Large stokes shift and near-infrared fluorescent probe for bioimaging and evaluating the HClO in an rheumatoid arthritis mouse model.

It is crucial to identify aberrant HClO levels in living things since they pose a major health risk and are a frequent reactive oxygen species (ROS) in living organisms. In order to detect HClO in various biological systems, we created and synthesized a near-infrared fluorescent probe with an oxime group (-C = N-OH) as a recognition unit. The probe DCMP1 has the advantages of fast response (10 min), near-infrared emission (660 nm), large Stokes shift (170 nm) and high selectivity. This probe DCMP1 not only detects endogenous HClO in living cells, but also enables further fluorescence detection of HClO in living zebrafish. More importantly, it can also be used for fluorescence imaging of HClO in an rheumatoid arthritis mouse model. This fluorescent probe DCMP1 is anticipated to be an effective tool for researching HClO.

Light-Sheet Microscopic Imaging of Whole-Mouse Vascular Network with Fluorescent Microsphere Perfusion.

Visualizing the whole vascular network system is crucial for understanding the pathogenesis of specific diseases and devising targeted therapeutic interventions. Although the combination of light sheet microscopy and tissue-clearing methods has emerged as a promising approach for investigating the blood vascular network, leveraging the spatial resolution down to the capillary level and the ability to image centimeter-scale samples remains difficult. Especially, as the resolution improves, the issue of photobleaching outside the field of view poses a challenge to image the whole vascular network of adult mice at capillary resolution. Here, we devise a fluorescent microsphere vascular perfusion method to enable labeling of the whole vascular network in adult mice, which overcomes the photobleaching limit during the imaging of large samples. Moreover, by combining the utilization of a large-scale light-sheet microscope and tissue clearing protocols for whole-mouse samples, we achieve the capillary-level imaging resolution (3.2 × 3.2 × 6.5 µm) of the whole vascular network with dimensions of 45 × 15 × 82 mm in adult mice. This method thus holds great potential to deliver mesoscopic resolution images of various tissue organs for whole-animal imaging.

Biodistribution Assessment of a Novel 68Ga-Labeled Radiopharmaceutical in a Cancer Overexpressing CCK2R Mouse Model: Conventional and Radiomics Methods for Analysis.

The aim of the present study consists of the evaluation of the biodistribution of a novel 68Ga-labeled radiopharmaceutical, [68Ga]Ga-NODAGA-Z360, injected into Balb/c nude mice through histopathological analysis on bioptic samples and radiomics analysis of positron emission tomography/computed tomography (PET/CT) images. The 68Ga-labeled radiopharmaceutical was designed to specifically bind to the cholecystokinin receptor (CCK2R). This receptor, naturally present in healthy tissues such as the stomach, is a biomarker for numerous tumors when overexpressed. In this experiment, Balb/c nude mice were xenografted with a human epidermoid carcinoma A431 cell line (A431 WT) and overexpressing CCK2R (A431 CCK2R+), while controls received a wild-type cell line. PET images were processed, segmented after atlas-based co-registration and, consequently, 112 radiomics features were extracted for each investigated organ / tissue. To confirm the histopathology at the tissue level and correlate it with the degree of PET uptake, the studies were supported by digital pathology. As a result of the analyses, the differences in radiomics features in different body districts confirmed the correct targeting of the radiopharmaceutical. In preclinical imaging, the methodology confirms the importance of a decision-support system based on artificial intelligence algorithms for the assessment of radiopharmaceutical biodistribution.

Development of an Add-on 23Na-MRI Radiofrequency Platform for a 1H-MRI System Using a Crossband Repeater: Proof-of-concept.

23Na-MRI provides information on Na+ content, and its application in the medical field has been highly anticipated. However, for existing clinical 1H-MRI systems, its implementation requires an additional broadband RF transmitter, dedicated transceivers, and RF coils for Na+ imaging. However, a standard medical MRI system cannot often be modified to perform 23Na imaging. We have developed an add-on crossband RF repeater system that enables 23Na-MRI simply by inserting it into the magnet bore of an existing 1H MRI. The three axis gradient fields controlled by the 1H-MRI system were directly used for 23Na imaging without any deformation. A crossband repeater is a common technique used for amateur radio. This concept was proven by a saline solution phantom and in vivo mouse experiments. This add-on RF platform is applicable to medical 1H MRI systems and can enhance the application of 23Na-MRI in clinical usage.

