Cellular Bioluminescence Imaging of Circadian Clocks
Apr 26, 2016
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Join Us for a Free Webinar on Cellular Bioluminescence Imaging of Circadian Clocks
With David K. Welsh, M.D. and Colin Coates
Bioluminescence imaging of live cells has recently been recognized as an important alternative to fluorescence imaging that is particularly well suited to the study of circadian rhythms. Fluorescent probes are brighter than bioluminescent probes (luciferase enzymes), and therefore provide better spatial and temporal resolution and better contrast for delineating cell structure.
However, with bioluminescence imaging there is virtually no background or toxicity. As a result, bioluminescence can be superior to fluorescence for detecting and quantifying molecules and their interactions in living cells, particularly in long-term studies.
Structurally diverse luciferases from beetle and marine species have been used for a variety of applications, including tracking cells in vivo and reporting circadian clock gene expression. Such applications can be optimized by the use of brighter and variously colored luciferases, brighter microscope optics, and ultrasensitive, low-noise cameras.
In this presentation, David Welsh and Colin Coates review how bioluminescence imaging differs from fluorescence imaging, its application to the study of circadian rhythms in single cells, and some available probes, optics, and types of detectors. The presenters also offer practical suggestions for optimal bioluminescence imaging of single cells.
Choosing the Right Detector Technology for Bioluminescence
Understanding the distinctions between different imaging detector technologies is key to making the right detector choice for your microscopy set-up and target applications. The presenters demystify some of these specification subtleties and relate them to the distinct requirements of bioluminescence versus fluorescence imaging.
David K. Welsh, M.D. earned his undergraduate degree at Stanford University, where he began a long-standing interest in circadian rhythms and sleep. In graduate work at Harvard Medical School, he used multielectrode arrays to show that individual neurons can generate circadian rhythms. He is currently an associate professor at the UC San Diego Department of Psychiatry and a staff psychiatrist at the VA San Diego Healthcare System. Dr. Welsh studies circadian rhythms in single cells using bioluminescence imaging to monitor clock gene expression.
Colin Coates is product manager at Andor Technology, responsible for camera technologies. His Ph.D. and postdoctoral research background at Queen’s University of Belfast, Ireland focused primarily on the use of laser spectroscopic studies to elucidate the detailed photophysics of molecules. He has also worked in industry R&D, developing novel DNA microarray technology.
Click here for more information about Andor Technology.
Audience questions and presenter David Welsh's responses are provided below.
1. What kinds of applications are best suited to bioluminescent vs. fluorescent reporters?
Live cell applications in which high spatial and temporal resolution is not critical, particularly long-term studies of biological processes with slower dynamics or light-sensitive components. These include in vivo cell tracking, measuring circadian clock gene expression, and studies of living retina.
2. What kinds of technical improvements do you foresee for bioluminescence imaging?
Brighter luciferases (e.g. NanoLuc), variously colored luciferases used as dual reporters, luciferases engineered to respond to particular signaling molecules (e.g., cAMP), improved variants of coelenterazine, caged luciferins, more sensitive detectors, 3D imaging.
3. Can nitrated protein be monitored?
I'm not sure how this could be done.
4. Can this (technology) be easily adapted to investigate lower metazoans and cnidarians' circadian clocks and if so do you have any suggestions?
I don't see why not, if you make your own reporters. I found this helpful review article: Circadian Clocks in the Cnidaria: Environmental Entrainment, Molecular Regulation, and Organismal Outputs, Oxford Journal.