Miniaturized Devices for Bioluminescence Imaging in Freely Behaving Animals

Background

Microscopic imaging of fluorescent indicators to track neural activity is an increasingly important tool in systems neuroscience.Since almost a decade, fluorescence miniature microscopy in vivo has been a major advance, enabling single-cell resolution imaging in freely behaving animals. Since its release it has been used to study questions in domains ranging from memory representation to olfactory processing. Some limitations of fluorescence-based microscopy include photobleaching and phototoxicity; and that the signal-to-noise ratio for localizing discrete signals in the tissue is limited, due to autofluorescence from unintended sources, and non-homogeneous illumination due to the scattering of excitation light. Bioluminescence provides an alternative kind of activity-dependent light indicator, whereby light is generated via chemiluminescent reaction between luciferase (an enzyme) and luciferin (a substrate). Bioluminescent cells do not suffer from photobleaching, phototoxicity, or excitation scattering, and these point sources do not create autofluorescence. Further, bioluminescent indicators also do not require excitation light optics: the removal of this component should make lighter and lower cost microscope with fewer assembly parts. While there has been progress in making brighter and faster bioluminescence indicators, parallel advances in imaging hardware have not followed. In their work, Celinskis et al (1) investigate whether fluorescent miniature microscopes can be rendered sensitive enough to detect bioluminescence, and tested its capacity for detecting bioluminescent signals in vitro and in vivo.

Key findings and developments

In this work, Celinskis et al modified the miniscope v3.2 ((2), referred to as the UCLA miniscope) to create the Bioluminescence Miniscope (BLmini) removing the components necessary for excitation light optics, allowing simplification of the microscope’s architecture. They configured parameters including power setup, acquisition speed, gain, and exposure time. They then performed an in vivo comparison of the UCLA miniscope, comparing signals emanating from a mouse expressing mNeonGreen tethered to EkL9H luciferase (i.e. a shrimp-based luciferase variant also referred to as NCS2). Both miniscopes were able to detect bioluminescence from both constructs, but the BLmini showed significantly stronger signal. Other aspects compared included mass, number of components, cost, and power consumption. BLmini is 22% lighter in weight, has 45% fewer components, is up to 58% less expensive, offers up to 15 times stronger signal (as dichroic filtering is not required) and is sensitive enough to capture spatiotemporal dynamics of bioluminescence in the brain with a signal-to-noise ratio of 34 dB. The BLmini was also able to capture the temporal dynamics of in vivo bioluminescence. A limitation discussed is that as BLmini does not rely on any form of cooling, it consequently suffers from significantly stronger thermoelectric background noise.

This progress represents a key step in the refinement of this approach, which can prove transformative to an important imaging approach.

 

What I like about this preprint

I think this is an important methodological advance with the potential for multiple applications in many research fields. As the authors discussed, the miniscope was revolutionary, and so far nothing similar had been produced for bioluminescence. The authors provide that opportunity now, which offers complementary advantages to fluorescence imaging.

Open questions

1. You used for testing, EkL9H. What is the range of wavelengths that the BLmini can detect? Would the signal-to-noise ratio become limiting for probes such as NanoLuc, Renilla Luciferase, or Gaussia Luciferase?
2. Is there a chance to do dual-bioluminescence imaging?
3. Since you used the miniscope as a basis, is there a chance to have a platform that uses both fluorescence and bioluminescence, without much increased cost or weight, in order to use the advantages of both imaging modalities?
4. Is it only possible to use the BLmini in the brain of free-moving animals? Has it been attempted in the spinal cord or other organs accessible dorsally?
5. Are there any specific limitations to bioluminescence imaging associated with free- movement?

References

1. Celinskis D et al, Miniaturized devices for bioluminescence imaging in freely behaving animals, bioRxiv 2020.
2. Cai DJ et al, A shared neural ensemble links distinct contextual memories encoded close in time, Nature, 2016

 

doi: https://doi.org/10.1242/prelights.22918

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