Table 2.
Advantages and disadvantages of common in vivo and ex vivo cell tracking methodologies
| Technique | Common imaging agents | Limit of detection | Depth of penetration | Spatial resolution | Temporal resolution | Useful applications advantages | Limitations | References |
|---|---|---|---|---|---|---|---|---|
| In vivo techniques | ||||||||
| BLI | D‐luciferin/Firefly luciferase, Coelenterazine/Renilla luciferase | 10–10,000 cells (depending on tissue depth) | ∼3 cm (∼10‐fold loss of signal intensity per cm) | 50 μm | 1 min | Whole‐body imaging;Reporter enzyme expressed only inmetabolically activelive cells; Cheap; Ease of use | 2D image; Limited to small animal studies;Signal attenuation in deeper tissues; Signal quantitation not comparable across tissues; Susceptible to vascular disrupting agents | 21, 56, 65, 76, 95, 102, 106 |
| MRI | Ferumoxide;Ferumoxytol;Ferritin;Gd chelates | 1,000–10,000 cells (depending on imaging agent and instrumentation) | No limit | 100–200 μm | 1 min | Whole‐body imaging;Anatomical imaging;Ferritin reporter gene enables detection exclusively of live cells;Clinical integration | T2 hypointensity indistinguishablefrom tumor hemorrhage;False positives due to macrophage uptake;2D image; Label diluted with each cell division; Potential cytotoxicity and ROS generation; Sophisticated equipment and expertise needed | 26, 55, 56 |
| PET | 18F‐FDG;Hsv‐TK + 18F‐FDG;64Cu‐PTSM;89Zr;124I | 100–25,000 cells (depending on imaging agent and instrumentation) | No limit | 1–2 mm | 15 min | Whole‐body imaging; 3D imaging; Multiple radioisotopes and labeling chemistries available; Hsv‐TK reporter gene enables detection exclusively of live cells; Clinical integration | Use of radioisotopes;Hazardous transfection reagents; Potential false positives aftercell death/label efflux; Sophisticated equipment and expertise needed | 33, 48 |
| In vivo microscopy | GFP;Vital dyes (DID/DiL/DiD/DiR);CFDA/CFSE | Single cell | 150–250 μm | Single cell | Video‐rate | Imaging in context of natural microenvironment;Visualization of dynamic processes(migration, vascular adhesion, TEM) | Limited to preselected areas with low required path length; Tissue exteriorization may influence MSC recruitment/retention; Tissue auto‐fluorescence; Sophisticated equipment and expertise needed | 18, 23, 46, 58, 60, 61, 99, 100 |
| In vivo flow cytometry | GFP;Vital dyes (DID/DiL/DiD/DiR);CFDA/CFSE;PKH26 | 1–10 cells/mL | 150–250 μm | Single cell | Continuous | Enumeration of circulating cells; Tracking dynamic entry and exit from circulation; Detection of cell flow velocities;Analysis of large blood volume; No artifacts from cell isolation and processing | Quantification influenced by flow velocityand vessel size; Tissue auto‐fluorescence;Antibody labeling can lead to target cell depletion; Sophisticated equipment and expertise needed | 62, 63, 64 |
| Ex vivo techniques | ||||||||
| Histology | X‐gal/β‐gal;Prussian blue/Iron oxide | Single cell | N/A | Single cell | Single time point/sample | In situ visualization within contextual tissue structure | Loss of signal from gene silencing or cell division; Sampling bias | 26, 34, 47, 94 |
| IHC | GFP/anti‐GFP;CFDA/anti‐fluorescein; PKH26; Vital dyes (DiI/DiL/DiD/DiR); Quantum dots;Fluc/anti‐FLuc | Single cell | N/A | Single cell | Single time point/sample | In situ visualization within contextual tissue structure | Loss of signal from gene silencing or cell division; Sampling bias; Tissue auto‐fluorescence;Potential transfer of membrane dyes | 18, 43, 44, 45, 46, 56, 71, 72, 73, 77 |
| FISH | Y‐Chromosome;Alu sequences | Single cell | N/A | Single cell | Single time point/sample | In situ visualization within contextual tissue structure;No loss of signal | Sampling bias;Tissue auto‐fluorescence | 71 |
| Ex vivo flow cytometry | GFP | 1–10 cells/mL | N/A | Single cell | Single time point/sample | Enumeration of circulating cells | Potential artifacts from cell isolationand processing; Cell auto‐fluorescence;Sophisticated equipment and expertise needed | 26, 34, 63, 64 |
| qRT‐PCR | Y‐Chromosome;Alu sequences | 1/600,000 cells (∼0.0002%) | N/A | Single cell | Single time point/sample | Quantification from whole tissue | Rare transcripts difficult to detect due to stochasticity of PCR amplification | 25, 28, 40 |
| ddPCR/BEAMing | Y‐Chromosome;Alu sequences | 1/10,000 cells (0.01%) | N/A | Single cell | Single time point/sample | Quantification from whole tissue; Identification of rare transcripts/cell populations | Sophisticated equipment and expertise needed | 41 |
| Scintigraphy | 111In‐oxine; 18F‐FDG;64Cu‐PTSM; 51Cr; 125I;99mTc‐HMPAO | Single cell (depending on labeling efficiency) | N/A | Single cell | Single time point/sample | Quantification from whole tissue | Use of radioisotopes | 20, 26, 33, 47, 51, 52 |
Abbreviations: BLI, bioluminescence imaging; FISH, fluorescence in situ hybridization; Gd, gadolinium; Hsv‐tk, herpes simplex virus‐thymidine kinase; IHC, fluorescence imaging and immunohistochemistry; MRI, magnetic resonance imaging; PET, positron emission tomography; qRT‐PCR, quantitative real‐time PCR.