and luminous source reconstruction. Specifically, slices of the original anatomy (provided by computed tomography) and boundary optical measurement of imaged objects serve as inputs of a recurrent convolutional neural network encoded parallel to extract multimodal features, and 2D information from a few axial planes within the samples is explicitly incorporated, which enables 3DOL to recognize different imaged objects. Subsequently, the optical field is recovered under the constraint of the object geometry, and then the luminous source is segmented by a learnable Laplace operator from the recovered optical field, which obtains stable and high-quality reconstruction results with extremely few parameters. This strategy enable 3DOL to better understand the relationship between the boundary optical measurement, optical field, and luminous source to improve 3DOL’s ability to work in a wide range of spectra. The results of numerical simulations, physical phantoms, and in vivo experiments demonstrate that 3DOL is a compatible deep-learning approach to tomographic imaging diverse objects. Moreover, the fully trained 3DOL under specific wavelengths can be generalized to other spectra in the 620–900 nm NIR-I window.
... Fluorescence molecular tomography (FMT) is a preclinical optical tomographic imaging technique that reveals the distribution of fluorophores in vivo through the detection of visible or near-IR light emitted by fluorophores located within living bodies [1][2][3][4]. Different from anatomical tomographic imaging techniques such as X-ray computed tomography (XCT), FMT produces functional information of living bodies rather than structural information. Thanks to the great many varieties of fluorophores, FMT can trace various physiological and pathological processes at the cellular or even molecular level through the combination of fluorophores and biomolecules. ...
Fluorescence molecular tomography (FMT) is a preclinical optical tomographic imaging technique that can trace various physiological and pathological processes at the cellular or even molecular level. Reducing the number of FMT projection views can improve the data acquisition speed, which is significant in applications such as dynamic problems. However, a reduction in the number of projection views will dramatically aggravate the ill-posedness of the FMT inverse problem and lead to significant degradation of the reconstructed images. To deal with this problem, we have proposed a deep-learning-based reconstruction method for sparse-view FMT that only uses four perpendicular projection views and divides the image reconstruction into two stages: image restoration and inverse Radon transform. In the first stage, the projection views of the surface fluorescence are restored to eliminate the blur derived from photon diffusion through a fully convolutional neural network. In the second stage, another convolutional neural network is used to implement the inverse Radon transform between the restored projections from the first stage and the reconstructed transverse slices. Numerical simulation and phantom and mouse experiments are carried out. The results show that the proposed method can effectively deal with the image reconstruction problem of sparse-view FMT.
... Employing fluorescent nanoparticles (FNPs), fluorescence molecular imaging (FMI) particularly fluorescence molecular tomography (FMT) can noninvasively identify the molecular processes of interest and has been widely applied in pre-/co-clinical studies in drug development, disease detection, screening, diagnosis, and treatment evaluation (1)(2)(3)(4). ...
Using active tumor-targeting nanoparticles, fluorescence imaging can provide highly sensitive and specific tumor detection, and precisely guide radiation in translational radiotherapy study. However, the inevitable presence of non-specific nanoparticle uptake throughout the body can result in high levels of heterogeneous background fluorescence, which limits the detection sensitivity of fluorescence imaging and further complicates the early detection of small cancers. In this study, background fluorescence emanating from the baseline fluorophores was estimated from the distribution of excitation light transmitting through tissues, by using linear mean square error estimation. An adaptive masked-based background subtraction strategy was then implemented to selectively refine the background fluorescence subtraction. First, an in vivo experiment was performed on a mouse intratumorally injected with passively targeted fluorescent nanoparticles, to validate the reliability and robustness of the proposed method in a stringent situation wherein the target fluorescence was overlapped with the strong background. Then, we conducted in vivo studies on 10 mice which were inoculated with orthotopic breast tumors and intravenously injected with actively targeted fluorescent nanoparticles. Results demonstrated that active targeting combined with the proposed background subtraction method synergistically increased the accuracy of fluorescence molecular imaging, affording sensitive tumor detection.
... Fluorescence molecular tomography allows for detection and quantification of fluorescence signals in three dimensions (3D) in deep tissues. However, it involves protracted tomographic scans and suffers from poor spatial resolution, typically 1 mm or worse in living tissues 2 . In contrast, scanning intravital confocal 3 and multi-photon [4][5][6] microscopy can achieve diffraction-limited optical resolution in 3D at the expense of restricted FOV and/or slow volume rates, making these techniques suboptimal for studying fast biodynamics on a large (e.g., whole-cortex) scale. ...
Wide-field fluorescence imaging is an indispensable tool for studying large-scale biodynamics. Limited space-bandwidth product and strong light diffusion make conventional implementations incapable of high-resolution mapping of fluorescence biodistribution in three dimensions. We introduce a volumetric wide-field fluorescence microscopy based on optical astigmatism combined with fluorescence source localization, covering 5.6×5.6×0.6 mm ³ imaging volume. Two alternative configurations are proposed exploiting multifocal illumination or sparse localization of point emitters, which are herein seamlessly integrated in one system. We demonstrate real-time volumetric mapping of the murine cortical microcirculation at capillary resolution without employing cranial windows, thus simultaneously delivering quantitative perfusion information across both brain hemispheres. Morphological and functional changes of cerebral vascular networks are further investigated after an acute ischemic stroke, enabling cortex-wide observation of concurrent collateral recruitment events occurring on a sub-second scale. The reported technique thus offers a wealth of unmatched possibilities for non- or minimally invasive imaging of biodynamics across scales.
... Fluorescence has gained its universal value as a powerful tool in analytical sensing and optical imaging. In in vivo fluorescence imaging, small-molecule probes that fluorescing at the near-infrared (NIR) region (650-900 nm) are preferred to overcome the photon attenuation in living tissue [6][7][8]. In vivo imaging with NIR probes has great advantages of deep tissue penetration, avoidance of tissue autofluorescence, and noninvasive real-time imaging. ...
