We would like to acknowledge Nadine Henn and Arend Heerschap for their valuable assistance. This work was supported by the Netherlands Institute of Regenerative Medicine (NIRM, FES0908), the EU FP7 program ENCITE (HEALTH-F5-2008-201842) and TargetBraIn (HEALTH-F2-2012-279017), a grant from the Volkswagen Foundation (I/83 443), the Netherlands Organization for Scientific Research (VENI 700.10.409 and Vidi 917.76.363), ERC (Advanced Grant 269019), and Radboud University Nijmegen Medical Centre (AGIKO-2008-2-4).

Note: All experiments and procedures involving animals must be carried out in accordance with relevant ethical guidelines and conform with standard animal care and humane requirements.
1. Cell Labeling (Standard Protocol with Coincubation)
<ol>
	<li>Add 19F label8 to immature DCs at a concentration of 4 mg/106 cells (in 2 ml medium). Swirl gently to mix.</li>
	<li>Incubate the cells with the label for 3 days before maturing and harvesting the DCs.</li>
	<li>Collect the DCs and remove excess label by washing at least 3x in PBS. Count the cells.</li>
	<li>Resuspend in a small volume of PBS for injection or further testing.</li>
</ol>
Note: This protocol is relevant for these specific DCs and label. The procedure was optimized in another publication8 and included here are only the steps to prepare a sample to image, since the focus of this protocol is on the imaging. Hence, the protocol for isolation, culture and labeling of a specific cell type will either need to be found in the literature or optimized by the researcher. A summary of all other 19F labels that have been used in the literature is presented elsewhere6.
2. Determination of 19F/cell Using 19F NMR
<ol>
	<li>Place a known number of labeled cells (typically more than 0.5 x 106, or a number that results in sufficient signal) in a NMR tube.</li>
	<li>Add 10 &#956;l of 5% trifluoroacetic acid (TFA). If the resonance frequency of TFA is unsuitable due to overlap with the label, use an alternative soluble compound, preferably with a single 19F resonance.</li>
	<li>Place the sample in an NMR spectrometer and obtain the 19F spectrum. Ensure that the bandwidth is sufficient for both the TFA (reference) and the agent.</li>
</ol>
Note: The TR should be long enough for full relaxation of the reference and the sample spins (around 5 times T1 relaxation time of the longest T1 component in the tube, typically TR ~5-8 sec). Some spectrometers require D2O for a frequency lock signal. A standard 19F spectrum is sufficient. Extensive averaging or higher cell numbers will be required if cell uptake of the label is poor&#160;or if cell numbers are low. The spectrum should be centered between the reference and relevant label peaks, unless corrections are made to account for partial excitation.
<ol start="4">
	<li>Calculate the relative areas under the peaks of the reference and the sample (or the main peak of the sample), and then calculate the average number of 19F&#39;s/cell in the sample.</li>
</ol>
Note: The entire process must be repeated and averaged extensively enough to reach statistical significance. This can also be done using spectroscopy directly at the MRI scanner. However, note that this procedure reveals only the average 19F loading for a large number of cells, not the variability between cells. Checking uniformity of cell uptake requires the presence of a fluorescent component at the 19F agent, so that cell uptake can be analyzed using microscopy or flow cytometry.
3. Considerations for Experimental Design - Cell Injection
Due to the relatively poor sensitivity of 19F MRI for cell tracking, an animal model where the cells will localize in large numbers is necessary for successful in vivo imaging. Typical sensitivity values range from 1,000-100,000 cells/voxel/1 hr scan time6, with a cell loading in the range of 1011-1013 fluorine atoms.
The number and frequency of imaging sessions must be carefully planned, as this influences the choice of anesthetic. Note that isoflurane may not be suitable for 19F MRI if the 19F resonance frequency of isoflurane is close to that of the label compound used. isoflurane may still be suitable if the imaging sessions are short enough to prevent significant build-up. Several alternative anesthetics have been used in the literature10,11; an example is included in the imaging protocol. Anesthetics may need to be optimized for different mouse strains and disease models.
4. Design of an External 19F Reference
<ol>
	<li>Use an external reference containing a known amount of 19F signal for quantification12. Choose the signal intensity of the reference to be roughly comparable to that expected from the subject.</li>
</ol>
Note: Generally, it is simplest if the reference compound is the same as the cell label, to match the relaxation parameters. If spectroscopic sequences or sequences without spatial localization are used, then a compound with a different chemical shift may be necessary.
<ol start="2">
	<li>Modify the placement, size and shape of the reference as necessary, dependent on the region of interest (ROI). Note: It is generally easiest to use tubes with a uniform cross-section for the reference.</li>
</ol>
5. Imaging and Mouse Preparation
<ol>
	<li>Dilute commercially available ketamine and xylazine 1:10 v/v with physiological saline to yield stock solutions of 10 mg/ml ketamine and 2 mg/ml xylazine.</li>
	<li>Turn on the heating system in the scanner (typically warm air) to ensure the bore will be warm before the mouse is imaged.</li>
