Advances in molecular and cellular imaging are largely due to the development of major types of light-emitting probes and ingenious ways of labeling them for use in living laboratory animals and, in a few instances, in endoscopic imaging in humans. Bioluminescent and fluorescent probes are two major types.
Bioluminescent probes use “luciferase,” an enzyme, to generate and emit light by an organism, providing real-time analyses of disease processes at the molecular level in living organisms, including laboratory animals. Luciferase is the enzyme in fireflies and glowworms that makes them light up. These are the best known examples of organisms that naturally produce bioluminescence, but deep sea marine organisms and some bacteria and fungi also produce bioluminescence. A bioluminescent probe is prevalently used in studying infections and cancer progression. There are many organic fluorescent probes, such as fluorescein, rhodamine, acridine dyes, phycoerythin and others. There also are bioluminescent protein probes. In fact, it was during isolation of a bioluminescent protein that fluorescent probes were discovered.
Fluorescent protein probes are green fluorescent protein, its yellow, blue and cyan-colored mutants, and red fluorescent proteins. In addition, there are hundreds of other fluorescent probes that are not fluorescent proteins. Fluorescent probes are introduced into an animal and visualized in the animal or its tissue cultures when excited by ultraviolet or visible light and viewed with optical imaging techniques. Fluorescence is the absorption and subsequent re-radiation of light by an organism. Fluorescence was known and used for microscopy, including intravital imaging, for many years prior to the discovery of fluorescent proteins.
The discovery of fluorescent proteins occurred after scientists, isolating a blue bioluminescent protein from a specific type of jellyfish, observed another protein that produced green fluorescence when illuminated with ultraviolet light. The gene for green fluorescent protein was cloned in the early 1990s; but its utility as a molecular probe occurred later, after scientists used fusion products to track gene expression in bacteria and nematodes.
The colored proteins, plus fusion proteins and biosensors—all of which are referred to as fluorescent proteins—are used primarily to visualize molecules in living cells. Moreover, multiple (different colored) fluorescent probes can simultaneously identify several target molecules within a cell and show their actions. The fluorescent proteins are fused to specific proteins and enzymes in the laboratory and primarily introduced into the animal through production of “transgenic” strains, whereby the fluorescent protein is introduced into the germline (sequence of germ cells containing genetic material), often under the control of tissue-specific or cell type-specific promoters. In another approach, engineered genes that encode fluorescent protein fusions, rather than the proteins themselves, are introduced into the animals by attaching them to harmless viruses, which serve as vectors to carry the fluorescent protein fusions into the animal. The fluorescently labeled cells in tissues of interest are then imaged.
To introduce bioluminescent and fluorescent probes into the animal, a widely used technique is genetic transfer. The gene that produces bioluminescence or fluorescence is cloned in the laboratory and introduced into a laboratory animal in one of two ways. The gene may be inserted into a harmless virus (called a vector) and introduced into the animal. Or, the gene is inserted into a stem cell and introduced into the animal so that the differentiated cell that the stem cell develops into will express the luminescence or fluorescent protein. The scientists at the forefront of this technology were awarded a Nobel Prize in Physiology or Medicine.
Adoptive transfer, another technique, involves tagging specific cells, such as in an animal model of a disease, and transferring those tagged cells into another laboratory animal to see how they work. Adaptive transfer is used to label cells that are “naturally occurring probes” in the body, in that they migrate to specific targets.
An example is lymphocytes (a family of white blood cells) which are immune “T” or “B” cells. These immune cells produce antibodies, which are proteins. Subsets of immune T or B cell lymphocytes are taught by immune dendritic cells to attack a specific foreign invader, such as an infectious agent, or a cancer. The newly educated lymphocytes then travel to the site of the infection or tumor to attack it. The antibodies (proteins) produced by the subset of T or B cells bind to specific “antigens.” These are surface molecules that are selectively expressed on specific cell types, including immune cells and also on tumor cells. Antibodies can be used, therefore, to identify immune cells or tumors in tissue samples or in live laboratory animals. Scientists label the antibody protein with a fluorescent tag and inject the material into another laboratory animal; or, they "stain" tissue sections or isolated cells within tissues and image the tissues or cells using a technique called flow cytometry.
Use of “Dendrimers” with fluorescent probes is another recent approach to labeling particles that travel to a target, especially a tumor. Unlike antibodies, which are proteins produced by living cells, dendrimer nanoparticles are compounds that are synthesized in the laboratory. They can be made from various materials (often polymers) that are synthesized and assemble into high molecular weight spherical particles. Scientists can add to the dendrimers a fluorescent probe that targets moieties (two divisible parts) and a “payload,” such as a drug. In this way, dendrimer nanoparticles can be targeted to tumor cells and emit light and/or deliver a therapeutic agent that kills the tumors. Dendrimer nanoparticles can cross the blood brain barrier and enter the brain, so they are being intensively studied as a potential treatment approach for brain tumors.
Another technique for imaging and targeting drug delivery is the use of aptamers, which are made from nucleic acids. Aptamers can be produced to have exquisite binding specificity for defined molecular targets, similar to antibodies. They can be attached to the surface polymeric particles for imaging and to enable targeted drug delivery.
The variations in the ways that different bioluminescent and fluorescent probes provide information are influenced by the type of microscope or other imaging technologies used.