Over the past few decades, many ways of tracking cells have emerged. From early simple experiments to the increasingly powerful and complex studies of late, this article provides a general overview of a few techniques for tracking cells in vivo. So, if one day you find yourself wondering, “how can I find my cells once I put them into a mouse?” or, “I wish there was a way to see where my cells go while my mice are still living!”, you may be able to use some of these techniques.

Over numerous decades, scientists have been developing methods to follow intracellular events and to identify how cells move within a living organism. Some initial tracking studies in the field of immunology include those that investigated the development of T cells as they migrated from the bone marrow to the thymus. Other early studies used radioactive tracers including isotopes of iron (59Fe) and phosphorous (32P) to follow transfused red blood cells in humans. Some of these isotopes, especially 32P, are quite dangerous and would not be permitted for use in any human cell tracing experiments today.

Radioactive tracers have also been used in non-human experimental models, but not as extensively. Instead, fluorescent dyes have been the tool of choice for tracking cell migration. Pioneering work by Coons and Kaplan in 1941 was the first to demonstrate conjugation of a fluorescent dye, fluorescein isothiocynate (FITC), to antibodies and led to the rise of immunofluorescence. Subsequent studies by others in 1980 utilized FITC and other dyes to label lymphocytes that were transferred into mice. Fluorescently labeled cells had the advantage of allowing for further analysis by flow cytometry, which permitted a more granular investigation of cell migration than by radioisotope analysis alone.

Immunogold labeling has also been used to track cells. Some studies used a colloidal gold label for antibodies directed against cell-specific antigens. At the time, the authors suggested that immunogold labelling could replace fluorescence and that the counting of labelled cells could be automated such that a flow cytometer need not be used. However, based on the volume of literature published using fluorescence over the past few decades, it is clear that fluorescent dyes have won out over immunogold labelling. The high cost of colloidal gold most likely made this technique impractical for many researchers.

Current methods
The current gold standard for visualizing cell interactions in vivo is two-photon (or multiphoton) microscopy. Multiphoton microscopy (MPM) is a powerful technique that enables real-time, in vivo or ex vivo visualization of cell migration in three-dimensional space. MPM relies on the principle of multiphoton excitation, a phenomenon that occurs when two or more photons arrive simultaneously at a specific location. For multiphoton excitation to occur, these photons must have a total sum energy equal to the transition energy required to promote a fluorophore (e.g. AlexaFluor, GFP, etc.) from its ground to excited state. The relaxation energy of the fluorophore is emitted as visible light, which is captured by a detector. The microscope stage is adjusted up or down and the sample is imaged again at a different focal plane (i.e. a different depth). Hundreds of images can be acquired over a single z-space stack, and the process is repeated for the duration of the imaging session. The resulting stack of images is reconstructed in silico to provide a three-dimensional view of cell migration over time.

MPM utilizes near-infrared lasers for the excitation energy source, allowing for deeper imaging of tissues while reducing light scatter. The near-infrared laser provides very short (femtosecond) but high-powered pulses of light to minimize photodamage and improve imaging quality, especially over long timecourses or repeated imaging experiments.
Some rather beautiful studies in mouse lymph nodes, brains, bone marrow, and thymus have been performed. These techniques have also been applied using transgenic mice where the genes for specific transcription factors or cell surface markers have been modified to contain GFP, RFP or YFP. These mice are useful for following the movement of specific cell populations or for doing fate-mapping studies, to determine what types of cells migrate to specific locations during development. Additionally, induction of luminescence or photoactivatable fluorescent proteins provides insights into the function of cells by using multiphoton microscopy.

In addition to microscopy, fluorescently-labeled cells can be imaged using epifluorescence and bioluminescence systems (e.g. Perkin-Elmer IVIS Lumina) where an animal is placed inside a fluorescence imager under anesthetic, and the particular region(s) of interest can be targeted for imaging based on the presence of fluorescent cells. These instruments have shown success in imaging mice with tumors and arthritis. Tumor metastases can be monitored over time in living mice – a valuable tool in this field.

Functional analyses can also be performed in mice by injecting fluorescent dyes that are activated enzymatically. For example, metalloproteinase (MMP) activity, which is often used as a marker of inflammation associated with tumors or atherosclerosis, can be assessed by using a fluorescent probe. MMP activity results in the activation of the fluorescent probe and concurrent imaging can reveal the location of the disease site. This technique can be combined with cells labelled on a different fluorescence channel to correlate possible migration of the labelled cells to the site of interest.

More recently, MRI has been used to detect single cells in transfer studies. The advantages to MRI tracking are that the technology is non-invasive, widely available, and routinely used in both clinical and research settings. Transplanted cells may be labeled with iron oxide particles, which are already FDA-approved, and the fate of the cells can be observed by a simple MRI scan.

In mice, extensive studies to show trafficking of cells from an injection site to a draining lymph node, or into the site of an induced spinal cord injury have been performed. The results are quite encouraging, and for animals that cannot be imaged using fluorescence, MRI is a valuable alternative. Mice can be imaged frequently throughout the course of the study and different scan parameters can be used to isolate the tissue of interest.

Drawbacks
Of course, there are also a few disadvantages to any kind of imaging technology. The first major drawback is cost. As many of these technologies are fairly recent, they can be fairly expensive to use. While in some studies cells can be labelled and tracked by injecting a fluorescent dye, techniques such as multiphoton microscopy and MRI require expensive equipment that is both costly to purchase and maintain.

Another drawback to some of these imaging techniques is poor resolution. Large-scale imaging systems such as MRI or whole body fluorescence imaging allow for a broad survey of cell migration throughout the body. However, the large field of view afforded by these systems comes at a cost of resolution and sensitivity. Neither technique can provide the level of detail and precision that intravital microscopy can. Multiphoton microscopes, despite their power to image within a tissue or sample, are still limited to a practical depth of approximately 0.5-1mm. Imaging at greater depths will require a combination of more powerful lasers, improved fluorochromes, and better detectors.

One thing is for certain: we have come a long way in the past few decades with respect to cell tracking technologies. Our ability to image cell-cell interactions and trafficking in vivo is now at an unprecedented resolution and accuracy. Further developments in fluorochromes in regards to brightness, durability, and variety will improve signal to noise ratios and enable simultaneous imaging of many parameters. Refinement of emerging technologies such as multiphoton microscopy and the development of new imaging techniques will also augment our ability to track cells. Watching immunological processes occurring in real time is extraordinarily fascinating and will help reveal new insights into the fluid and dynamic nature of cell migration and interaction.

-by Leesa Pennell and Charles Tran

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