	<li>Inject 100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneal&#160;to initiate anesthesia. Note: These doses have been tested for CD1(ICR) and NU-Foxn1nu 8-week old male mice. Other strains may need slightly different doses, which should be optimized in a separate experiment. This dose will keep the mouse anesthetized for about 45 min. Note: The depth of anesthesia is normally sufficient if the animal shows no reaction to a foot pinch.</li>
	<li>Once anesthetized, place the animal in an animal cradle and immobilize to prevent motion during MRI. If required, position the external reference next to the animal. Both the reference and the ROI (expected location of the injected cells) should be in the center of the coil. The animal should be connected to temperature and breathing control for physiological monitoring during scanning.</li>
	<li>Cover the eyes of the mouse with sterile&#160;eye ointment to prevent drying&#160;out during long imaging sessions.</li>
	<li>If the imaging time exceeds 40 min, prepare additional catheters for injection of additional ketamine/xylazine before the animal may wake up. Position two separate subcutaneous&#160;catheters with the working solutions of ketamine and xylazine. Use this catheter to give the mouse an additional 2.5 mg of ketamine every 20 min after the first 40 min. If necessary, further prolong anesthesia by injection of additional 0.5 mg xylazine through the catheter, 100 min after the initial injection. In all cases, experimental time should not exceed 3 hr, except possibly under terminal anesthesia.</li>
	<li>Ensure that the mouse is warmed directly after the first injection and maintained at 37 &#176;C body temperature. With this protocol, &#60;80% blood oxygenation was detected under atmosphere air breathing. To prevent side effects from the lowered blood oxygenation, use 100% oxygen (0.3 L/min). Body temperature and breathing should be controlled throughout the whole experiment and until full recovery of the animal. Note that the spontaneous breathing rate under ketamine/xylazine is very high (200-250/min). Irregularities in the breathing pattern, rather than the breathing rate, can indicate that a higher dose is necessary to maintain a suitable state of anesthesia.&#160;&#160;The mouse will awaken spontaneously within 20-30 min&#160;after the last dose of anesthetic.</li>
</ol>
Note: For cell tracking, it is necessary to image the same animal more than once. In that case, the imaging sessions should be scheduled with consideration to the time necessary for clearance of anesthesia from the system and animal use guidelines of the institute.
6. Imaging
1H adjustments and imaging 
<ol>
	<li>Position the animal inside the scanner so that the ROI is in the isocenter using a low-resolution, anatomical scan with different slice orientations (a localizer).</li>
	<li>Use the regular shimming protocol on 1H, e.g. a global shim on the whole volume inside the coil.</li>
	<li>Adjust the reference pulse gain (RG), i.e. the transmitter power measured in dB for a 1 msec Hermite 90&#176; RF pulse, using the adjustment scan provided by the vendor.</li>
</ol>
Note: This adjustment will vary with the type of RF coil and is essential for 19F MRI since the signal on the 19F frequency is usually too low for direct adjustments. As a consequence, an estimate of the RG must be made from measurements on the 1H signal. For RF transmission with a surface coil, the 1H RG should be adjusted to a plane parallel to the coil through the part of interest, for a volume coil with homogenous B1 profile, the exact settings of the adjustment are less important.
<ol start="4">
	<li>The following protocol of 19F reference pulse gain adjustment works only if both 1H and 19F RF transmission is carried out with the same (single or double-tuned) RF coil. For such an imaging setup, the coil profile (B1 transmit field) on 1H should be proportional to the 19F coil profile, when a fixed RG is applied, i.e. B1,1H=C*B1,19F with a proportionality constant C.
	<ol>
		<li>To confirm that coil profiles are proportional for a specific setup, acquire a 1H and a 19F flip angle map (see following section on B1 correction) on a tube with highly concentrated 19F, e.g. TFA in water (1:1 v/v) using identical fields-of-view (FOVs), beforehand (Figure 3).</li>
		<li>Using the same RG, check that both maps are identical except for the global factor C.</li>
		<li>Once the 1H RG is determined, note this number down.</li>
	</ol>
	</li>
	<li>Carry out a conventional 1H MRI scan as an anatomical reference for the 19F imaging. Tune the system to the 19F frequency of the cell marker and carry out a 19F MR scan. Ideally the 19F MRI scan will have the same field-of-view and slice selection as the 1H MRI, although usually with lower resolution.&#160;The animal can be revived as soon as the imaging is complete. Image processing can then be carried out offline.&#160;</li>
	<li>Determine the 19F RG via the relation dB19F=dB1H-20*log10(C). Estimate the 19F agent frequency in a separate in vitro experiment.</li>
</ol>
Note: In vivo, the exact frequency can vary slightly from experiment to experiment due to different shimming conditions. To prevent chemical shift artifacts the exact frequency should therefore be determined in each experiment by 19F MRS, if possible. A typical scan for very low 19F signal would be global (pulse-acquire) spectroscopy (TR=200 msec, NEX=3,000, TA=10 min, BW=50 kHz). NEX, thus TA, can be dramatically reduced for high 19F signal. The 19F agent frequency is determined from the spectrum after Fourier transformation and phase correction. NEX refers to the number of excitations, TA is the acquisition time and BW is the bandwidth.
