Intravital

Fluorescence acquisition and image display over a high dynamic range is highly desirable. However, the limited dynamic range of current photodetectors and imaging charge-coupled devices impose a limit on the fluorescence intensities that can be simultaneously captured during a single image acquisition. This is particularly troublesome when imaging biological samples, where protein expression fluctuates considerably. As a result, biological images will often contain regions with signal that is either saturated or hidden within background noise, causing information loss. In this paper, we summarize recent work from our group and others, to extended conventional to high dynamic range fluorescence imaging. These strategies have many biological applications, such as mapping of neural connections, vascular imaging, biodistribution studies or pharmacologic imaging at the single cell and organ level.

Innate immune cells recruited to inflammatory sites have short life spans and originate from the marrow, which is distributed throughout the long and flat bones. While bone marrow production and release of leukocyte increases after stroke, it is currently unknown whether its activity rises homogeneously throughout the entire hematopoietic system. To address this question, we employed spectrally resolved in vivo cell labeling in the murine skull and tibia. We show that in murine models of stroke and aseptic meningitis, skull bone marrow-derived neutrophils are more likely to migrate to the adjacent brain tissue than cells that reside in the tibia. Confocal microscopy of the skull-dura interface revealed myeloid cell migration through microscopic vascular channels crossing the inner skull cortex. These observations point to a direct local interaction between the brain and the skull bone marrow through the meninges.

Rationale: Inflammatory stress induced by exposure to bacterial lipopolysaccharide causes hematopoietic stem cell expansion in the bone marrow niche, generating a cellular immune response. As an integral component of the hematopoietic stem cell niche, the bone marrow vasculature regulates the production and release of blood leukocytes, which protect the host against infection but also fuel inflammatory diseases. Objective: We aimed to develop imaging tools to explore vascular changes in the bone marrow niche during acute inflammation. Methods and Results: Using the TLR (Toll-like receptor) ligand lipopolysaccharide as a prototypical danger signal, we applied multiparametric, multimodality and multiscale imaging to characterize how the bone marrow vasculature adapts when hematopoiesis boosts leukocyte supply. In response to lipopolysaccharide, ex vivo flow cytometry and histology showed vascular changes to the bone marrow niche. Specifically, proliferating endothelial cells gave rise to new vasculature in the bone marrow during hypoxic conditions. We studied these vascular changes with complementary intravital microscopy and positron emission tomography/magnetic resonance imaging. Fluorescence and positron emission tomography integrin alpha V beta 3 imaging signal increased during lipopolysaccharide-induced vascular remodeling. Vascular leakiness, quantified by albumin-based in vivo microscopy and magnetic resonance imaging, rose when neutrophils departed and hematopoietic stem and progenitor cells proliferated more vigorously. Conclusions: Introducing a tool set to image bone marrow either with cellular resolution or noninvasively within the entire skeleton, this work sheds light on angiogenic responses that accompany emergency hematopoiesis. Understanding and monitoring bone marrow vasculature may provide a key to unlock therapeutic targets regulating systemic inflammation.

Quantitation of drug target engagement in single cells has proven to be difficult, often leaving unanswered questions in the drug development process. We found that intracellular target engagement of unlabeled new therapeutics can be quantitated using polarized microscopy combined with competitive binding of matched fluorescent companion imaging probes. We quantitated the dynamics of target engagement of covalent BTK inhibitors, as well as reversible PARP inhibitors, in populations of single cells using a single companion imaging probe for each target. We then determined average in vivo tumor concentrations and found marked population heterogeneity following systemic delivery, revealing single cells with low target occupancy at high average target engagement in vivo.

Respiratory- and cardiac-induced motion artifacts pose a major challenge for in vivo optical imaging, limiting the temporal and spatial imaging resolution in fluorescence laser scanning microscopy. Here, we present an imaging platform developed for in vivo characterization of physiologically induced axial motion. The motion characterization system can be straightforwardly implemented on any conventional laser scanning microscope and can be used to evaluate the effectiveness of different motion stabilization schemes. This method is particularly useful to improve the design of novel tissue stabilizers and to facilitate stabilizer positioning in real time, therefore facilitating optimal tissue immobilization and minimizing motion induced artifacts. (C) 2017 Society of Photo-Optical Instrumentation Engineers (SPIE).

