Fluorescence Molecular Tomography: An Imaging Technique for In Vivo Imaging of Fluorescent Protein-tagged Glioblastoma Xenografts in Mouse Model

Protokoll

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1.FMT Imaging

NOTE: According to the experimental aim, the iRFP-tagged glioblastoma cells can be monitored in vivo before, during, or after treatment. For imaging purpose, anesthetize animals using an isoflurane induction chamber, maintain in an imaging cassette during the scanning, and image in the docking station of the FMT imager.

1. Remove the animal from the cage and place in the isoflurane induction chamber. Close the chamber and turn on oxygen flow and vaporizer to 3–5%.
2. Monitor the animal until it is completely anesthetized, usually within 1–2 min. Unconscious animals should not respond to external stimuli.
3. Remove the anesthetized animal from the chamber and put the animal in the imaging cassette by placing the head adaptor first, followed by placing the animal facing down. Make sure that the head is located in the center of the cassette.
4. Place the top plate of the cassette on top and tighten the adjustment knobs until 17 mm.
5. Once the animal is secure in the imaging cassette, proceed to imaging by inserting the cassette into the internal docking station of the FMT imager, where the isoflurane is pumped to keep the animal anesthetized.
6. Run the imager and analyzer software by double-clicking the software icon.
7. In the "Experimental tab" window, select the "Database" and "Study group".
8. Go to the "Scan tab" window and select the subject to image by clicking "select subject". Also, select the laser channel in the "Laser channel" panel. For the FP635-labeled cells, select "laser channel 635 nm", and for FP720-labeled cells, select "laser channel 680 nm".
9. Acquire an image by clicking the "Capture" option in the "Scan tab" window. Then, adjust the scan field by clicking and dragging the scan field in the captured image to a determined number of source locations, usually within 20–25 sources for the ipsilateral side.
10. Check the "Add to reconstruction queue" option and hit "Scan" in the "Scan tab" window.
11. Wait until the scanning is completed and then remove the imaging cassette from the docking station.
12. Remove the top plate of the cassette and place the animal back into the cage.
13. Repeat steps 1.1–1.12 for the rest of the animals.

2. Image Analysis

1. From the imager and analyzer software, select the "Analysis tab" window.
2. Load the dataset and the subject to analyze by clicking the "+" button in the "Dataset selection" panel of the "Analysis tab" window.
3. Once the image has been loaded in the "Analysis tab" window, perform the region of interest (ROI) analysis by selecting the ellipsoid icon, located in the upper left corner of the "Analysis tab" window.
4. Adjust the threshold to zero in the "statistic data" panel by right clicking the "threshold" column in the "Analysis tab" window.
5. The total value in the "Statistic data" panel represents the signal from the fluorescent-tagged glioblastoma cells implanted in the mouse brain.
6. Repeat steps 2.2–2.4 for the rest of the animals. Load or remove a new subject by clicking the "+" or "−" button, respectively, in the "Dataset selection" panel of the "Analysis tab" window.

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Materialien

Name	Company	Catalog Number	Comments
Fluorescence Tomography System	PerkinElmer	FMT 2500 XL
TrueQuant Imaging Software	Perkin Elmer	7005319
Athymic nude mice	Charles River Laboratories	Strain Code 490 (Homozygous)	Prior approval by the Institutional Animal Care Program and by the Institutional Biosafety Committee required.