Note: Typical imaging parameters9 on a 11.7T/16 cm dedicated animal scanner are: An anatomic 1H imaging scan using a turbo spin echo (TSE) sequence with TR/effective echo time (TEeff) = 2,200 msec/42.8 msec, 8 echoes per excitation, NEX = 2, 10 consecutive, 1 mm thick slices, FOV = 1.92 x 1.92 cm2, 128 x 128 matrix, i.e. a resolution of 150 x 150 x 1,000 &#956;m3, TA = 1 min, BW = 50 kHz and a linear phase encoding scheme. 19F images were acquired with the same sequence and matching geometry, but at lower in-plane resolution and lower BW (NEX = 256, 48 x 48 matrix, i.e. a resolution of 400 x 400 x 1,000 &#956;m3, TA = 57 min, BW = 10 kHz).
<ol start="7">
	<li>B1 correction 
	Using an RF coil with inhomogeneous B1 profile, e.g. a surface coil, can hamper quantification of 19F data since the signal depends not only on 19F concentration but also on the distance from the coil. Acquire a B1 map on the 1H channel in order to retrospectively correct 19F data. This requires that 1H and 19F coil profiles are identical, except for the proportionality factor C, and that sufficient 1H background signal is provided in regions of 19F. An example protocol for correction of a 19F spin echo sequence is presented, using a single-tuned Tx/Rx surface coil tunable to both 1H and 19F and with proportional B1 profiles13.
	<ol>
		<li>Acquire a 1H flip angle map with the two flip angle method 14. Acquire a gradient echo scan with very long TR (&#62;3 times 1H T1) with an estimated flip angle of just below 90&#176;. Close to the coil. Acquire a second gradient echo scan with a RG=dB1H-6, i.e. twice the flip angle of the first scan.</li>
		<li>Calculate the 1H flip angle, &#945;, at voxel (x,y,z) via the signal intensities (SI) of the first and second gradient echo scans: &#945;(x,y,z)=arcos(SIGE,1(x,y,z)/2SIGE,2(x,y,z))14. The 19F flip angle is identical (except for the factor 1/C) due to proportional B1 profiles. According to Faraday&#39;s principle of reciprocity15, the attenuation of 19F signal intensity from a spin echo sequence (excitation flip angle &#945;, refocusing flip angle 2&#945;) during signal reception is attRx(x,y,z)~&#945;(x,y,z). The attenuation due to imperfect excitation is attTx(x,y,z)~sin3(&#945;(x,y,z))16. The overall attenuation is written as att=attRx*attTx= &#945;*sin3(&#945;)/90&#176;. Note that it is normalized to 1 in case of perfect 90&#176;/180&#176; excitation/refocusing flip angle. The B1 corrected 19F image signal intensities SI19F,corr are calculated via SI19F,corr(x,y,z)= SI19F(x,y,z)/att(x,y,z).</li>
		<li>In order to compare signal intensities from different experiments, place a reference tube with defined 19F concentration in the field of view, and normalize the SI19F,corr to the mean signal in the reference.</li>
	</ol>
	</li>
</ol>
Note: Other 1H B1/flip angle mapping methods can be used and different 19F pulse sequences require modifications of attTx,19F. Any kind of B1 correction scheme will introduce additional error in the cell quantification procedure. If accurate cell quantification is the main premise, an RF coil with homogenous B1 profile can be used to circumvent this part of the protocol.
7. Image Processing
<ol>
	<li>Making 1H and 19F overlay images
	<ol>
		<li>Export the image data for the final 1H and 19F magnitude images from the scanner.</li>
		<li>Open the files in an image processing program, such as ImageJ (freeware, NIH).</li>
		<li>Carry out image adjustments as necessary (cropping, brightness, contrast) keeping the adjustments the same for all images. The 19F images will typically need to be upscaled to a higher resolution to match the corresponding 1H images.</li>
		<li>Render the 19F images in false color (select a different look-up table, under the &#34;image&#34; menu in Image J) and then overlay on the corresponding 1H images in order to localize the signal.</li>
	</ol>
	</li>
	<li>19F Quantification
	<ol>
		<li>Select a 19F image (magnitude image) with the relevant cells and the reference visible.</li>
		<li>In a program such as Image J, draw ROIs over the relevant cells, the reference and a region outside the subject (noise).</li>
		<li>Use the command Analyze and then Measure (or simply Ctrl+M) to calculate the mean pixel intensity within these regions, and their area. Let S(cells) refer to the mean pixel intensity in the ROI over the cells. Multiply that by the volume (= area x slice thickness) to calculate the total signal.</li>
		<li>Calculate the SNR, for example using the formula SNR = 0.65 x S(cells) / &#963;, where &#963; is the standard deviation of pixel intensity in an ROI outside the sample which is free of any chemical shift artifact (typically in the background air)17. If the SNR is under 5, a correction for the signal due to the noise may be necessary and can be carried out as described elsewhere11 10.</li>
		<li>Otherwise, calculate the total amount of 19F responsible for the signal in the ROI over the reference by multiplying the area of the reference in the slice by slice thickness (to calculate the volume) by the concentration of 19F in the reference, which is known and assumed to be homogeneous.</li>
		<li>Multiply that value by the total signal in the ROI over the cells, divided by the total signal over the reference to calculate the amount of 19F in the cells.</li>
		<li>Calculate the number of cells by dividing that number by the value for 19F/cell, which was calculated in section 2.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interests to disclose.

