We acknowledge support from the National Cancer Institute (NCI) R01 CA128908 and Stanford Medical Scholar Research Fellowship. No other potential conflict of interest relevant to this article was reported.

1. Tumor Model
 <ol>
 <li>Culture H460 cells (American Type Culture Collection) in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen Life Technologies). It should be noted that the choices of cell lines, culture mediums, locations of inoculation, number of xenografts per mouse, and other considerations are all to be tailored to the goals of a particular study. Here we will only present one specific project design to serve as an illustration.</li>
 <li>Maintain cell lines in a humidified atmosphere of 5% CO2 at 37 &deg;C and change to fresh medium every other day.</li>
 <li>When a 75% confluent monolayer of cells is formed, detach the monolayer with trypsin and dissociate cells into a single-cell suspension for further cell culture.</li>
 <li>Suspend approximately 1 &times; 106 H460 cells in phosphate-buffered saline (PBS; Invitrogen) and implant subcutaneously in both left and right shoulders of nude mice (female athymic nude mice (nu/nu), 4 - 6 weeks old, Charles River Laboratories, Inc.). </li>
 <li>Allow tumors to grow to 150 - 200 mm3. It takes approximately 2 weeks for H460 tumor xenografts to grow to this size. Standard caliper measurement is carried out to track tumor sizes.</li>
 <li>When tumors reach the ideal size the tumor bearing mice are now ready for treatment and in vivo imaging via both PET and CLI.</li>
 </ol>
 2. PET
 <ol>
 <li>Perform the PET studies according to this schedule or any variation of it depending on the specific project (Figure 1)12. A number of factors could influence the design of the schedule, including, but not limited to, choice of tumor xenograft cell lines, anticancer drugs, and dosing regimens. Here we will only present one specific imaging schedule. The CLI studies are to be performed according to the same schedule as those of the PET studies, with CLI performed immediately after the corresponding PET. It should be also noted here that the purpose of the PET studies is mainly for validation of the CLI results. For common users who just wish to use OI instruments for imaging radiolabeled probes, no PET is necessary. However, if one does desire PET validation it should be stressed that PET and CLI instruments must be located within very close proximity for the validation to be successful because of the short half life of 18F (109.77 min).</li>
 <li>Divide mice into treatment and control groups (n &ge; 3 each). Treat mice in the treatment group with 2 injections of bevacizumab of 20 mg/kg at days 0 and 2. Day 0 is defined by the first injection. Note that at Day -1 a pre-scan should be performed via both PET and CLI.</li>
 <li>Small-animal PET of tumor-bearing mice is to be performed with an R4 rodent model scanner (Siemens Medical Solutions USA, Inc.).</li>
 <li>Anesthetize all mice with 2% isoflurane (Aerrane; Baxter) and inject with 3'-deoxy-3'-18F-fluorothymidine (18F-FLT; 7.3 - 8.0 MBq [198 - 215 &mu;Ci]) via the tail vein. The PET probe is to be diluted in PBS before injection. </li>
 <li>After 1 hr, anesthetize mice again and place anesthetized mice prone and near the center of the field of view of the small-animal PET scanner.</li>
 <li>Obtain three-minute static scans and reconstruct the images by a 2-dimensional ordered-subsets expectation maximum algorithm. Background correction is not necessary.</li>