The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPEPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

Here we present the first generation of two-photon beta cell specific in vivo imaging probes based on GLP1R targeting peptides. Among the three compounds of potential interest, we found quite unexpectedly that a squarine-rotaxane conjugate (2PEx-647) had near ideal in vivo imaging characteristics.

Epithelial cells are generally considered to be static relative to their neighbours. Basal cells in pseudostratified epithelia display a single long cytoplasmic process that can cross the tight junction barrier to reach the lumen. Using in vivo microscopy to visualize the epididymis, a model system for the study of pseudostratified epithelia, we report here the surprising discovery that these basal cell projections-which we call axiopodia-periodically extend and retract over time. We found that axiopodia extensions and retractions follow an oscillatory pattern. This movement, which we refer to as periodic axial motility (PAM), is controlled by c-Src and MEK1/2-ERK1/2. Therapeutic inhibition of tyrosine kinase activity induces a retraction of these projections. Such unexpected cell motility may reflect a novel mechanism by which specialized epithelial cells sample the luminal environment.

Small molecule therapeutic drugs must reach their intended cellular targets (pharmacokinetics) and engage them to modulate therapeutic effects (pharmacodynamics). These processes are often difficult to measure in vivo due to their complexities and occurrence within single cells. It has been particularly difficult to directly measure cellular drug target binding. Fluorescence polarization is commonly used in pharmacological screening assays to measure drug-protein or protein-protein interactions. We hypothesized that fluorescence polarization imaging could be adapted and used with fluorescently labeled drugs to measure drug target engagement in vivo. Here, we summarize recent results using two photon fluorescence anisotropy microscopy. Our imaging technique offers quantitative pharmacological binding information of diverse molecular interactions at themicroscopic level, differentiating between bound, and unbound states. Used in combination with other recent advances in the development of novel fluorescently labeled drugs, we expect that the described imaging modality will provide a window into the distribution and efficacy of drugs in real time and in vivo at the cellular and subcellular level.

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

Measuring key pharmacokinetic and pharmacodynamic parameters in vivo at the single cell level is likely to enhance drug discovery and development. In this review, we summarize recent advances in this field and highlight current and future capabilities.

Intravital imaging microscopy (i.e., imaging in live animals at microscopic resolution) has become an indispensable tool for studying the cellular micro-dynamics in cancer, immunology and neurobiology. High spatial and temporal resolution, combined with large penetration depth and multi-reporter visualization capability make fluorescence intravital microscopy compelling for heart imaging. However, tissue motion caused by cardiac contraction and respiration critically limits its use. As a result, in vitro cell preparations or non-contracting explanted heart models are more commonly employed. Unfortunately, these approaches fall short of understanding the more complex host physiology that may be dynamic and occur over longer periods of time. In this review, we report on novel technologies, which have been recently developed by our group and others, aimed at overcoming motion-induced artifacts and capable of providing in vivo subcellular resolution imaging in the beating mouse heart. The methods are based on mechanical stabilization, image processing algorithms, gated/triggered acquisition schemes or a combination of both. We expect that in the immediate future all these methodologies will have considerable applications in expanding our understanding of the cardiac biology, elucidating cardiomyocyte function and interactions within the organism in vivo, and ultimately improving the treatment of cardiac diseases.

Knowledge of cardiomyocyte biology is limited by the lack of methods to interrogate single-cell physiology in vivo. Here we show that contracting myocytes can indeed be imaged with optical microscopy at high temporal and spatial resolution in the beating murine heart, allowing visualization of individual sarcomeres and measurement of the single cardiomyocyte contractile cycle. Collectively, this has been enabled by efficient tissue stabilization, a prospective real-time cardiac gating approach, an image processing algorithm for motion-artifact-free imaging throughout the cardiac cycle, and a fluorescent membrane staining protocol. Quantification of cardiomyocyte contractile function in vivo opens many possibilities for investigating myocardial disease and therapeutic intervention at the cellular level.