The protocol outlined here describes the general procedure for in vivo 19F MRI cell tracking. Despite the described co-incubation method, there are several different protocols for labeling cells with a 19F agent. However, the co-incubation is most often used and can be further optimized e.g. by addition of transfection agents6. The actual cell labeling protocol will depend on the cell type. Only key labeling steps are described in the protocol here. Generally, the label ...

In vivo cell tracking is essential for the optimization and monitoring of cellular therapeutics1. Due to its noninvasive nature, imaging offers excellent opportunities to monitor cells in vivo. Magnetic Resonance Imaging (MRI) is independent of ionizing radiation and allows a superior imaging resolution and intrinsic soft tissue contrast. MRI-based cell tracking has already been used clinically to follow dendritic cells in melanoma patients2. Conventional clinical MRI is carried out on the 1H nucleus, present in mobile water in tissues. It is also possible to carry out MRI on other active nuclei, such as 13C, 19F and 23Na. However, only 19F MRI has been applied successfully to in vivo cell tracking as it offers the highest sensitivity after 1H. The absence of MRI-detectable endogenous 19F in tissues permits high signal selectivity for the detection of exogenous 19F contrast agents and allows quantification of fluorine concentration directly from the image data. For a detailed discussion on 19F MRI, see3-5. A key issue with 19F MRI is the need to develop and optimize suitable 19F cell labels, although several labels have been developed, with a trend towards multimodal agents6.
The protocol we describe here is based on studies by our groups7-9, including the first articles that described in vivo quantitative 19F MRI-based cell tracking10,11. The general procedure of cell tracking using 19F MRI is summarized in Figure 1. We describe a general protocol for labeling and imaging of dendritic cells (DCs) using a custom-made perfluorocarbon contrast agent8. The imaging protocol is generally applicable to different cell types, labels and animal models. The cell type and animal model described here should only be taken as an example, and thus we do not provide details on the cell isolation and labeling, but rather focus on the imaging protocol. Modifications will be necessary for each label, cell type, animal model, and imaging setup, and these can be found in the literature or may need to be optimized by the researchers. Some common modifications are included in the discussion.

