Trends in Immunology
ReviewIlluminating Epigenetics and Inheritance in the Immune System with Bioluminescence
Section snippets
Bioluminescence and Luciferase-Based Assays
Bioluminescence is a process in which light is emitted from a living organism during oxygenation of a substrate (a luciferin, see Glossary) by an enzyme (a luciferase) [1]. Descriptions of this extraordinary phenomenon date back thousands of years, with bioluminescence occurring extensively in marine life and in a range of terrestrial organisms, including fungi and soft-bodied beetles, such as fireflies. Bioluminescent systems encompass a huge variety of enzymes and substrates, each of which
Using Bioluminescence for In Vivo Imaging
A variety of technologies now exist for in vivo reporter imaging [6,7] (Box 1); however, BLI offers a relatively low-cost and accessible approach with several distinct advantages over other modalities, including over other optical techniques, such as fluorescence imaging (FLI) [1,3,4]. In particular, BLI offers an exceptionally high signal-to-noise ratio, since no external illumination is required, and background light emission is essentially zero. This contrasts with FLI, which relies on
Using BLI to Monitor Gene Expression In Vivo
Advances in BLI technology are now allowing researchers to expand applications beyond cell tracking and revisit luciferase as a reporter for transcriptional dynamics, in an in vivo setting. Although BLI can be used to report in vivo gene expression in a variety of different contexts, as exemplified later, imaging requires three basic components: a luciferase enzyme (encoded genetically), a luciferin substrate (often administered by injection), and a sensitive imaging system for light detection,
Applying BLI Approaches to the Immune System
In the body, immune cells do not operate in isolation, but as part of a spatially and temporally complex and dynamic system, influenced by multiple cellular interactions and environmental factors. However, much of our current molecular understanding of the immune system is derived from ex vivo studies of individual cells and factors [43]. BLI offers a complementary and non-invasive approach to study immune responses in a physiological setting in vivo.
Seeing More with Bioluminescence
BLI provides a powerful tool for studying biology in vivo and new technological advances are on the horizon. There has been significant progress in the discovery and synthetic development of new luciferase enzymes and improved substrates for in vivo imaging [1., 2., 3., 4.] (Table 1). Despite the sensitivity of BLI, one of the biggest challenges remains imaging deeper internal sites, due to absorption and scattering of light by host tissue, especially for shorter wavelengths below 600 nm [69].
Concluding Remarks
As researchers strive to reach a more complete understanding of the immune system upon challenge and across organism lifespans, non-invasive gene reporters promise to make increasingly important contributions. The intrinsic advantages of BLI, in particular its low background and high sensitivity, make this approach ideal for studying complex, spatially and temporally dynamic processes that are hallmarks of immunology. The BLI toolkit continues to grow and further innovations for imaging deep
Acknowledgments
This work was funded by core support from the Medical Research Council UK to the London Institute of Medical Sciences.
Glossary
- 2A self-cleaving peptide sequences
- (e.g., T2A, P2A) allow production of separate proteins from a single open reading frame.
- Autonomous bioluminescence
- light production from cells or an organism that does not require external addition of luciferin or other components, as is the case in naturally occurring bioluminescence. This requires that both the luciferase and the pathways for substrate synthesis be genetically encoded, which is currently rare for artificial reporter systems.
- Bacterial
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2022, iScienceCitation Excerpt :However, our in vivo evaluations of these candidates against LCMV-GFP or LCMV-mCherry were hampered by the low-resolution images produced using GFP and mCherry. BLI has been reported to offer much higher sensitivity than fluorescence imaging (approximately 500-fold more sensitive than fluorescence imaging) (Dimond et al., 2020). The ATP-independent Nluc, characterized with smaller size (19 kDa), high physical stability, and higher brightness (∼150-fold greater than that of either firefly or Renilla luciferases) (Hall et al., 2012) was eventually selected.
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2022, Radiation Dosimetry Phosphors: Synthesis, Mechanisms, Properties and AnalysisA bioluminescence reporter mouse strain for in vivo imaging of CD8<sup>+</sup> T cell localization and function
2021, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Such reporter mice are especially valuable if the cells of interest are to be studied in various physiological and pathological states. BLI technology is widely used to analyze the immune system in vivo, due to the fact that it allows a real-time visualization of the immune cell dynamics [21]. T cells, among many other immune cells, often change locations and expand or contract in numbers under different states.