The presence of Aβ plaques in the brain is a hallmark of Alzheimer's disease. Here, we designed and synthesized a series of molecular rotors with various bi-aromatic rings and investigated their applications as near-infrared (NIR) probes for Aβ plaques. We found that the interaction with Aβ aggregates hindered the rotational freedom of the molecular rotors, which brought about a noticeable enhancement in fluorescence intensity. Among them, probe 4b (Kd = 8.5 nM) with a phenyl-pyridine ring showed a 98-fold increase in fluorescence intensity upon binding with Aβ aggregates. In addition, 4b could identify Aβ plaques in brain sections of both a transgenic (Tg) mouse and an AD patient. Furthermore, 4b could readily penetrate the blood-brain barrier (brain2min = 10.11% ID/g) and washed out rapidly. Finally, the NIR imaging with Tg mice confirmed the practical application of 4b in detecting Aβ plaques in vivo. Altogether, our work widens the landscape of Aβ NIR probes and offers a new tool for Aβ detection.
... As a valuable molecular imaging technology, fluorescence imaging has many unique advantages, such as high sensitivity, low price, no radiation, and convenience, which has been attracting more and more attention and applied in many fields [3][4][5][6]. In recent years, BLT [7][8][9] and FMT [11,12] has become research focuses due to their excellent performance and made significant progress. Now, most of them are based on diffusion equation. ...
... Therefore, to overcome the limitations of FMI, fluorescence molecular tomography (FMT), a powerful non-invasive imaging technology capable of reconstructing 3D spatial information and the concentration distribution of internal fluorescent targets in small animals, was further developed (Tan and Jiang 2008a, Han et al 2010a, Liu et al 2012a, Zhu and Li 2014, Zhang et al 2015b. Up to now, FMT has been popular in numerous physiological and clinical studies owing to its advantages of no radiation, low cost, high specificity and sensitivity, and has been extensively applied in various small animal model-based studies, including the early detection and diagnosis of tumors, the analysis of pathological tumor characteristics and efficacy evaluation of anticancer drug discovery (Ntziachristos et al 2002, Graves et al 2004, Guo et al 2011. ...
Objective:
Fluorescence molecular tomography (FMT), as an encouraging and non-invasive optical molecular imaging technology with strong specificity and sensitivity, has great potential for preclinical and clinical studies in tumor diagnosis, drug development, and therapeutic evaluation. However, the strong scattering of photons and the insufficient surface measurements make it very challenging to improve the quality of FMT reconstruction and practical application for early tumor detection. Therefore, continuous efforts have been made to explore more effective approaches or solutions in the pursuit of obtaining high-quality FMT reconstructions.
Approach:
This review takes a comprehensive overview of imaging methodology advances of FMT, mainly focusing on two critical issues in FMT reconstructions: improving the accuracy of solving the forward physical model and mitigating the ill-posed nature of the inverse problem from a methodological point of view. More importantly, numerous impressive and practical strategies and methods for improving the FMT reconstruction quality are summarized. Notably, the deep learning methods have been elaborately discussed to illustrate the advantages in promoting the imaging performance of FMT owing to the practicality of large datasets, the emergence of optimized algorithms, and the applications of innovative networks.
Main results:
The results demonstrate that the imaging quality of FMT can be effectively promoted by improving the accuracy of optical parameter modeling, combining with prior knowledge, and reducing dimensionality. In addition, the traditional regularization-based methods and the deep neural network-based methods, especially the end-to-end deep network, can enormously alleviate the ill-posedness of the inverse problem and improve the quality of FMT image reconstruction.
Significance:
This review aims at illustrating a variety of effective and practical methods for FMT image reconstruction, from which future research may benefit. Furthermore, it may provide some valuable research ideas and directions for FMT in the future, and could promote the development of FMT and other optical tomography.
... Fluorescence imaging is one of the most important imaging modalities in biology, therefore a number of techniques for deep tissue fluorescence imaging were developed. Fluorescence molecular tomography can reach several centimeters deep by detecting diffuse fluorescent light, but does not achieve single-cell resolution 2,12,13 . Mesoscopic fluorescence molecular tomography is able to image a few millimeter thick biological tissues with resolution in the order of 100 μm 14,15 . ...
Optical microcavities and microlasers were recently introduced as probes inside living cells and tissues. Their main advantages are spectrally narrow emission lines and high sensitivity to the environment. Despite numerous novel methods for optical imaging in strongly scattering biological tissues, imaging at single-cell resolution beyond the ballistic light transport regime remains very challenging. Here, we show that optical microcavity probes embedded inside cells enable three-dimensional localization and tracking of individual cells over extended time periods, as well as sensing of their environment, at depths well beyond the light transport length. This is achieved by utilizing unique spectral features of the whispering-gallery modes, which are unaffected by tissue scattering, absorption, and autofluorescence. In addition, microcavities can be functionalized for simultaneous sensing of various parameters, such as temperature or pH value, which extends their versatility beyond the capabilities of standard fluorescent labels. Optical microcavities are useful as probes in cells due to their narrow emission spectra and high sensitivity to environment. Here, the authors use the unique spectral features of microcavities, which are unaffected by tissue scattering, and show 3D localisation and tracking of cells deep in tissues.
... In quite early works, fluorescence molecular tomography (FMT) has been used to image brain tumors in mice [129]. This trend has continued in very recent works, as FMT has been used to study the pathophysiology of glioblastoma in an intracranial tumor xenograft model of mice [130]. ...