A New Preclinical Decision Support System Based on PET Radiomics: A Preliminary Study on the Evaluation of an Innovative 64Cu-Labeled Chelator in Mouse Models.

The 64Cu-labeled chelator was analyzed in vivo by positron emission tomography (PET) imaging to evaluate its biodistribution in a murine model at different acquisition times. For this purpose, nine 6-week-old female Balb/C nude strain mice underwent micro-PET imaging at three different time points after 64Cu-labeled chelator injection. Specifically, the mice were divided into group 1 (acquisition 1 h after [64Cu] chelator administration, n = 3 mice), group 2 (acquisition 4 h after [64Cu]chelator administration, n = 3 mice), and group 3 (acquisition 24 h after [64Cu] chelator administration, n = 3 mice). Successively, all PET studies were segmented by means of registration with a standard template space (3D whole-body Digimouse atlas), and 108 radiomics features were extracted from seven organs (namely, heart, bladder, stomach, liver, spleen, kidney, and lung) to investigate possible changes over time in [64Cu]chelator biodistribution. The one-way analysis of variance and post hoc Tukey Honestly Significant Difference test revealed that, while heart, stomach, spleen, kidney, and lung districts showed a very low percentage of radiomics features with significant variations (p-value < 0.05) among the three groups of mice, a large number of features (greater than 60% and 50%, respectively) that varied significantly between groups were observed in bladder and liver, indicating a different in vivo uptake of the 64Cu-labeled chelator over time. The proposed methodology may improve the method of calculating the [64Cu]chelator biodistribution and open the way towards a decision support system in the field of new radiopharmaceuticals used in preclinical imaging trials.

Multiple-mouse magnetic resonance imaging with cryogenic radiofrequency probes for evaluation of brain development.

Multiple-mouse magnetic resonance imaging (MRI) increases scan throughput by imaging several mice simultaneously in the same magnet bore, enabling multiple images to be obtained in the same time as a single scan. This increase in throughput enables larger studies than otherwise feasible and is particularly advantageous in longitudinal study designs where frequent imaging time points result in high demand for MRI resources. Cryogenically-cooled radiofrequency probes (CryoProbes) have been demonstrated to have significant signal-to-noise ratio benefits over comparable room temperature coils for in vivo mouse imaging. In this work, we demonstrate implementation of a multiple-mouse MRI system using CryoProbes, achieved by mounting four such coils in a 30-cm, 7-Tesla magnet bore. The approach is demonstrated for longitudinal quantification of brain structure from infancy to early adulthood in a mouse model of Sanfilippo syndrome (mucopolysaccharidosis type III), generated by knockout of the Hgsnat gene. We find that Hgsnat-/- mice have regionally increased growth rates compared to Hgsnat+/+ mice in a number of brain regions, notably including the ventricles, amygdala and superior colliculus. A strong sex dependence was also noted, with the lateral ventricle volume growing at an accelerated rate in males, but several structures in the brain parenchyma growing faster in females. This approach is broadly applicable to other mouse models of human disease and the increased throughput may be particularly beneficial in studying mouse models of neurodevelopmental disorders.

Movement correction method for laser speckle contrast imaging of cerebral blood flow in cranial windows in rodents.

Laser speckle contrast imaging (LSCI) is used in clinical research to dynamically image blood flow. One drawback is its susceptibility to movement artifacts. We demonstrate a new, simple method to correct motion artifacts in LSCI signals measured in awake mice with cranial windows during sensory stimulation. The principle is to identify a region in the image in which speckle contrast (SC) is independent of blood flow and only varies with animal movement, then to regress out this signal from the data. We show that (1) the regressed signal correlates well with mouse head movement, (2) the corrected signal correlates better with independently measured blood volume and (3) it has a (59 ± 6)% higher signal-to-noise ratio. Compared to three alternative correction methods, ours has the best performance. Regressing out flow-independent global variations in SC is a simple and accessible way to improve the quality of LSCI measurements.

Development of a new advanced animal cradle for small animal multiple imaging modalities: acquisition and evaluation of high-throughput multiple-mouse imaging.