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>MFCD00131855</td><td></td></tr><tr><td>TFA</td><td>Sigma-Aldrich</td><td>76-05-1</td><td></td></tr><tr><td>Ketamine (Ketavet)</td><td>Pfizer</td><td>778-551</td><td></td></tr><tr><td>Xylazine (Rompun)</td><td>Bayer</td><td>QN05 cm92</td><td></td></tr><tr><td>Ophtosan </td><td>Produlab Pharma</td><td>2702</td><td>eye ointment</td></tr><tr><td colspan="4">&lt;strong&gt;Material name&lt;/strong&gt; </td></tr><tr><td>MRI scanner</td><td>Bruker Biospec</td><td></td><td></td></tr><tr><td>NMR spectrometer</td><td>Bruker Biospec</td><td></td><td></td></tr></tbody></table>

in vivo 19f mri for cell tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

Here we show the typical results for a protocol involving the transfer of 19F-labeled cells homing to a draining lymph node. Figure 2 shows a 19F NMR spectrum of 106 labeled cells using a TFA reference. The imaging setup was carried out as described in the protocol (Figure 3). For the in vivo imaging, we used a reference consisting of a sealed cylinder of the same label used for the cells placed between the feet of the mouse. A representative pro...

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In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

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14.6K Views.  Radboud University Medical Center. We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

Video: In vivo 19F MRI for Cell Tracking

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Cancer Research

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Immunology and Infection

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Neuroscience

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

Biology

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

In vivo ¹⁹F MRI for Cell Tracking

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Chapters in this video

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Monitoring Dendritic Cell Migration using ¹⁹F / ¹H Magnetic Resonance Imaging

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Tracking Mouse Bone Marrow Monocytes In Vivo

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur