Fluorescence Molecular Tomography: An Imaging Technique for In Vivo Imaging of Fluorescent Protein-tagged Glioblastoma Xenografts in Mouse Model
JoVE Encyclopedia of Experiments
Cancer Research
0 views • 3:09 min • July 8th, 2025
Fluorescence molecular tomography or FMT is an imaging technique that helps determine the distribution of fluorescent proteins present in tumors. To perform FMT, begin by taking an anesthetized mouse. This mouse model is engineered to express near-infrared fluorescent proteins in the glioblastoma cells - the cancerous cells of the brain.
Now, place the mouse in the prone position in the center of an imaging cassette. Fasten the cassette lid and insert it into the docking station of an FMT imager. Once inside, identify an appropriate tumor area containing labeled glioblastoma cells. The near-infrared light penetrates deeply through the brain tissue reaching the target cells.
These light waves scan the entire tumor area at multiple points. Consequently, the fluorescent proteins within the glioblastoma cells produce fluorescent emission signals. These signals are then detected by the transillumination system from above, which generates high-quality 2D images from various locations within the selected tumor area.
Finally, use the software algorithms to combine these images and reconstruct a 3D-tomographic model that indicates tumor localization in the brain. After scanning, remove the mouse from the imaging cassette and allow it to recover.
To image the injected cells, place the anesthetized recipient animal in the imaging cassette of a fluorescence molecular tomography imager - head adapter first in the prone position, with the head in the center of the cassette. With the cassette closed, tighten the adjustment knobs to 17 millimeters.
When the animal is secure, insert the cassette into the internal docking station and open the imager and analyzer software. In the "Experimental tab" window, select the appropriate "Database" and "Study group" and open the "Scan tab" window. Click "Select subject" to select the subject to image and select the laser channel in the "Laser channel" panel.
Click "Capture" to acquire an image, and click and drag the scan field in the captured image to identify the source locations. Check the "Add to reconstruction queue" option and click "Scan" in the "Scan tab" window. When the scanning is complete, remove the imaging cassette from the docking station and return the animal to its cage, with monitoring, until full recovery.
To analyze the images in the imager and analyzer software, open the "Analysis tab" window and click the "+" sign button in the "Dataset selection" panel to load the dataset and subject for analysis. Then, use the ellipsoid icon to select the region of interest and right-click the "Threshold" column to adjust the threshold to 0 in the "Statistic data" panel.
Overview
Fluorescence molecular tomography (FMT) is an advanced imaging technique used to visualize the distribution of fluorescent proteins in tumors, particularly glioblastoma. This method involves the use of near-infrared light to penetrate brain tissue, allowing for the detection and imaging of cancerous cells.
Key Study Components
Area of Science
- Neuroscience
- Imaging Techniques
- Oncology
Background
- FMT enables the visualization of fluorescent proteins in live subjects.
- This technique is particularly useful for studying tumor biology.
- Near-infrared light is effective in penetrating biological tissues.
- Mouse models are commonly used for glioblastoma research.
Purpose of Study
- To demonstrate the use of FMT in imaging glioblastoma tumors.
- To provide a detailed protocol for conducting FMT.
- To highlight the advantages of using fluorescent proteins for tumor localization.
Methods Used
- Preparation of anesthetized mouse models expressing fluorescent proteins.
- Placement of the mouse in an imaging cassette for FMT.
- Use of near-infrared light to scan tumor areas.
- Image reconstruction using software algorithms to create 3D models.
Main Results
- Successful imaging of glioblastoma cells using FMT.
- High-quality 2D images generated from multiple scanning points.
- 3D-tomographic models accurately indicating tumor localization.
- Effective recovery of the animal post-imaging.
Conclusions
- FMT is a valuable tool for studying tumor dynamics in vivo.
- The technique enhances understanding of glioblastoma biology.
- Future applications may include monitoring treatment responses.
Frequently Asked Questions
Executive Industry Relevance
Fluorescence molecular tomography (FMT) enables non-invasive, longitudinal monitoring of tumor burden in preclinical glioblastoma models, supporting target validation and therapeutic response assessment. By providing quantitative 3D localization of fluorescently tagged cancer cells, FMT enhances predictive confidence in early discovery and reduces biological de-risking timelines. This imaging modality informs go/no-go decisions by delivering reproducible, spatially resolved data on tumor dynamics in vivo.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by visualizing fluorescent protein-tagged glioblastoma xenografts in vivo.
- Operational Value: Supports biological de-risking through longitudinal tracking of tumor localization and growth patterns.
- Predictive Value: Generates quantitative 3D tomographic outputs that inform target confidence and portfolio triage.
Screening & Assay Development
- Assay Readiness: Prepares validated biological systems for downstream compound evaluation via standardized tumor imaging.
- Reproducibility: Delivers consistent 2D emission signals from multiple scan points, enabling reliable data capture.
- Scalability: Facilitates platform reuse across studies through defined imaging cassette positioning and laser channel selection.
Translational & Preclinical Research
- Disease Relevance: Models human glioblastoma biology using NIR fluorescent protein-tagged xenografts in mouse brains.
- Translational Continuity: Bridges discovery and preclinical validation by monitoring tumor response to anti-tumor therapeutics.
- Risk-Adjusted Advancement: Provides mechanistic de-risking via spatial tumor mapping that supports go/no-go decisions.
Pipeline & Workflow Integration
FMT integrates into the discovery continuum from early target validation through preclinical efficacy testing, enabling iterative assessment of therapeutic candidates.
- Discovery Biology: Supports hypothesis testing by tracking distribution and expression of fluorescently labeled glioblastoma cells over time.
- Screening: Enables assay readiness through reproducible fluorescence emission detection and region-of-interest selection.
- Analytics: Generates quantitative threshold-adjusted readouts and 3D reconstructions that allow comparative analysis across treatment groups.
- Translational Research: Connects to preclinical workflows by monitoring tumor localization changes indicative of therapeutic response.
- Enterprise Reuse: Functions as a reusable imaging platform via standardized subject selection, database configuration, and scan protocol execution.
Operational & Enterprise Impact
- Scientific Value: Enhances predictive confidence by reducing mechanistic ambiguity in tumor localization and therapeutic response.
- Operational Value: Ensures standardization and reproducibility through defined cassette positioning, laser channel selection, and threshold adjustment protocols.
- Strategic Value: Improves go/no-go decision-making by providing spatially resolved, quantitative tumor burden data.
- Portfolio Impact: Enables risk-adjusted prioritization through longitudinal imaging data that informs advancement criteria.
Implementation Considerations
- Requires expertise in small animal imaging, fluorescent protein expression, and near-infrared tomography systems.
- Dependent on FMT imager with transillumination capability, anesthesia equipment, and image reconstruction software.
- Necessitates cross-team standardization of subject handling, cassette positioning (17 mm head adjustment), and scan field selection.
- Involves adaptation considerations for different fluorescent protein emission spectra and tissue penetration depths.
- Limited by the need for genetically engineered models expressing near-infrared fluorescent proteins and access to dedicated imaging hardware.
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Last updated: 22 August 2026