The physiological conditions of small animals are an essential component to be considered when acquiring images for pre-clinical studies, and they play a vital role in the overall results of a study. However, several previous studies did not consider these conditions. In this study, a new animal cradle that can be modified and adjusted to suit multiple imaging modalities such as positron emission tomography (PET)/computed tomography (CT) and magnetic resonance imaging (MRI) was developed. Unlike previous cradles where only one mouse can be imaged at a time, a total of four mice can be imaged simultaneously using this new cradle. Additionally, fusion images with high-throughput multiple-mouse imaging (MMI) of PET/MRI and PET/CT images can be acquired using this newly developed cradle. The dynamic brain images were also acquired simultaneously by applying PET dynamic imaging technology to high-throughput MMI methods. The results of this study suggest that the newly developed small animal cradle can be widely used in pre-clinical studies.

Design and 3D-printing of MRI-compatible cradle for imaging mouse tumors.

BACKGROUND: The ability of 3D printing using plastics and resins that are magnetic resonance imaging (MRI) compatible provides opportunities to tailor design features to specific imaging needs. In this study an MRI compatible cradle was designed to fit the need for repeatable serial images of mice within a mouse specific low field MRI. METHODS: Several designs were reviewed which resulted in an open style stereotaxic cradle to fit within specific bore tolerances and allow maximum flexibility with interchangeable radiofrequency (RF) coils. CAD drawings were generated, cradle was printed and tested with phantom material and animals. Images were analyzed for quality and optimized using the new cradle. Testing with multiple phantoms was done to affirm that material choice did not create unwanted image artifact and to optimize imaging parameters. Once phantom testing was satisfied, mouse imaging began. RESULTS: The 3D printed cradle fit instrument tolerances, accommodated multiple coil configurations and physiological monitoring equipment, and allowed for improved image quality and reproducibility while also reducing overall imaging time and animal safety. CONCLUSIONS: The generation of a 3D printed stereotaxic cradle was a low-cost option which functioned well for our laboratory.

Micro-endoscopy for Live Small Animal Fluorescent Imaging.

Intravital microscopy has emerged as a powerful technique for the fluorescent visualization of cellular- and subcellular-level biological processes in vivo. However, the size of objective lenses used in standard microscopes currently makes it difficult to access internal organs with minimal invasiveness in small animal models, such as mice. Here we describe front- and side-view designs for small-diameter endoscopes based on gradient-index lenses, their construction, their integration into laser scanning confocal microscopy platforms, and their applications for in vivo imaging of fluorescent cells and microvasculature in various organs, including the kidney, bladder, heart, brain, and gastrointestinal tracts, with a focus on the new techniques developed for each imaging application. The combination of novel fluorescence techniques with these powerful imaging methods promises to continue providing novel insights into a variety of diseases.

Live Intravital Intestine with Blood Flow Visualization in Neonatal Mice Using Two-photon Laser Scanning Microscopy.

This protocol describes a novel technique to investigate the microcirculation dynamics underlying the pathology in the small intestine of neonatal mice using two-photon laser-scanning microscopy (TPLSM). Recent technological advances in multi-photon microscopy allow intravital analysis of different organs such as the liver, brain and intestine. Despite these advances, live visualization and analysis of the small intestine in neonatal rodents remain technically challenging. We herein provide a detailed description of a novel method to capture high resolution and stable images of the small intestine in neonatal mice as early as postnatal day 0. This imaging technique allows a comprehensive understanding of the development and blood flow dynamics in small intestine microcirculation.

Multifunctional Near-Infrared Fluorescent Probes with Different Ring-Structure Trigger Groups for Cell Health Monitoring and In Vivo Esterase Activity Detection.

A series of multifunctional ratiometric near-infrared fluorescent probes (CYOH-3, CYOH-4, CYOH-5, and CYOH-6) for esterase detection are designed by gradually changing the deflection of the plane twist in the molecule. These probes are composed of different ring-structure trigger groups (from three-membered ring to six-membered ring) and the same luminescent group CYOH. These probes show maximum absorption at ∼585 nm and a fluorescence emission peak at ∼655 ± 5 nm. In the presence of esterase, the probes were hydrolyzed to expose the fluorophore CYOH (λabs = 690 nm, λem = 710 ± 5 nm), thus exhibiting ratiometric near-infrared fluorescence. The probe CYOH-6 has lower plane deflection angle and better ratiometric (R = I710±5nm/I657±4nm) fluorescence properties than probes CYOH-3, CYOH-4, and CYOH-5. CYOH-6 (six-membered ring) has been successfully used to target esterase in mitochondria and distinguish between dead cells (esterase inactivation) and live cells. In addition, CYOH-6 has been well used for monitoring of esterase activity in zebrafish and mice, which proves that these probes have good prospects for clinical biomedical applications.