The possibility of measuring binding of small-molecule drugs to desired targets in live cells could provide a better understanding of drug action. However, current approaches mostly yield static data, require lysis or rely on indirect assays and thus often provide an incomplete understanding of drug action. Here, we present a multiphoton fluorescence anisotropy microscopy live cell imaging technique to measure and map drug-target interaction in real time at subcellular resolution. This approach is generally applicable using any fluorescently labelled drug and enables high-resolution spatial and temporal mapping of bound and unbound drug distribution. To illustrate our approach we measure intracellular target engagement of the chemotherapeutic Olaparib, a poly(ADP-ribose) polymerase inhibitor, in live cells and within a tumour in vivo. These results are the first generalizable approach to directly measure drug-target binding in vivo and present a promising tool to enhance understanding of drug activity.

Intravital microscopy has emerged in the recent decade as an indispensible imaging modality for the study of the microdynamics of biological processes in live animals. Technical advancements in imaging techniques and hardware components, combined with the development of novel targeted probes and new mice models, have enabled us to address long-standing questions in several biology areas such as oncology, cell biology, immunology, and neuroscience. As the instrument resolution has increased, physiological motion activities have become a major obstacle that prevents imaging live animals at resolutions analogue to the ones obtained in vitro. Motion compensation techniques aim at reducing this gap and can effectively increase the in vivo resolution. This paper provides a technical review of some of the latest developments in motion compensation methods, providing organ specific solutions.

Observing drug responses in the tumor microenvironment in vivo can be technically challenging. As a result, cellular responses to molecularly targeted cancer drugs are often studied in cell culture, which does not accurately represent the behavior of cancer cells growing in vivo. Using high-resolution microscopy and fluorescently labeled genetic reporters for apoptosis, we developed an approach to visualize drug-induced cell death at single-cell resolution in vivo. Stable expression of the mitochondrial intermembrane protein IMS-RP was established in human breast and pancreatic cancer cells. Image analysis was then used to quantify release of IMS-RP into the cytoplasm upon apoptosis and irreversible mitochondrial permeabilization. Both breast and pancreatic cancer cells showed higher basal apoptotic rates in vivo than in culture. To study drug-induced apoptosis, we exposed tumor cells to navitoclax (ABT-263), an inhibitor of Bcl-2, Bcl-xL, and Bcl-w, both in vitro and in vivo. Although the tumors responded to Bcl-2 inhibition in vivo, inducing apoptosis in around 20% of cancer cells, the observed response was much higher in cell culture. Together, our findings show an imaging technique that can be used to directly visualize cell death within the tumor microenvironment in response to drug treatment. Cancer Res; 72(12); 2949-56. (C)2012 AACR.

The environments that harbor hematopoietic stem and progenitor cells are critical to explore for a better understanding of hematopoiesis during health and disease. These compartments often are inaccessible for controlled and rapid experimentation, thus limiting studies to the evaluation of conventional cell culture and transgenic animal models. Here we describe the manufacture and image-guided monitoring of an engineered microenvironment with user-defined properties that recruits hematopoietic progenitors into the implant. Using intravital imaging and fluorescence molecular tomography, we show in real time that the cell homing and retention process is efficient and durable for short-and long-term engraftment studies. Our results indicate that bone marrow stromal cells, precoated on the implant, accelerate the formation of new sinusoidal blood vessels with vascular integrity at the micro-capillary level that enhances the recruitment hematopoietic progenitor cells to the site. This implantable construct can serve as a tool enabling the study of hematopoiesis.

