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Bioluminescence Basics: Study Setup & Methods

Jun 30, 2022 12:45:00 PM / by Champions Oncology


Bioluminescence imaging (BLI) is a non-invasive technique that allows for real-time imaging of biological processes in living animals. This method has revolutionized intravital imaging in mouse models, especially in the fields of immunology, oncology, and molecular genetics. Several elements are required to perform BLI studies and are outlined here.

Luciferase and Luciferin

BLI methods are based on light emitted due to bioluminescence reactions mediated by luciferase enzymes in the presence of luciferins. Luciferase enzymes are naturally expressed in some insects, crustaceans, fish, and bacteria and a subset of these enzymes have been engineered for expression in mammalian tissue for BLI studies. The most widely used luciferase is derived from the North American firefly (Photinus pyralis), and exposure to its complementary luciferin results in light being emitted at 600 nm, thus making it appropriate for detection in mammalian tissues with little light being lost to background absorption and scattering[1]. This and other luciferase genes have been codon-optimized for expression in mammalian cells. Photinus and Renilla-based luciferases, as well luciferases from other animals and bacteria, have been optimized to emit light at different wavelengths, which allows for combinations of these luciferases to be used for simultaneous visualization of multiple cell types in the same animal. Luciferase genes are delivered into animals via replication-competent viral vectors, like serotype 5 human adenovirus[2] or lentiviruses[3]. These viral vectors can be delivered into mice in different manners, including intracardiac, intravenous, intraperitoneal, or intratracheal routes. Vectors enter cells that express the appropriate viral receptors, where they replicate and express luciferase in the cytosol[4]. Several hours or days after vector delivery, mice are treated with luciferin substrate, and light emission can be detected several minutes to several hours after treatment. Cancer cell lines have also been engineered to express luciferase, and xenografted tumors comprised of these cell lines can be visualized over time as tumor volume changes. This approach has been particularly useful for preclinical drug and immunotherapy studies for breast, ovarian, and prostate cancers. In some cases, luciferase-expressing cell lines display different growth and metastasis rates in vivo compared with their parental cell line, and immune responses to these modified tumor cell lines can also alter outcomes[5].

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3D illustration of internal frame/structure of the portable 3CCD camera system for BLI detection


Optical Imaging Hardware

BLI visualization requires highly sensitive light detectors in charge-coupled device (CCD) cameras. These CCD cameras have been optimized for intravital imaging and may have intensified detectors that detect light at narrow wavelengths. This type of CCD setup can work well for a known luciferase system but may not be appropriate for detecting wavelengths outside of its range[6]. CCD cameras can also be cooled to -120˚C to reduce thermal noise and improve the signal-to-noise ratio[7]. CCD cameras are built into intravital imaging platforms in which sedated mice are placed into a chamber that allows for sufficient detection of light from tissues and organs just under the skin or at greater depths. Improvements in both CCD cameras and intravital set-ups have allowed for the detection of immune cells as they migrate to different tissues or tumors. Tumor metastases can also be visualized using luciferase-expressing tumor cell lines.


BLI techniques are now widely used for preclinical oncology studies because mouse models can be readily visualized over time as different experimental therapies are evaluated. Changes in tumor volume or metastases can be monitored without sacrificing mice, thus providing critical data on experimental treatment efficacy.


Preclinical In Vivo Imaging in Systemic Tumor Models - download now


[1] De Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 1987 Feb;7(2):725-37.

[2] Johnson M, Huyn S, Burton J, Sato M, Wu L. Differential biodistribution of adenoviral vector in vivo as monitored by bioluminescence imaging and quantitative polymerase chain reaction. Human Gene Ther. 2006 Dec 1;17(12):1262-9.

[3] Iyer M, Salazar FB, Wu L, Carey M, Gambhir SS. Bioluminescence imaging of systemic tumor targeting using a prostate-specific lentiviral vector. Human Gene Ther. 2006 Jan 1;17(1):125-32.

[4] Zinn KR, Chaudhuri TR, Szafran AA, O'Quinn D, Weaver C, Dugger K, Lamar D, Kesterson RA, Wang X, Frank SJ. Noninvasive bioluminescence imaging in small animals. ILAR Journal. 2008 Jan 1;49(1):103-15.

[5] Baklaushev VP, Kilpeläinen A, Petkov S, Abakumov MA, Grinenko NF, Yusubalieva GM, Latanova AA, Gubskiy IL, Zabozlaev FG, Starodubova ES, Abakumova TO. Luciferase expression allows bioluminescence imaging but imposes limitations on the orthotopic mouse (4T1) model of breast cancer. Sci. Rep. 2017 Aug 10;7(1):1-7.

[6] Oshiro M. Cooled CCD Versus Intensified Cameras for Low-Light Video---Applications and Relative Advantages. Methods Cell Biol. 1998 Jan 1;56:45-62.

[7] Contag CH, Bachmann MH. Advances in in vivo bioluminescence imaging of gene expression. Ann. Rev. Biomed. Eng. 2002 Aug;4(1):235-60.


Tags: Preclinical In Vivo Imaging