The Future is Bright: Imaging with Luciferase in Whole Animals

Long before Star Trek introduced the Tricorder scanner for whole-organism, non-invasive medical diagnostics, scientists were working on ways to understand cellular and molecular processes in vivo—in an essentially intact, living organism. As early as the 1940s, researchers used surgically introduced “skin folds” as a way of visualizing biological processes in an intact organism⁽¹⁾ . Cells or tissues can be transplanted into these skin folds, fluorescently labeled and visualized using epifluorescence or now, even laser scanning. However, only those tissues optically accessible through the skin fold can be studied.

Another technique for in vivo imaging involves detecting externally administered light-emitting molecules, such as a fluorescent vital dye, without disturbing the animal skin. However, as the cells divide, the amount of dye inside them is diluted, and this repeated dilution limits the length of time the signal can be detected.

Long before Star Trek introduced the Tricorder scanner for whole-organism, non-invasive medical diagnostics, scientists were working on ways to understand cellular and molecular processes in vivo.

Researchers are now adapting reporter assays, and even luminescent enzyme assays, traditionally used with cultured cells or extracts, to gain temporal and spatial information about biological processes within an entire organism. In this issue, we are presenting a paper that uses luciferase to image events within intact mice. Such imaging of luminescence in whole organisms, bioluminescence imaging (BLI), is the focus of this editorial. Ideally a reporter system for in vivo imaging would allow a researcher to follow a particular cellular or molecular process over an extended time frame, would be sensitive enough to reveal small changes over time, and would maintain physiological conditions within the animal so that naturally occurring processes are not disrupted [by over-expression of a protein, for instance⁽¹⁾ ]. The imaging technology also would need to produce signal with very low background and be able to detect an optical signal that passes through the tissues of a living organism⁽¹⁾ .

Researchers have demonstrated repeatedly that luciferase-based assays are exquisitely sensitive^{(1) (2) (3) (4) (5)} . Since the luciferase gene can be stably transfected into cells under the control of most any promoter, signal will not be lost following dilution by cell division. Luciferase assays in mammalian systems are particularly sensitive because they are not subject to high background as a result of tissue autofluorescence^{(1) (2) (3) (4) (5)} . This is a distinct advantage of luminescence over fluorescence. Most mammalian tissues absorb light in the blue/green and yellow wavelengths of the visible spectrum, which means that most of the detectable signal for an optical assay in an intact mammalian organism will be in the red (or near infrared) wavelengths. Naturally occurring luciferases so far characterized emit from 460–630nm; the longest wavelength of emission is found in a luciferase from the railroad worm⁽¹⁾ . Recombinant luciferase can be engineered so that the light emitted from the reaction falls within the red wavelengths of the visible spectrum⁽³⁾ , and technologies such as Bioluminescence resonance energy transfer (BRET) are being developed to take advantage of red-shifted fluorescent proteins that can absorb light from luciferase reactions.

Luciferase assays in mammalian systems are particularly sensitive because they are not subject to high background as a result of tissue autofluorescence.

Like in vivo fluorescence imaging, bioluminescent imaging is dependent on highly sensitive detectors. Typically a charged-coupled device (CCD) camera is used to image luciferase signal in whole animals. CCD cameras can be cooled to reduce thermal noise, and therefore background. Additionally, CCD systems detect the entire visible spectrum and near infrared wavelengths, allowing them to detect the light that is not absorbed by mammalian tissues⁽¹⁾ .

Bioluminescence imaging of intact small animals is a burgeoning field, with many gene reporter studies of BLI currently in the literature. BLI has been used to describe the distribution of cytotoxic T lymphocytes over time⁽²⁾ . In this first issue of the Promega Online Technical Publications Portal, we feature an article in which performance in vivo of different promoters and regulatory sequences was assessed by evaluating luciferase activity in mouse liver. Additionally we highlight two other examples of BLI: one in fish and a second one in mice.

The application of BLI to research problems extends beyond the typical gene reporter assay. BRET has been used to study protein interactions. In BRET, light emitted by luciferase activity can be absorbed by a partner GFP and emitted in a narrow emission peak that is shifted toward longer wavelengths. In one study, insulin-like growth factor II was fused to an engineered gene for green fluorescent protein (GFP). A second fusion construct was made in which insulin-like growth factor binding protein 6 was linked to Renilla luciferase. BRET was observed in cells transfected with both constructs in which the fusion proteins interacted in the presence of the Renilla substrate coelenterazine. It’s possible that BRET could be used to detect protein interactions in whole animals, especially if the partner fluorescent protein emits in the red wavelength⁽¹⁾ .

Luminescent enzyme activity assays can also be performed and monitored in vivo⁽⁵⁾ . In a proof-of-concept paper, Zhang and colleagues demonstrate induction and inhibition of CYP3A4 and 3A7 activity in a reporter mouse line using BLI and proluciferin substrates that are converted to luciferins through reactions catalyzed by specific cytochromes P450. With proluciferin substrates available for a variety of enzymes including several CYP450 isozymes, capsases, and other proteases, BLI could become a standard for studying drug effects in vivo, provided that substrates are soluble and not toxic to the test animal.

Although researchers have been working since the 1940s to study cellular and molecular events in vivo, BLI began to be a viable technique only in the late 20th century. However, as luciferases continue to be better engineered, as methods such as BRET are developed and improved, and as detection instrumentation increases in sensitivity, BLI will certainly become a laboratory standard. The future is bright.