Objectives The aim of this study was to explore post-myocardial infarction (MI) myocardial inflammation. Background Innate immune cells are centrally involved in infarct healing and are emerging therapeutic targets in cardiovascular disease; however, clinical tools to assess their presence in tissue are scarce. Furthermore, it is currently not known if the nonischemic remote zone recruits monocytes. Methods Acute inflammation was followed in mice with coronary ligation by 18-fluorodeoxyglucose ((18)FDG) positron emission tomography/magnetic resonance imaging, fluorescence-activated cell sorting, polymerase chain reaction, and histology. Results Gd-DTPA-enhanced infarcts showed high (18)FDG uptake on day 5 after MI. Cell depletion and isolation data confirmed that this largely reflected inflammation; CD11b(+) cells had 4-fold higher (18)FDG uptake than the infarct tissue from which they were isolated (p < 0.01). Surprisingly, there was considerable monocyte recruitment in the remote myocardium (approximately 10(4)/mg of myocardium, 5.6-fold increase; p < 0.01), a finding mirrored by macrophage infiltration in the remote myocardium of patients with acute MI. Temporal kinetics of cell recruitment were slower than in the infarct, with peak numbers on day 10 after ischemia. Quantitative polymerase chain reaction showed a robust increase of recruiting adhesion molecules and chemokines in the remote myocardium (e.g., 12-fold increase of monocyte chemoattractant protein-1), although levels were always lower than in the infarct. Finally, matrix metalloproteinase activity was significantly increased in noninfarcted myocardium, suggesting that monocyte recruitment to the remote zone may contribute to post-MI dilation. Conclusions This study shed light on the innate inflammatory response in remote myocardium after MI. (J Am Coll Cardiol 2012; 59: 153-63) (C) 2012 by the American College of Cardiology Foundation

Real-time imaging of moving organs and tissues at microscopic resolutions represents a major challenge in studying the complex biology of live animals. Here we present a technique based on a novel stabilizer setup combined with a gating acquisition algorithm for the imaging of a beating murine heart at the single-cell level. The method allows serial in vivo fluorescence imaging of the beating heart in live mice in both confocal and nonlinear modes over the course of several hours. We demonstrate the utility of this technique for in vivo optical sectioning and dual-channel time-lapse fluorescence imaging of cardiac ischaemia. The generic method could be adapted to other moving organs and thus broadly facilitate in vivo microscopic investigations.

A major challenge in high-resolution intravital confocal and multiphoton microscopy is physiologic tissue movement during image acquisition. Of the various physiological sources of movement, respiration has arguably the largest and most wide-ranging effect. We describe a technique for achieving stabilized microscopy imaging using a dual strategy. First, we designed a mechanical stabilizer for constraining physical motion; this served to simultaneously increase the in-focus range over which data can be acquired as well as increase the reproducibility of imaging a certain position within each confocal imaging plane. Second, by implementing a retrospective breathing-gated imaging modality, we performed selective image extraction gated to a particular phase of the respiratory cycle. Thanks to the high reproducibility in position, all gated images presented a high degree of correlation over time. The images obtained using this technique not only showed significant improvements over images acquired without the stabilizer, but also demonstrated accurate in vivo imaging during longitudinal studies. The described methodology is easy to implement with any commercial imaging system, as are used by most biological imaging laboratories, and can be used for both confocal and multiphoton laser scanning microscopy. c 2012 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.JBO.17.9.096018]

For centuries, biological discoveries were based on optical imaging, in particular microscopy but also several chromophoric assays and photographic approaches. With the recent emergence of methods appropriate for bio-marker in vivo staining, such as bioluminescence, fluorescent molecular probes and proteins, as well as nanoparticle-based targeted agents, significant attention has been shifted toward in vivo interrogations of different dynamic biological processes at the molecular level. This progress has been largely supported by the development of advanced tomographic imaging technologies suitable for obtaining volumetric visualization of bio-marker distributions in small animals at a whole-body or whole-organ scale, an imaging frontier that is not accessible by the existing tissue-sectioning microscopic techniques due to intensive light scattering beyond the depth of a few hundred microns. Major examples of such recently developed optical imaging modalities are reviewed here, including bioluminescence tomography (BLT), fluorescence molecular tomography (FMT), and optical projection tomography (OPT). The pharmaceutical imaging community has quickly appropriated itself of these novel forms of optical imaging, since they come with very compelling advantages, such as quantitative three-dimensional capabilities, direct correlation to the biological cultures, easiness and cost-effectiveness of use, and the use of safe non-ionizing radiation. Some multi-modality approaches, combining light with other imaging modalities such as X-Ray CT or MRI, giving the ability to acquire both an optical contrast reconstruction along with a hi-fidelity anatomical images, are also reviewed. A separate section is devoted to the hybrid imaging techniques based on the optoacoustic phenomenon, such as multispectral optoacoustic tomography (MSOT), which are poised to leverage the traditional contrast and specificity advantages of optical spectrum by delivering an ever powerful set of capabilities, including real-time operation and high spatial resolution, not affected by the scattering nature of biological tissues.

A number of small-molecule poly (ADP-ribose) polymerase (PARP) inhibitors are currently undergoing advanced clinical trials. Determining the distribution and target inhibitory activity of these drugs in individual subjects, however, has proven problematic. Here, we used a PARP agent for positron emission tomography-computed tomography (PET-CT) imaging (F-18-BO), which we developed based on the Olaparib scaffold using rapid bioorthogonal conjugation chemistries. We show that the bioorthogonal F-18 modification of the parent molecule is simple, highly efficient, and well tolerated, resulting in a half maximal inhibitory concentration (IC50) of 17.9 +/- 1.1 nM. Intravital imaging showed ubiquitous distribution of the drug and uptake into cancer cells, with ultimate localization within the nucleus, all of which were inhibitable. Whole-body PET-CT imaging showed tumoral uptake of the drug, which decreased significantly, after a daily dose of Olaparib. Standard F-18-fludeoxyglucose imaging, however, failed to detect such therapy-induced changes. This research represents a step toward developing a more generic approach for the rapid codevelopment of companion imaging agents based on small-molecule therapeutic inhibitors.

Motion artifacts continue to present a major challenge to single cell imaging in cardiothoracic organs such as the beating heart, blood vessels or lung. In this study, we present a new water-immersion suctioning stabilizer that enables minimally invasive intravital fluorescence microscopy using water-based stick objectives. The stabilizer works by reducing major motion excursions and can be used in conjunction with both prospective or retrospective gating approaches. We show that the new approach offers cellular resolution in the beating murine heart without perturbing normal physiology. In addition, because this technique allows multiple areas to be easily probed, it offers the opportunity for wide area coverage at high resolution.

Recent studies have revealed asymmetries between the left and right sides of the brain in invertebrate species. Here we present a review of a series of recent studies from our laboratories, aimed at tracing asymmetries at different stages along the honeybee’s (Apis mellifera) olfactory pathway. These include estimates of the number of sensilla present on the two antennae, obtained by scanning electron microscopy, as well as electroantennography recordings of the left and right antennal responses to odorants. We describe investigative studies of the antennal lobes, where multi-photon microscopy was used to search for possible morphological asymmetries between the two brain sides. Moreover, we report on recently published results obtained by two-photon calcium imaging for functional mapping of the antennal lobe aimed at comparing patterns of activity evoked by different odours. Finally, possible links to the results of behavioural tests, measuring asymmetries in single-sided olfactory memory recall, are discussed.

Due to the honey bee’s importance as a simple neural model, there is a great need for new functional imaging modalities. Herein we report on the development and new finding of a combined two-photon microscope with a synchronized odor stimulus platform for in-vivo functional and morphological imaging of the honey bee’s olfactory system focusing on its primary centers, the antennal lobes (ALs). Our imaging platform allows for simultaneously obtaining both morphological measurements of the AL’s functional units, the glomeruli, and in-vivo calcium recording of their neural activities. By applying external odor stimuli to the bee’s antennae, we were able to record the characteristic glomerular odor response maps. Compared to previous works where conventional fluorescenc microscopy was used, our approach has been demonstrated to offer all the advantages of multi-photon imaging, providing substantial enhancement in both spatial and temporal resolutions while minimizing photo-damages. In addition, compared to previous full-fiel microscopy calcium recordings, a four-fold improvement in the functional signal has been achieved. Finally, the multi-photon associated extended penetration depth allows for functional imaging of profound glomeruli. (C) 2010 Optical Society of America