Quantification of Mouse Renal Perfusion Using Arterial Spin Labeled MRI at 1 T.

RATIONALE AND OBJECTIVES: Quantitative measurement of renal perfusion in murine models provides important information on the organ physiology and disease states. The 1-T desktop magnetic resonance imaging has a small footprint and a self-contained fringe field. This resultant flexibility in siting makes the system ideal for preclinical imaging research. Our objective was to evaluate the capability of the 1-T desktop magnetic resonance imaging to measure mouse renal perfusion without the administration of exogenous contrast agents. MATERIALS AND METHODS: We implemented a flow-sensitive alternating inversion recovery (FAIR)-based arterial spin labeling sequence with a mouse volume coil on a 1-T desktop magnetic resonance scanner. The validity of the implementation was tested by comparing obtained renal perfusion results with literature values for normal mice and challenging the technique with mice treated with furosemide, a blood vessel vasoconstrictor drug. RESULTS: The measured cortical and medullary perfusions were quantified to be 402 ± 95 and 184 ± 52 mL/100 g/min, respectively, in agreement with literature values. The ratio of cortical to medullary renal blood flow was between 2 and 3 and was independent of the mouse weight. As expected, upon furosemide injection, a decrease (~50%) in cortical perfusion was observed in the mice population, at 1 hour post injection compared to baseline (P < 0.0001), which returned to baseline after 24 hours (P = 0.68). CONCLUSIONS: We reported the successful application of FAIR-based arterial spin labeling for noncontrast perfusion measurement of mouse kidneys using a 1-T desktop scanner. The easy implementation of FAIR sequence on a 1-T desktop scanner offers the potential for longitudinal perfusion studies in limited access areas such as behind the barrier in mouse facilities and in multimodality preclinical imaging laboratories without the administration of exogenous contrast agents.

The importance of imaging strategies for pre-clinical and clinical in vivo distribution of oncolytic viruses.

Oncolytic viruses (OVs) are an emergent and unique therapy for cancer patients. Similar to chemo- and radiation therapy, OV can lyse (kill) cancer cell directly. In general, the advantages of OVs over other treatments are primarily: a higher safety profile (as shown by less adverse effects), ability to replicate, transgene(s) delivery, and stimulation of a host's immune system against cancer. The latter has prompted successful use of OVs with other immunotherapeutic strategies in a synergistic manner. In spite of extended testing in pre-clinical and clinical setting, using biologically derived therapeutics like virus always raises potential concerns about safety (replication at non-intended locations) and bio-availability of the product. Recent advent in in vivo imaging techniques dramatically improves the convenience of use, quality of pictures, and amount of information acquired. Easy assessing of safety/localization of the biotherapeutics like OVs became a new potential weapon in the physician's arsenal to improve treatment outcome. Given that OVs are typically replicating, in vivo imaging can also track virus replication and persistence as well as precisely mapping tumor tissues presence. This review discusses the importance of imaging in vivo in evaluating OV efficacy, as well as currently available tools and techniques.

Correlation between positron emission tomography and Cerenkov luminescence imaging in vivo and ex vivo using 64Cu-labeled antibodies in a neuroblastoma mouse model.

Antibody-based therapies gain momentum in clinical therapy, thus the need for accurate imaging modalities with respect to target identification and therapy monitoring are of increasing relevance. Cerenkov luminescence imaging (CLI) are a novel method detecting charged particles emitted during radioactive decay with optical imaging. Here, we compare Position Emission Tomography (PET) with CLI in a multimodal imaging study aiming at the fast and efficient screening of monoclonal antibodies (mAb) designated for targeting of the neuroblastoma-characteristic epitope disialoganglioside GD2. Neuroblastoma-bearing SHO mice were injected with a 64Cu-labeled GD2-specific mAb. The tumor uptake was imaged 3 h, 24 h and 48 h after tracer injection with both, PET and CLI, and was compared to the accumulation in GD2-negative control tumors (human embryonic kidney, HEK-293). In addition to an in vivo PET/CLI-correlation over time, we also demonstrate linear correlations of CLI- and γ-counter-based biodistribution analysis. CLI with its comparably short acquisition time can thus be used as an attractive one-stop-shop modality for the longitudinal monitoring of antibody-based tumor targeting and ex vivo biodistribution.These findings suggest CLI as a reliable alternative for PET and biodistribution studies with respect to fast and high-throughput screenings in subcutaneous tumors traced with radiolabeled antibodies. However, in contrast to PET, CLI is not limited to positron-emitting isotopes and can therefore also be used for the visualization of mAb labeled with therapeutic isotopes like electron emitters.