... In particular, over-thousand-nanometer NIR (OTN-NIR), which is less scattered by biological tissues than commonly used NIR-I (700-1000 nm) [2], has an expanded observation depth ranging from several millimeters to 1-2 cm [3]. Despite the diffuse nature of photon propagation in tissues, NIR fluorescence from inside tissues can be reconstructed into three-dimensional (3D) images of molecular localization and activity [4]. The intensity distribution of fluorescent probes can be mapped in 3D images via fluorescence tomography [5][6][7]. ...
The refraction of fluorescence from the inside of a sample at the surface results in fluctuations in fluorescence computed tomography (CT). We evaluated the influence of the difference in refractive index (RI) between the sample body and the surroundings on fluorescence CT results. The brightest fluorescent point is away from the correct point on the tomograms owing to the refraction. The speculated position is determined as the exact point if the RI ratio ranges between 0.97 and 1.03 by immersing the body in an RI matching liquid. The results can help in experimental settings of fluorescence CT for acquiring three-dimensional positional information.
... Fluorescence-mediated tomography (FMT) is an imaging technique used to assess the three-dimensional distribution of fluorescent probes in preclinical studies [1][2][3]. In recent years, the sensitivity and accuracy of FMT have notably increased, resulting in improved detection of fluorescent probes in deep tissue regions [4][5][6][7]. ...
Purpose
Pharmacokinetic modeling can be applied to quantify the kinetics of fluorescently labeled compounds using longitudinal micro-computed tomography and fluorescence-mediated tomography (μCT-FMT). However, fluorescence blurring from neighboring organs or tissues and the vasculature within tissues impede the accuracy in the estimation of kinetic parameters. Contributions of elimination and retention activities of fluorescent probes inside the kidneys and liver can be hard to distinguish by a kinetic model. This study proposes a deconvolution approach using a mixing matrix to model fluorescence contributions to improve whole-body pharmacokinetic modeling.
Procedures
In the kinetic model, a mixing matrix was applied to unmix the fluorescence blurring from neighboring tissues and blood vessels and unmix the fluorescence contributions of elimination and retention in the kidney and liver compartments. Accordingly, the kinetic parameters of the hepatobiliary and renal elimination routes and five major retention sites (the kidneys, liver, bone, spleen, and lung) were investigated in simulations and in an in vivo study. In the latter, the pharmacokinetics of four fluorescently labeled compounds (indocyanine green (ICG), HITC-iodide-microbubbles (MB), Cy7-nanogels (NG), and OsteoSense 750 EX (OS)) were evaluated in BALB/c nude mice.
Results
In the simulations, the corrected modeling resulted in lower relative errors and stronger linear relationships (slopes close to 1) between the estimated and simulated parameters, compared to the uncorrected modeling. For the in vivo study, MB and NG showed significantly higher hepatic retention rates (P<0.05 and P<0.05, respectively), while OS had smaller renal and hepatic retention rates (P<0.01 and P<0.01, respectively). Additionally, the bone retention rate of OS was significantly higher (P<0.01).
Conclusions
The mixing matrix correction improves pharmacokinetic modeling and thus enables a more accurate assessment of the biodistribution of fluorescently labeled pharmaceuticals by μCT-FMT.
... Comparable to the already described data in the results part of the third manuscript (Chapter 6.3, Results), the LNP showed an efficient uptake by the tumor tissue compared to healthy tissue. These data were also confirmed by FMT imaging using LNP that contained a near-infrared fluorescent dye, which allowed not only an epifluorescence measurement but a three-dimensional measurement of the mTHPC uptake in the tumor tissue (Ntziachristos, Tung et al. 2002;Jacquart, Keramidas et al. 2013). ...
... Целесообразен поиск более доступных и близких к человеческим протеиназам протеолитических ферментов животных [12]. Используя флуоресцентные молекулярные маяки ближнего инфракрасного диапазона и методы инверсии, удалось получить трех мерные изображения протеазы in vivo (метод молекулярной томографии протеолитической активности) [13]. ...
The actual problem of experimental medicine is the substantiation of new model organisms that meet modern requirements of bioethics, cost and conditions of detention. The aim of this work was a comparative analysis of the homology degree of proteolytic enzymes in humans and pulmonary freshwater mollusks. The homology of enzymes in nucleotide sequences in humans and pulmonary freshwater mollusks in the analysis of unregulated proteolysis is 66–68 %; regulated proteolysis – 69–76 %; ubiquitin-like modifiers – 78–83 %; extracellular enzymes – 67–76 %; and intracellular enzymes – 65–72 %. The evolutionary conservatism of proteolytic enzymes and the presence of an open blood circulation, which allows the substances under study to be delivered from the hemolymph directly to target cells, make it possible to use these animals as cheap and convenient test organisms. The practical importance of a sufficiently high homology degree of proteolytic enzymes in humans and pulmonary freshwater mollusks justifies the expediency of forming mollusk aquaculture to obtain proteolytic enzyme protein preparations from their tissues within the framework of the tasks of biopharmaceuticals, cosmetics and the food industry.
Simultaneous spatial mapping of the activity of multiple enzymes in a living system can elucidate their functions in health and disease. However, methods based on monitoring fluorescent substrates are limited. Here, we report the development of nitrile (C≡N)-tagged enzyme activity reporters, named nitrile chameleons, for the peak shift between substrate and product. To image these reporters in real time, we developed a laser-scanning mid-infrared photothermal imaging system capable of imaging the enzymatic substrates and products at a resolution of 300 nm. We show that when combined, these tools can map the activity distribution of different enzymes and measure their relative catalytic efficiency in living systems such as cancer cells, Caenorhabditis elegans, and brain tissues, and can be used to directly visualize caspase–phosphatase interactions during apoptosis. Our method is generally applicable to a broad category of enzymes and will enable new analyses of enzymes in their native context.