The hallmark of type 1 diabetes is autoimmune destruction of the insulin-producing beta-cells of the pancreatic islets. Autoimmune diabetes has been difficult to study or treat because it is not usually diagnosed until substantial beta-cell loss has already occurred. Imaging agents that permit noninvasive visualization of changes in beta-cell mass remain a high-priority goal. We report on the development and testing of a near-infrared fluorescent beta-cell imaging agent. Based on the amino acid sequence of exendin-4, we created a neopeptide via introduction of an unnatural amino acid at the K(12) position, which could subsequently be conjugated to fluorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4(x12)-VT750) had a high binding affinity (similar to 3 nM). Its in vivo properties were evaluated using high-resolution intravital imaging, histology, whole-pancreas visualization, and endoscopic imaging. According to intravital microscopy, the probe rapidly bound to beta-cells and, as demonstrated by confocal microscopy, it was internalized. Histology of the whole pancreas showed a close correspondence between fluorescence and insulin staining, and there was an excellent correlation between imaging signals and beta-cell mass in mice treated with streptozotocin, a beta-cell toxin. Individual islets could also be visualized by endoscopic imaging. In short, E4(x12)-VT750 showed strong and selective binding to glucose-like peptide-1 receptors and permitted accurate measurement of beta-cell mass in both diabetic and nondiabetic mice. This near-infrared imaging probe, as well as future radioisotope-labeled versions of it, should prove to be important tools for monitoring diabetes, progression, and treatment in both experimental and clinical contexts.

The honeybee, Apis mellifera L (Hymenoptera: Apidae), has recently become a model for studying brain asymmetry among invertebrates. A strong lateralization favouring the right antenna was discovered in odour learning and short-term memory recall experiments, and a lateral shift favouring the left antenna for long-term memory recall. Corresponding morphological asymmetries have been found in the distribution of olfactory sensilla between the antennae and confirmed by electrophysiological odour response measurements in isolated right and left antennae. The aim of this study was to investigate whether a morphological asymmetry can be observed in the volume of the primary olfactory centres of the central nervous system, the antennal lobes (ALs). Precise volume measurements of a subset of their functional units, the glomeruli, were performed in both sides of the brain, exploiting the advantages of two-photon microscopy. This novel method allowed minimal invasive acquisition of volume images of the ALs, avoiding artefacts from brain extraction and dehydration. The study was completed by a series of behavioural experiments in which response asymmetry in odour recall following proboscis extension reflex conditioning was assessed for odours, chosen to stimulate strong activity in the same glomeruli as in the morphological study. The volumetric measurements found no evidence of lateralization in the investigated glomeruli within the experimental limits. Instead, in the behavioural experiments, a striking odour dependence of the lateralization was observed. The results are discussed on the basis of recent neurophysiological and ethological experiments in A. mellifera. (C) 2011 Elsevier BM. All rights reserved.

New high-resolution molecular and structural imaging strategies are needed to visualize high-risk plaques that are likely to cause acute myocardial infarction, because current diagnostic methods do not reliably identify at-risk subjects. Although molecular imaging agents are available for low-resolution detection of atherosclerosis in large arteries, a lack of imaging agents coupled to high-resolution modalities has limited molecular imaging of atherosclerosis in the smaller coronary arteries. Here, we have demonstrated that indocyanine green (ICG), a Food and Drug Administration-approved near-infrared fluorescence (NIRF)-emitting compound, targets atheromas within 20 min of injection and provides sufficient signal enhancement for in vivo detection of lipid-rich, inflamed, coronary-sized plaques in atherosclerotic rabbits. In vivo NIRF sensing was achieved with an intravascular wire in the aorta, a vessel of comparable caliber to human coronary arteries. Ex vivo fluorescence reflectance imaging showed high plaque target-to-background ratios in atheroma-bearing rabbits injected with ICG compared to atheroma-bearing rabbits injected with saline. In vitro studies using human macrophages established that ICG preferentially targets lipid-loaded macrophages. In an early clinical study of human atheroma specimens from four patients, we found that ICG colocalized with plaque macrophages and lipids. The atheroma-targeting capability of ICG has the potential to accelerate the clinical development of NIRF molecular imaging of high-risk plaques in humans.

New imaging methods are urgently needed to identify high-risk atherosclerotic lesions prior to the onset of myocardial infarction, stroke, and ischemic limbs. Molecular imaging offers a new approach to visualize key biological features that characterize high-risk plaques associated with cardiovascular events. While substantial progress has been realized in clinical molecular imaging of plaques in larger arterial vessels (carotid, aorta, iliac), there remains a compelling, unmet need to develop molecular imaging strategies targeted to high-risk plaques in human coronary arteries. We present recent developments in intravascular near-IR fluorescence catheter-based strategies for in vivo detection of plaque inflammation in coronary-sized arteries. In particular, the biological, light transmission, imaging agent, and engineering principles that underlie a new intravascular near-IR fluorescence sensing method are discussed. Intravascular near-IR fluorescence catheters appear highly translatable to the cardiac catheterization laboratory, and thus may offer a new in vivo method to detect high-risk coronary plaques and to assess novel atherosclerosis biologics. (C) 2010 Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.3280282]

Fusion imaging of radionuclide-based molecular (PET) and structural data [x-ray computed tomography (CT)] has been firmly established. Here we show that optical measurements [fluorescence-mediated tomography (FMT)] show exquisite congruence to radionuclide measurements and that information can be seamlessly integrated and visualized. Using biocompatible nanoparticles as a generic platform (containing a F-18 isotope and a far red fluorochrome), we show good correlations between FMT and PET in probe concentration (r(2) > 0.99) and spatial signal distribution (r(2) > 0.85). Using a mouse model of cancer and different imaging probes to measure tumoral proteases, macrophage content and integrin expression simultaneously, we demonstrate the distinct tumoral locations of probes in multiple channels in vivo. The findings also suggest that FMT can serve as a surrogate modality for the screening and development of radionuclide-based imaging agents.

New optical and optoacustic methods are solving the bioscientist’s conundrum of imaging deep into living tissue at high resolution.

Fluorescent proteins have become essential reporter molecules for studying life at the cellular and sub-cellular level, re-defining the ways in which we investigate biology. However, because of intense light scattering, most organisms and tissues remain inaccessible to current fluorescence microscopy techniques at depths beyond several hundred micrometres. We describe a multispectral opto-acoustic tomography technique capable of high-resolution visualization of fluorescent proteins deep within highly light-scattering living organisms. The method uses multiwavelength illumination over multiple projections combined with selective-plane opto-acoustic detection for artifact-free data collection. Accurate image reconstruction is enabled by making use of wavelength-dependent light propagation models in tissue. By performing whole-body imaging of two biologically important and optically diffuse model organisms, Drosophila melanogaster pupae and adult zebrafish, we demonstrate the facility to resolve tissue-specific expression of eGFP and mCherrry fluorescent proteins for precise morphological and functional observations in vivo.

Mesoscopic-scale living organisms (i.e. 1 mm to 1 cm sized) remain largely inaccessible by current optical imaging methods due to intensive light scattering in tissues. Therefore, imaging of many important model organisms, such as insects, fishes, worms and similarly sized biological specimens, is currently limited to embryonic or other transparent stages of development. This makes it difficult to relate embryonic cellular and molecular mechanisms to consequences in organ function and animal behavior in more advanced stages and adults. Herein, we have developed a selective-plane illumination optoacoustic tomography technique for in vivo imaging of optically diffusive organisms and tissues. The method is capable of whole-body imaging at depths from the sub-millimeter up to centimeter range with a scalable spatial resolution in the order of magnitude of a few tenths of microns. In contrast to pure optical methods, the spatial resolution here is not determined nor limited by light diffusion; therefore, such performance cannot be achieved by any other optical imaging technology developed so far. The utility of the method is demonstrated on several whole-body models and small-animal extremities.

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (>1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery. We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism. The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size. We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

We report on a systematic study of upconverting fluorescence signal generation within turbid phantoms and real tissues. An accurate three-point Green’s function, describing the forward model of photon propagation, is established and experimentally validated. We further demonstrate, for the first time to our knowledge, autofluorescence-free transillumination imaging of mice that have received biocompatible upconverting nanoparticles. The method holds great promise for artifact-free whole-body visualization of optical molecular probes. (C) 2009 Optical Society of America

Eosinophils are multifunctional leukocytes that degrade and remodel tissue extracellular matrix through production of proteolytic enzymes, release of proinflammatory factors to initiate and propagate inflammatory responses, and direct activation of mucus secretion and smooth muscle cell constriction. Thus, eosinophils are central effector cells during allergic airway inflammation and an important clinical therapeutic target. Here we describe the use of an injectable MMP-targeted optical sensor that specifically and quantitatively resolves eosinophil activity in the lungs of mice with experimental allergic airway inflammation. Through the use of real-time molecular imaging methods, we report the visualization of eosinophil. responses in vivo and at different scales. Eosinophil responses were seen at single-cell resolution in conducting airways using near-infrared fluorescence fiberoptic bronchoscopy, in lung parenchyma using intravital microscopy, and in the whole body using fluorescence-mediated molecular tomography. Using these real-time imaging methods, we confirmed the immunosuppressive effects of the glucocorticoid drug dexamethasone in the mouse model of allergic airway inflammation and identified a viridin-derived prodrug that potently inhibited the accumulation and enzyme activity of eosinophils in the lungs. The combination of sensitive enzyme-targeted sensors with noninvasive molecular imaging approaches permitted evaluation of airway inflammation severity and was used as a model to rapidly screen for new drug effects. Both fluorescence-mediated tomography and fiberoptic bronchoscopy techniques have the potential to be translated into the clinic.

Background-To enable intravascular detection of inflammation in atherosclerosis, we developed a near-infrared fluorescence ( NIRF) catheter-based strategy to sense cysteine protease activity during vascular catheterization. Methods and Results-The NIRF catheter design was based on a clinical coronary artery guidewire. In phantom studies of NIRF plaques, blood produced only a mild (<30%) attenuation of the fluorescence signal compared with saline, affirming the favorable optical properties of the NIR window. Catheter evaluation in vivo used atherosclerotic rabbits (n=11). Rabbits received an injection of a cysteine protease-activatable NIRF imaging agent (Prosense750; excitation/emission, 750/770 nm) or saline. Catheter pullbacks through the blood-filled iliac artery detected NIRF signals 24 hours after injection of the probe. In the protease agent group, the in vivo peak plaque target-to-background ratio was 558% greater than controls (6.8 +/- 1.9 versus 1.3 +/- 0.3, mean +/- SEM; P<0.05). Ex vivo fluorescence reflectance imaging corroborated these results (target-to-background ratio, 10.3 +/- 1.8 for agent versus 1.8 +/- 0.3 for saline group; P<0.01). In the protease group only, saline flush-modulated NIRF signal profiles further distinguished atheromata from normal segments in vivo (P<0.01). Good correlation between the in vivo and ex vivo plaque target-to-background ratio was present (r=0.82, P<0.01). Histopathological analyses demonstrated strong NIRF signal in plaques only from the protease agent group. NIRF signals colocalized with immunoreactive macrophages and the cysteine protease cathepsin B. Conclusions-An intravascular fluorescence catheter can detect cysteine protease activity in vessels the size of human coronary arteries in real time with an activatable NIRF agent. This strategy could aid in the detection of inflammation and high-risk plaques in small arteries. (Circulation. 2008; 118: 1802-1809.)

We report a technique for fluorescence tomography that operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The method uses multi-projection illumination and photon transport description in opaque tissues. We demonstrate whole-body three-dimensional visualization of the morphogenesis of GFP-expressing salivary glands and wing imaginal discs in living Drosophila melanogaster pupae in vivo and over time.

Fluorochromes have become essential reporter molecules in biological research. We show that the depth-resolved distribution of fluorochromes in small animals can be imaged with 25 fmol sensitivity and 150 Am spatial resolution by means of multispectral photoacoustic imaging. The major advantage of the multispectral approach is the sensitive differentiation of chromophores and fluorochromes of interest based on self-reference measurements, as evidenced in this study by resolving a commonly used fluorochrome (Alexa Fluor 750) in mouse. The suggested method is well suited for enhancing visualization of functional and molecular information in vivo and longitudinally. (c) 2007 Optical Society of America.