Three-Dimensional Optoacoustic and Laser-Induced Ultrasound Tomography System for Preclinical Research in Mice: Design and Phantom Validation.

In this work, we introduce a novel three-dimensional imaging system for in vivo high-resolution anatomical and functional whole-body visualization of small animal models developed for preclinical and other type of biomedical research. The system (LOUIS-3DM) combines a multiwavelength optoacoustic tomography (OAT) and laser-induced ultrasound tomography (LUT) to obtain coregistered maps of tissue optical absorption and speed of sound, displayed within the skin outline of the studied animal. The most promising applications of the LOUIS-3DM include 3D angiography, cancer research, and longitudinal studies of biological distributions of optoacoustic contrast agents.

Hepatic fat during fasting and refeeding by MRI fat quantification.

PURPOSE: To explore the sensitivity of high-field small animal magnetic resonance imaging to dynamic changes in fat content in the liver and to characterize the effect of prandial state on imaging studies of hepatic fat. MATERIALS AND METHODS: A total of three timepoints were acquired using asymmetric spin-echo acquisitions for 12 mice with 24-hour spacing. After the first scan, half of the cohort was placed on a water-only diet. The second half of the cohort continued to have access to their high-fat chow. The scans were repeated after 24 hours. All animals were then returned to the high-fat diet, and the scans were again repeated after 24 hours. Fat fraction maps were computed using previously described methods. Regions of interests were manually drawn in the livers and the patterns of the two groups over time were compared. RESULTS: Five out of six of the animals in the starved group showed an increase in hepatic fat fraction during the fasting period (average increase 0.54 ± 0.48), which decreased on refeeding. Analysis of variance indicated that the results significantly depended on both the group and the timepoint (P = 0.003). CONCLUSION: Fat-water imaging methods are able to measure hepatic fat changes caused by short-term dietary perturbations.

Performing label-fusion-based segmentation using multiple automatically generated templates.

Classically, model-based segmentation procedures match magnetic resonance imaging (MRI) volumes to an expertly labeled atlas using nonlinear registration. The accuracy of these techniques are limited due to atlas biases, misregistration, and resampling error. Multi-atlas-based approaches are used as a remedy and involve matching each subject to a number of manually labeled templates. This approach yields numerous independent segmentations that are fused using a voxel-by-voxel label-voting procedure. In this article, we demonstrate how the multi-atlas approach can be extended to work with input atlases that are unique and extremely time consuming to construct by generating a library of multiple automatically generated templates of different brains (MAGeT Brain). We demonstrate the efficacy of our method for the mouse and human using two different nonlinear registration algorithms (ANIMAL and ANTs). The input atlases consist a high-resolution mouse brain atlas and an atlas of the human basal ganglia and thalamus derived from serial histological data. MAGeT Brain segmentation improves the identification of the mouse anterior commissure (mean Dice Kappa values (κ = 0.801), but may be encountering a ceiling effect for hippocampal segmentations. Applying MAGeT Brain to human subcortical structures improves segmentation accuracy for all structures compared to regular model-based techniques (κ = 0.845, 0.752, and 0.861 for the striatum, globus pallidus, and thalamus, respectively). Experiments performed with three manually derived input templates suggest that MAGeT Brain can approach or exceed the accuracy of multi-atlas label-fusion segmentation (κ = 0.894, 0.815, and 0.895 for the striatum, globus pallidus, and thalamus, respectively).

Micro-ultrasound for preclinical imaging.

Over the past decade, non-invasive preclinical imaging has emerged as an important tool to facilitate biomedical discovery. Not only have the markets for these tools accelerated, but the numbers of peer-reviewed papers in which imaging end points and biomarkers have been used have grown dramatically. High frequency 'micro-ultrasound' has steadily evolved in the post-genomic era as a rapid, comparatively inexpensive imaging tool for studying normal development and models of human disease in small animals. One of the fundamental barriers to this development was the technological hurdle associated with high-frequency array transducers. Recently, new approaches have enabled the upper limits of linear and phased arrays to be pushed from about 20 to over 50 MHz enabling a broad range of new applications. The innovations leading to the new transducer technology and scanner architecture are reviewed. Applications of preclinical micro-ultrasound are explored for developmental biology, cancer, and cardiovascular disease. With respect to the future, the latest developments in high-frequency ultrasound imaging are described.