Fluorescence molecular tomography (FMT) remains challenging for accurate monitoring of fast biological processes in vivo due to the long data acquisition time and complex iterative computational process. The limited-view-based algorithms allow for three-dimensional (3D) FMT reconstructions using fewer projections by reducing data acquisition time. However, the previously limited-view FMT studies usually need to acquire at least three projections or more, and almost all are based on time-consuming regularization methods, which brings a huge bottleneck to fast imaging of living animals. Single-view FMT reconstruction has not been investigated due to extremely insufficient measurement data and the severely ill-conditioness of the inverse problem. To solve this intractable problem, we propose a novel single-view reconstruction network (SVRNet) to achieves FMT reconstruction. First, a global-local hybrid multi-head self-attention mechanism was carefully elaborated to capture both local and long-range pixel interactions simultaneously. Further, rich and accurate contextual associations in the spatial domain can be obtained to exploit practical projection information. Second, we integrated neural network priors as the regularizers to explore deep features within limited measurements and impose constraints on the solution space of the imaging domain, significantly mitigating the ill-conditioned nature of the inverse problem. Results from numerical simulations, physical phantoms, and in vivo mouse experimental results prove that the proposed SVRNet method using a single projection achieves excellent imaging quality in terms of target localization accuracy, target shape recovery, and spatial resolution. This single-view reconstruction method can enable ultrafast FMT imaging and may help simplify the hardware design of FMT systems.
A computational three‐dimensional (3D) microscopy technique, termed programmable aperture light‐field microscopy (PALFM), for motion‐free, high‐resolution volumetric imaging of fluorescent or self‐luminous samples is proposed. The well‐known Fourier slice theorem is extended to incoherent tomographic imaging, which states that a detected image under an ideal aperture corresponds to a central slice in the 3D object spectrum, so the spectrum coverage can be accomplished based on the motion‐free aperture modulation. When further considering frequency extension and coverage, a hybrid aperture modulation scheme is designed consisting of non‐centrosymmetric circular and annular apertures for high‐efficiency, non‐ambiguous depth discrimination. A PALFM system with an easy‐to‐build programmable aperture module attached to an off‐the‐shelf inverted fluorescence microscope is constructed, where annular apertures can manipulate Bessel‐like beams for spectrum modulation. Experimental results on near‐diffraction‐limited imaging of a resolution target across a large depth range and high‐resolution, multi‐color 3D imaging of a mouse kidney section verify the validity and effectiveness of PALFM. High‐speed, long‐term time‐lapse volumetric imaging of HeLa cells in vitro further demonstrates that PALFM is a promising tomographic imaging tool for studying dynamic cellular processes and events without requiring complicated sample rotation or beam scanning.
Urinary sensing of synthetic biomarkers that are released into urine after specific activation in an in vivo disease environment is an emerging diagnosis strategy to overcome the insensitivity of a previous biomarker assay. However, it remains a great challenge to achieve sensitive and a specific urinary photoluminescence (PL) diagnosis. Herein, we report a novel urinary time-resolved PL (TRPL) diagnosis strategy by exploiting europium complexes of diethylenetriaminepentaacetic acid (Eu-DTPA) as synthetic biomarkers and designing the activatable nanoprobes. Notably, TRPL of Eu-DTPA in the enhancer can eliminate the urinary background PL for ultrasensitive detection. We achieved sensitive urinary TRPL diagnosis of mice kidney and liver injuries by using simple Eu-DTPA and Eu-DTPA-integrated nanoprobes, respectively, which cannot be realized by traditional blood assays. This work demonstrates the exploration of lanthanide nanoprobes for in vivo disease-activated urinary TRPL diagnosis for the first time, which might advance the noninvasive diagnosis of diverse diseases via tailorable nanoprobe designs.
Significance: Fluorescence molecular lifetime tomography (FMLT) plays an increasingly important role in experimental oncology. The article presents and experimentally verifies an original method of mesoscopic time domain FMLT, based on an asymptotic approximation to the fluorescence source function, which is valid for early arriving photons. Aim: The aim was to justify the efficiency of the method by experimental scanning and reconstruction of a phantom with a fluorophore. The experimental facility included the TCSPC system, the pulsed supercontinuum Fianium laser, and a three-channel fiber probe. Phantom scanning was done in mesoscopic regime for three-dimensional (3D) reflectance geometry. Approach: The sensitivity functions were simulated with a Monte Carlo method. A compressed sensing like reconstruction algorithm was used to solve the inverse problem for the fluorescence parameter distribution function, which included the fluorophore absorption coefficient and fluorescence lifetime distributions. The distributions were separated directly in the time domain with the QR-factorization least square method. Results: 3D tomograms of fluorescence parameters were obtained and analyzed using two strategies for the formation of measurement data arrays and sensitivity matrices. An algorithm is developed for the flexible choice of optimal strategy in view of attaining better reconstruction quality. Variants on how to improve the method are proposed, specifically, through stepped extraction and further use of a posteriori information about the object. Conclusions: Even if measurement data are limited, the proposed method is capable of giving adequate reconstructions but their quality depends on available a priori (or a posteriori) information. Further research aims to improve the method by implementing the variants proposed.
Significance:
Fluorescence molecular lifetime tomography (FMLT) plays an increasingly important role in experimental oncology. The article presents and experimentally verifies an original method of mesoscopic time domain FMLT, based on an asymptotic approximation to the fluorescence source function, which is valid for early arriving photons.
Aim:
The aim was to justify the efficiency of the method by experimental scanning and reconstruction of a phantom with a fluorophore. The experimental facility included the TCSPC system, the pulsed supercontinuum Fianium laser, and a three-channel fiber probe. Phantom scanning was done in mesoscopic regime for three-dimensional (3D) reflectance geometry.
Approach:
The sensitivity functions were simulated with a Monte Carlo method. A compressed-sensing-like reconstruction algorithm was used to solve the inverse problem for the fluorescence parameter distribution function, which included the fluorophore absorption coefficient and fluorescence lifetime distributions. The distributions were separated directly in the time domain with the QR-factorization least square method.
Results:
3D tomograms of fluorescence parameters were obtained and analyzed using two strategies for the formation of measurement data arrays and sensitivity matrices. An algorithm is developed for the flexible choice of optimal strategy in view of attaining better reconstruction quality. Variants on how to improve the method are proposed, specifically, through stepped extraction and further use of a posteriori information about the object.
Conclusions:
Even if measurement data are limited, the proposed method is capable of giving adequate reconstructions but their quality depends on available a priori (or a posteriori) information. Further research aims to improve the method by implementing the variants proposed.
In this chapter, we discuss the applications of FMT to several clinically significant areas and demonstrate its validity in animals or in humans. As we see from the contents presented below, the unique high molecular specificity coupled with the large depth resolution render FMT an excellent modality for various tissue types/dimensions and organ sites ranging from fruit flies to human breasts, and from breast/ovarian/brain tumors to Alzheimer’s and cardiovascular diseases.KeywordsFluorescence molecular tomographyDrosophila/fruit fliesMesoscopic imagingFluorescence microscopyConfocal microscopyCy5.5 dyeDsRed moleculesStem cellsContinuous-wave (cw)Diode laserCharge-coupled-device (CCD) camerasRadiative transfer equationMammary carcinoma (4 T1) xenograft tumorOvarian tumorLung tumorBrain tumorBreast tumorTargeted nanoparticlesEpidermal growth factor receptor (EGFR)Near-infrared dyesMagnetic iron oxide nanoparticlesOptical fibersTumor response to therapyReceptor densityBone remodelingAlzheimer’s diseaseStrokeMyocardial infarction
This work considers the time-domain fluorescence diffuse optical tomography (FDOT). We recover the distribution of fluorophores in biological tissue by the boundary measurements. With the Laplace transform and the knowledge of complex analysis, we build the uniqueness theorem of this inverse problem. After that, the numerical inversions are considered. We introduce an iterative inversion algorithm under the framework of regularizing scheme, then give several numerical examples in three-dimensional space illustrating the performance of the proposed inversion schemes.
Fluorescence Molecular Tomography (FMT), providing thethree-dimensional fluorescent distribution information of specific molecular probes in tumors, is widely applied to detect
in vivo
tumors. However, the ill-posedness of reconstruction greatly affects the resolution of FMT. Traditional methods have introduced different regularization terms to solve this problem, but there are still challenges for the high-resolution reconstruction of small tumors under complex conditions. In this paper, we proposed an elastic net method optimized by the relaxed Alternating Direction Method of Multipliers (EN-RADMM) to improve the reconstruction resolution for small tumors. The objective function consisted of the Least-Square term and elastic net regularization. Relaxation, equivalent deformation directing at ill-posed equations, and LU decomposition were applied to accelerate algorithm convergence and improve solution accuracy. Thereby, the light from small tumors can be precisely reconstructed. We designed a series of digital tumor models with different distances, sizes, and shapes to verify the performance of EN-RADMM, and utilized the real glioma-bearing mouse models to further verify its feasibility and accuracy. The simulation results demonstrated that EN-RADMM can achieve significantly higher resolution and reconstruction accuracy of morphology and position with less time compared with other advanced methods. Furthermore,
in vivo
experiments proved the broad prospect of EN-RADMM in pre-clinical application of FMT reconstruction.
Small animal models play a fundamental role in brain research by deepening the understanding of the physiological functions and mechanisms underlying brain disorders and are thus essential in the development of therapeutic and diagnostic imaging tracers targeting the central nervous system. Advances in structural, functional, and molecular imaging using MRI, PET, fluorescence imaging, and optoacoustic imaging have enabled the interrogation of the rodent brain across a large temporal and spatial resolution scale in a non-invasively manner. However, there are still several major gaps in translating from preclinical brain imaging to the clinical setting. The hindering factors include the following: (1) intrinsic differences between biological species regarding brain size, cell type, protein expression level, and metabolism level and (2) imaging technical barriers regarding the interpretation of image contrast and limited spatiotemporal resolution. To mitigate these factors, single-cell transcriptomics and measures to identify the cellular source of PET tracers have been developed. Meanwhile, hybrid imaging techniques that provide highly complementary anatomical and molecular information are emerging. Furthermore, deep learning-based image analysis has been developed to enhance the quantification and optimization of the imaging protocol. In this mini-review, we summarize the recent developments in small animal neuroimaging toward improved translational power, with a focus on technical improvement including hybrid imaging, data processing, transcriptomics, awake animal imaging, and on-chip pharmacokinetics. We also discuss outstanding challenges in standardization and considerations toward increasing translational power and propose future outlooks.
This work considers the time-domain fluorescence diffuse optical tomography (FDOT). We recover the distribution of fluorophores in biological tissue by the boundary measurements. With the Laplace transform and the knowledge of complex analysis, we build the uniqueness theorem of this inverse problem. After that, the numerical reconstructions are considered. We introduce a non-iterative inversion strategy by peak detection and an iterative inversion algorithm under the framework of regularizing scheme, then give several numerical examples in three-dimensional space illustrating the performance of the proposed inversion schemes.
DESCRIPTION
This chapter discusses the field of diffuse fluorescence tomography in terms of fluorescence diffuse optical tomography (FDOT) and fluorescence molecular tomography (FMT). A brief overview of the forward photon propagation model is given. In addition, the techniques and challenges associated with solving the inverse problem, required for successful reconstruction, are discussed. Moreover, special attention is given to the different instrumentation used in diffuse fluorescence tomography. This includes the instrumentation associated with adequate illumination of the sample as well as efficient detection. Furthermore, the diverse applications of diffuse fluorescence tomography are explored, ranging from its use in biomarkers to preclinical applications and translational imaging. Finally, the chapter looks at the emerging technologies, which will shape the field in the near future.
Luminescent iridium(III) complexes exhibit high phosphorescence quantum yields and photostability, good cell membrane permeability, rich and sensitive photophysical properties, and thus have been extensively studied as intracellular sensors for photoluminescence microscopy imaging. The microenvironmental parameters and interactions of the complexes with cellular biological species can be readily reflected by changes in luminescence properties. In many studies, such changes are limited to responses in the phosphorescence intensity and wavelength; the phosphorescence lifetime is often ignored because it is invisible and difficult to analysis in cellular conditions. In recent decades, photoluminescence lifetime imaging microscopy (PLIM) has been emerging and fast developed, which allows visualizing and mapping the decays of luminescent dyes in the time domain during cellular imaging. In this review article, we focus on the design and applications of lifetime-responsive phosphorescent iridium(III) complexes as imaging reagents for mapping environmental parameters and sensing analytes of interest in intracellular conditions. These parameters and analytes either interact with the probes in the excited state to affect their decay rates or directly react with the probes yielding new products. The utilization of the phosphorescent complexes to visualize intracellular labeling of biomolecules via PLIM are also discussed.
Fluorescence molecular tomography (FMT) is an optical molecular tomography technology with great promise, and it has broad application prospects for its high sensitivity. However, it is still a challenge in reconstruction because of its severe ill-posedness. In this study, a new extraction strategy to determine a permissible region of target is proposed for FMT, which can provide a definite central position and size of the permissible region. Numerical simulation experiments and an in vivo experiment have been carried out to verify the performances of the strategy. The experimental results demonstrated that the strategy can provide a permissible region of target with a definite position and size, which further allowed a steady and accurate reconstruction for FMT.
In the past decade, gene- and cell-based therapies have been translated into clinical application despite enormous costs for the establishment of an efficient gene therapeutic regimen. Technological advances were made with regards to smart vector design serving targeted, efficient, regulated, and sustained gene expression without interference of the host immune response. Great efforts have been made to serve safe delivery of vector particles to specific tissues and cells in vivo. Intriguing technologies for editing genes and correcting inherited mutations in vivo based on artificial zinc-finger nucleases and CRISPR/Cas9 technology have been developed. Stem- and T-cell technologies have moved the entire field of regenerative medicine and targeted engagement of immune cells to fight cancer a significant step forward. The developments in various aspects of molecular imaging (MI) technologies have enabled not only the possibility to follow the dynamics of disease-specific molecular alterations in vivo but also to visualize the location and dynamics of the expression of transduced genes or gene-modified stem- or T-cells on a quantitative level in vivo. In the past 20 years, various MI techniques for safe, repeated, and high-resolution in vivo imaging of gene expression have been successfully implemented in animals and humans. The MI technologies have been mostly applied in the field of gene therapy of highly dynamic diseases, such as cancer.
In this chapter, the basis of gene expression imaging is described and the different steps of a gene therapy protocol from gene transduction to assessment of therapy response are illustrated. Linking MI and gene therapy will help to design smart, efficient, and safe gene therapy protocols for human application. The chapter was phrased during the SARS-CoV-2 pandemic and aims to summarize the most important developments of MI for gene- and cell-based therapies in dedication to all those worldwide who suffered and fought COVID disease.
Intravital microscopy coupled with chronic animal window models has provided stunning insight into tumor pathophysiology, including gene expression, angiogenesis, cell adhesion and migration, vascular, interstitial and lymphatic transport, metabolic microenvironment and drug delivery. However, the findings to date have been limited to the tumor surface (< 150 microm). Here, we show that the multiphoton laser-scanning microscope can provide high three-dimensional resolution of gene expression and function in deeper regions of tumors. These insights could be critical to the development of novel therapeutics that target not only the tumor surface, but also internal regions.
Multicolor optical coding for biological assays has been achieved by embedding different-sized quantum dots (zinc sulfide-capped cadmium selenide nanocrystals) into polymeric microbeads at precisely controlled ratios. Their novel optical properties (e.g., size-tunable emission and simultaneous excitation) render these highly luminescent quantum dots (QDs) ideal fluorophores for wavelength-and-intensity multiplexing. The use of 10 intensity levels and 6 colors could theoretically code one million nucleic acid or protein sequences. Imaging and spectroscopic measurements indicate that the QD-tagged beads are highly uniform and reproducible, yielding bead identification accuracies as high as 99.99% under favorable conditions. DNA hybridization studies demonstrate that the coding and target signals can be simultaneously read at the single-bead level. This spectral coding technology is expected to open new opportunities in gene expression studies, high-throughput screening, and medical diagnostics.
We present a novel tomographer for three‐dimensional reconstructions of fluorochromes in diffuse media. Photon detection is based on charge‐coupled device technology that allows the implementation of a large parallel array of detection channels with high sensitivity. Using this instrument we studied the response and detection limits of near‐infrared fluorochromes in diffuse media as a function of light intensity and for a wide range of biologically relevant concentrations. We further examined the resolution of the scanner and the reconstruction linearity achieved. We demonstrate that the instrument attains better than 3 mm resolution, is linear within more than two orders of magnitude of fluorochrome concentration, and can detect fluorescent objects at femto‐mole quantities in small animal‐like geometries. These measurements delineate detection and reconstruction characteristics associated with imaging of novel classes of fluorescent probes developed for in vivo molecular and functional probing of tissues.
We predict the capacity of near-infrared fluorescent signals to propagate through human tissue for non-invasive medical imaging. This analysis employs experimental measurements of a biologically relevant local fluorochrome embedded in tissuelike media and predicts the equivalent photon counts expected from breast, lung, brain, and muscle as a function of diameter by use of an analytical solution of the diffusion equation that can take into account large arbitrary geometries. The findings address feasibility issues for clinical studies and are relevant to recent development of near-infrared fluorescent probes and molecular beacons for in vivo applications.
We present a review of methods for the forward and inverse problems in optical tomography. We limit ourselves to the highly scattering case found in applications in medical imaging, and to the problem of absorption and scattering reconstruction. We discuss the derivation of the diffusion approximation and other simplifications of the full transport problem. We develop sensitivity relations in both the continuous and discrete case with special concentration on the use of the finite element method. A classification of algorithms is presented, and some suggestions for open problems to be addressed in future research are made.
The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.
The use of green fluorescent protein (GFP) is a powerful technology that has recently enabled investigators to study dynamic molecular events within living cells. One method for detecting molecular interactions involves fluorescence resonance energy transfer (FRET) between two GFPs or between GFP and a second fluorophore. This review summarizes the use of GFP for FRET and illustrates the theme with specific examples on how GFP has been employed as an intracellular molecular sensor.
We have developed a method to image tumor-associated lysosomal protease
activity in a xenograft mouse model in vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous injection, the NIRF probe carrier accumulated in solid
tumors due to its long circulation time and leakage through tumor neovasculature. Intratumoral NIRF signal was generated by lysosomal proteases in tumor cells that cleave the macromolecule, thereby releasing previously quenched fluorochrome.
In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters. This strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.
Increased expression of cathepsin B has been reported in a number of human and animal tumors. This has also been observed in human gliomas where increases in cathepsin B mRNA, protein, activity and secretion parallel malignant progression. In the present study, we showed that cathepsin B was directly involved in glioma cell invasion. Activity of cathepsin B was an order of magnitude higher in glioma tissue than in matched normal brain. Inhibitors of cysteine proteases reduced invasion of glioma cells in two in vitro models: invasion through Matrigel and infiltration of a glioma spheroid into a normal brain aggregate. Glioma spheroids expressed higher levels of cathepsin B than did monolayers and the ability of subclones differing in cathepsin B activity to infiltrate normal brain aggregates paralleled their cathepsin B activity. We confirmed that intracellular staining for cathepsin B occurs at the cell periphery and in cell processes and observed extracellular staining on the cell surface. In addition, we demonstrated that intracellular cathepsin B located at the cell periphery and in processes was active. The cell surface cathepsin B colocalized with areas of degradation of an extracellular matrix component. We hypothesize that the increased expression of active cathepsin B in gliomas leads to increases in invasion in vitro and in vivo and have developed a xenotransplant model in which this hypothesis can be tested.
We present quantitative optical images of human breast in vivo. The images were obtained by using near-infrared diffuse optical tomography (DOT) after the administration of indocyanine green (ICG) for contrast enhancement. The optical examination was performed concurrently with a magnetic resonance imaging (MRI) exam on patients scheduled for excisional biopsy or surgery so that accurate image coregistration and histopathological information of the suspicious lesions was available. The ICG-enhanced optical images coregistered accurately with Gadolinium-enhanced magnetic resonance images validating the ability of DOT to image breast tissue. In contrast to simple transillumination, we found that DOT provides for localization and quantification of exogenous tissue chromophore concentrations. Additionally our use of ICG, an albumin bound absorbing dye in plasma, demonstrates the potential to differentiate disease based on the quantified enhancement of suspicious lesions.
Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.
The single biggest challenge facing in vivo imaging techniques is to develop biocompatible molecular beacons that are capable of specifically and accurately measuring in vivo targets at the protein, RNA, or DNA level. Our efforts have focused on developing activatable imaging probes to measure specific enzyme activities in vivo. Using cathepsin D as a model target protease, we synthesized a long-circulating, synthetic graft copolymer bearing near-infrared (NIR) fluorochromes positioned on cleavable substrate sequences. In its native state, the reporter probe was essentially nonfluorescent at 700 nm due to energy resonance transfer among the bound fluorochromes (quenching) but became brightly fluorescent when the latter were released by cathepsin D. NIR fluorescence signal activation was linear over at least 4 orders of magnitude and specific when compared with scrambled nonsense substrates. Using matched rodent tumor models implanted into nude mice expressing or lacking the targeted protease, it could be shown that the former generated sufficient NIR signal to be directly detectable and that the signal was significantly different compared with negative control tumors. The developed probes should find widespread applications for real-time in vivo imaging of a variety of clinically relevant proteases, for example, to detect endogenous protease activity in disease, to monitor the efficacy of protease inhibitors, or to image transgene expression.
DsRed, a brilliantly red fluorescent protein, was recently cloned from Discosoma coral by homology to the green fluorescent protein (GFP) from the jellyfish Aequorea. A core question in the biochemistry of DsRed is the mechanism by which the GFP-like 475-nm excitation and 500-nm emission maxima of immature DsRed are red-shifted to the 558-nm excitation and 583-nm emission maxima of mature DsRed. After digestion of mature DsRed with lysyl endopeptidase, high-resolution mass spectra of the purified chromophore-bearing peptide reveal that some of the molecules have lost 2 Da relative to the peptide analogously prepared from a mutant, K83R, that stays green. Tandem mass spectrometry indicates that the bond between the alpha-carbon and nitrogen of Gln-66 has been dehydrogenated in DsRed, extending the GFP chromophore by forming C==N==C==O at the 2-position of the imidazolidinone. This acylimine substituent quantitatively accounts for the red shift according to quantum mechanical calculations. Reversible hydration of the C==N bond in the acylimine would explain why denaturation shifts mature DsRed back to a GFP-like absorbance. The C==N bond hydrolyses upon boiling, explaining why DsRed shows two fragment bands on SDS/PAGE. This assay suggests that conversion from green to red chromophores remains incomplete even after prolonged aging.
Proteins provide the building blocks for multicomponent molecular units, or pathways, from which higher cellular functions emerge. These units consist of either assemblies of physically interacting proteins or dispersed biochemical activities connected by rapidly diffusing second messengers, metabolic intermediates, ions or other proteins. It will probably remain within the realm of genetics to identify the ensemble of proteins that constitute these functional units and to establish the first-order connectivity. The dynamics of interactions within these protein machines can be assessed in living cells by the application of fluorescence spectroscopy on a microscopic level, using fluorescent proteins that are introduced within these functional units. Fluorescence is sensitive, specific and non-invasive, and the spectroscopic properties of a fluorescent probe can be analysed to obtain information on its molecular environment. The development and use of sensors based on the genetically encoded variants of green-fluorescent proteins has facilitated the observation of 'live' biochemistry on a microscopic level, with the advantage of preserving the cellular context of biochemical connectivity, compartmentalization and spatial organization. Protein activities and interactions can be imaged and localized within a single cell, allowing correlation with phenomena such as the cell cycle, migration and morphogenesis.
A number of different matrix metalloproteinase (MMP) inhibitors have been developed as cytostatic and anti-angiogenic agents and are currently in clinical testing. One major hurdle in assessing the efficacy of such drugs has been the inability to sense or image anti-proteinase activity directly and non-invasively in vivo. We show here that novel, biocompatible near-infrared fluorogenic MMP substrates can be used as activatable reporter probes to sense MMP activity in intact tumors in nude mice. Moreover, we show for the first time that the effect of MMP inhibition can be directly imaged using this approach within hours after initiation of treatment using the potent MMP inhibitor, prinomastat (AG3340). The developed probes, together with novel near-infrared fluorescence imaging technology will enable the detailed analysis of a number of proteinases critical for advancing the therapeutic use of clinical proteinase inhibitors.
The number of known proteases is increasing at a tremendous rate as a consequence of genome sequencing projects. Although one can guess at the functions of these novel enzymes by considering sequence homology to known proteases, there is a need for new tools to rapidly provide functional information on large numbers of proteins. We describe a method for determining the cleavage site specificity of proteolytic enzymes that involves pooled sequencing of peptide library mixtures. The method was used to determine cleavage site motifs for six enzymes in the matrix metalloprotease (MMP) family. The results were validated by comparison with previous literature and by analyzing the cleavage of individually synthesized peptide substrates. The library data led us to identify the proteoglycan neurocan as a novel MMP-2 substrate. Our results indicate that a small set of libraries can be used to quickly profile an expanding protease family, providing information applicable to the design of inhibitors and to the identification of protein substrates.
The development of miniaturized imaging equipment and reporter probes has improved our ability to study animal models of disease, such as transgenic and knockout mice. These technologies can now be used to continuously monitor in vivo tumour development, the effects of therapeutics on individual populations of cells, or even specific molecules. If these techniques prove effective in mice, they might be translated into the clinic in the future, where they could be used to non-invasively detect and monitor treatment of human cancers.
We present a normalized Born expansion that facilitates fluorescence reconstructions in turbid, tissuelike media. The algorithm can be particularly useful for tissue investigations of fluorochrome distributionin vivo, since it does not require absolute photon-field measurements or measurements before contrast-agent administration. This unique advantage can be achieved only in fluorescence mode. We used this algorithm to three-dimensionally image and quantify an indocyanine fluorochrome phantom, using a novel fluorescence tomographic imager developed for animals.
The feasibility of employing fluorescent contrast agents to perform optical imaging in tissues and other scattering media has been examined through computational studies. Fluorescence lifetime and yield can give crucial information about local metabolite concentrations or environmental conditions within tissues. This information can be employed toward disease detection, diagnosis, and treatment if noninvasively quantitated from reemitted optical signals. However, the problem of inverse image reconstruction of fluorescence yield and lifetime is complicated because of the highly scattering nature of the tissue. Here a light propagation model employing the diffusion equation is used to account for the scattering of both the excitation and fluorescent light. Simulated measurements of frequency-domain parameters of fluorescent modulated ac amplitude and phase lag are used as inputs to an inverse image-reconstruction algorithm, which employs the diffusion model to predict frequency-domain measurements resulting from a modulated input at the phantom periphery. In the inverse image-reconstruction algorithm, a Newton-Raphson technique combined with a Marquardt algorithm is employed to converge on the fluorescent properties within the medium. The successful reconstruction of both the fluorescence yield and lifetime in the case of a heterogeneous fluorophore distribution within a scattering medium has been demonstrated without a priori information or without the necessity of obtaining absence images.
Imaging of metalloproteinase2 inhibi-tion in vivo
- C Bremer
- C Tung
- R Weissleder
Bremer, C., Tung, C. & Weissleder, R. Imaging of metalloproteinase2 inhibi-tion in vivo. Nature Med. 7, 743–748 (2001).
CCD-based scanner for three-dimensional fluorescence-mediated diffuse optical tomography of small animals
- V Ntziachristos
- R Weissleder
Ntziachristos, V. & Weissleder, R. CCD-based scanner for three-dimensional fluorescence-mediated diffuse optical tomography of small animals. Medical Phys. 29, 803–809 (2002).
In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy
- E B Brown
- EB Brown