 <li>Draw regions of interest (ROIs; 5 pixels for coronal and transaxial slices) over the tumors on the decay-corrected whole-body coronal images. Obtain the maximum counts per pixel per minute from the ROIs and convert to counts per milliliter per minute by use of a calibration constant. With the assumption of a tissue density of 1 g/ml, convert the ROIs to counts per gram per minute. Determine image ROI-derived %ID/g values by dividing counts per gram per minute by injected dose. Attenuation correction is not necessary.</li>
 </ol>
 3. CLI
 <ol>
 <li>CLI is to be performed with an IVIS Spectrum system (Caliper Life Sciences). Acquisition and analysis of images are to be carried out using Living Image 3.0 software (Caliper Life Sciences). Wavelength-resolved spectral imaging is to be performed using an 18-set narrow-band emission filter (490 - 850 nm). Again, for each mouse, perform CLI immediately after PET to minimize the amount of radioactive decay if the PET studies are included in the protocol.</li>
 <li>Place animals in a light-tight chamber under isoflurane anesthesia. Multiple mice can be placed simultaneously to increase throughput.</li>
 <li>Acquire images using 3 min exposure time (f/stop=1, binning=4). Use the same illumination settings (lamp voltage, filters, f/stop, fields of view, binning) to acquire all images. Use the dorsal skin area to calculate the signal intensity of background tissue. Normalize fluorescence emission to photons per second per centimeter squared per steradian (p/s/cm2/sr).</li>
 </ol>
 4. Representative Results
 Visual comparison between CLI and PET images can be easily carried out. After unifying the scale bar across images from the same modality and place CLI and PET images side by side one can see in this representative panel (Figure 2A) that both CLI and PET revealed significantly decreased signals from H460 xenografts in treated mice from pre-treatment to day 3, suggesting significant therapeutic effect. As a comparison, moderately increased to unchanged signals were observed in untreated mice during the same time period (data not shown). By visual inspection alone one can observe that there is a good consistence between tumor contrasts that are visualized from CLI and PET. In fact, this visual correlation has sufficient resolution to show central necrosis of the tumor secondary to the anticancer treatment regimen (please compare the CLI and PET images from Day 3). To validate the imaging findings quantifications and correlation analysis can be carried out.
 Quantifications of CLI and PET images and a simple fitting via linear regression showed that the two modalities indeed had an excellent correlation (Figure 2B, R2=0.9309 for 18F-FLT probed treatment group). Notably, in all of our CLI and PET imaging studies with different tumor models and different anticancer drugs the slopes of the fits are also remarkably close, thus suggesting an excellent fit of linear regression even of all data are conglomerated (data not shown). Both representative images are adapted from our previous publication12.
 <img src="/files/ftp_upload/4341/4341fig1.jpg" alt="Figure 1"/>
 Figure 1. Schematic of experimental design of PET and CLI studies. Tumors were implanted bilaterally in shoulder region and allowed to grow to 150-200 mm3, and tumor-bearing mice were subjected to in vivo imaging via PET and CLI at day -1, 1, and 3. Bevacizumab treatment was performed by 2 injections of 20 mg/kg at days 0 and 2.
 <img src="/files/ftp_upload/4341/4341fig2.jpg" alt="Figure 2"/>
 Figure 2. (A) In vivo CLI and PET images of mice bearing H460 xenografts treated with Bevacizumab before treatment (pre-scan) and after treatment (day 3). (B) Corresponding quantitative analysis of CLI and PET results (n=3) and their correlations. Images adapted from (6).<a href="https://www.jove.com/files/ftp_upload/4341/4341fig2large.jpg" target="_blank"> Click here to view larger figure</a>.

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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+in+cancer+research:+basic+principles,+tumor+detection,+and+therapeutic+monitoring.">Optical imaging in cancer research: basic principles, tumor detection, and therapeutic monitoring.</a> Med. Princ. Pract. 20 (5), 397 (2011).</li><li>Liu, H., Ren, G., Miao, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Optical+Imaging+with+Radioactive+Probes.">Molecular Optical Imaging with Radioactive Probes.</a> PLoS One. 5 (3), e9470 (2010).</li><li>Robertson, R., Germanos, M. 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No conflicts of interest declared.

CLI is emerging as a promising molecular imaging technique that has found potentials in many basic science research applications and even clinical use4,5,15,16,17. The major advantages of CLI over traditional nuclear imaging modalities such as PET stem from its use of OI instruments, which are easier to use, characterized by short acquisition time and high throughput, significantly less expensive, and more widely available to researchers. Additionally, what sets CLI apart from OI in general is its use of...

<table border="1">
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>H460 Cell Line</td>
 <td>American Type Culture Collection</td>
 <td>ATCC Number: HTB-177 </td>
 </tr>
 <tr>
 <td>RPMI 1640 Medium</td>
 <td>Invitrogen Life Technologies</td>
 <td>12633-012</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Invitrogen Life Technologies</td>
 <td>10091-148</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin</td>
 <td>Invitrogen Life Technologies</td>
 <td>15640-055</td>
 </tr>
 <tr>
 <td>Phosphate-Buffered Saline</td>
 <td>Invitrogen Life Technologies</td>
 <td>10010-023</td>
 </tr>
 <tr>
 <td>Female Athymic Nude Mice</td>
 <td>Charles River Laboratories, Inc.</td>
 <td>Strain Code: 088</td>
 </tr>
 <tr>
 <td>Bevacizumab (Avastin)</td>
 <td>Genentech/Roche</td>
 <td>N/A</td>
 </tr>
 <tr>
 <td>MicroPET Rodent R4</td>
 <td>Siemens Medical Solutions USA, Inc.</td>
 <td>N/A</td>
 </tr>
 <tr>
 <td>Isoflurane (Aerrane)</td>
 <td>Baxter</td>
 <td>Baxter Number: AHN3637</td>
 </tr>
 <tr>
 <td>IVIS Spectrum</td>
 <td>Caliper Life Sciences</td>
 <td>N/A</td>
 </tr>
 </table>

cerenkov luminescence imaging (cli) for cancer therapy monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Watch this Scientific Journal Video about Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring at JoVE.com

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

cerenkov-luminescence-imaging-cli-for-cancer-therapy-monitoring

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13.6K Views.  Stanford University. Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Video: Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Cancer therapies which are less toxic and invasive than their existing counterparts are highly desirable. The use of RF electric-fields that penetrate deep into the body, causing minimal toxicity, are currently being studied as a viable means of non-invasive cancer therapy. It is envisioned that the interactions of RF energy with internalized nanoparticles (NPs) can liberate heat which can then cause overheating (hyperthermia) of the cell, ultimately ending in cell necrosis.
In the case of non-biological systems, we present detailed protocols relating to quantifying the heat liberated by highly-concentrated NP colloids. For biological systems, in the case of in vitro experiments, we describe the techniques and conditions which must be adhered to in order to effectively expose cancer cells to RF energy without bulk media heating artifacts significantly obscuring the data. Finally, we give a detailed methodology for in vivo mouse models with ectopic hepatic cancer tumors.

We describe the protocols used to investigate the interactions of 13.56 MHz radiofrequency (RF) electric-fields with gold nanoparticle colloids in both non-biological and biological systems (in vitro/vivo). These interactions are being investigated for applications in cancer therapy.

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable risks for normal tissue injury and the hazards of physical tumor miss. A first-of-its-kind SBRT platform achieves high-precision ablative radiation treatment through a combination of versatile real-time imaging solutions and sophisticated tumor tracking capabilities. It uses dual-diagnostic kV x-ray units for stereoscopic open-loop feedback of cancer target intrafraction movement occurring as a consequence of respiratory motions and heartbeat. Image-guided feedback drives a gimbaled radiation accelerator (maximum 15 x 15 cm field size) capable of real-time &#177;4 cm pan-and-tilt action. Robot-driven &#177;60&#176; pivots of an integrated &#177;185&#176; rotational gantry allow for coplanar and non-coplanar accelerator beam set-up angles, ultimately permitting unique treatment degrees of freedom. State-of-the-art software aids real-time six dimensional positioning, ensuring irradiation of cancer targets with sub-millimeter accuracy (0.4 mm at isocenter). Use of these features enables treating physicians to steer radiation dose to cancer tumor targets while simultaneously reducing radiation dose to normal tissues. By adding respiration correlated computed tomography (CT) and 2-[18F] fluoro-2-deoxy-&#7429;-glucose (18F-FDG) positron emission tomography (PET) images into the planning system for enhanced tumor target contouring, the likelihood of physical tumor miss becomes substantially less1. In this article, we describe new radiation plans for the treatment of moving lung tumors.

Stereotactic ablative body radiation therapy (SBRT) involves the precise delivery of high-dose radiation to cancer tumor targets. A novel SBRT platform offers a first-of-its-kind gimbaled radiation accelerator mounted within a pivoting O-ring gantry capable of dynamic image-guided tumor tracking. Here, we describe dynamic tumor tracking for lung targets.

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...