Instant FLIM enables 4D in vivo lifetime imaging of intact and injured zebrafish and mouse brains
Abstract
Traditional fluorescence microscopy is blind to molecular microenvironment information that is present in a fluorescence lifetime, which can be measured by fluorescence lifetime imaging microscopy (FLIM). However, most existing FLIM techniques are slow to acquire and process lifetime images, difficult to implement, and expensive. Here we present instant FLIM, an analog signal processing method that allows real-time streaming of fluorescence intensity, lifetime, and phasor imaging data through simultaneous image acquisition and instantaneous data processing. Instant FLIM can be easily implemented by upgrading an existing two-photon microscope using cost-effective components and our open-source software. We further improve the functionality, penetration depth, and resolution of instant FLIM using phasor segmentation, adaptive optics, and super-resolution techniques. We demonstrate through-skull intravital 3D FLIM of mouse brains to depths of 300 µm and present the first in vivo 4D FLIM of microglial dynamics in intact and injured zebrafish and mouse brains for up to 12 h.
Additional Information
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement. Received 5 April 2021; revised 10 May 2021; accepted 12 May 2021 (Doc. ID 426870); published 14 June 2021. We thank V. Mannam, X. Yuan, and O. Abdalsalam for discussion. We also thank L. Li for proofreading this paper. Funding: National Institutes of Health (R01 CA194697, R01 CA222405, R01 NS107553); National Science Foundation (CBET-1554516); University of Notre Dame; Elizabeth and Michael Gallagher Family; Alfred P. Sloan Foundation; Berry Family Foundation Graduate Fellowship of Advanced Diagnostics & Therapeutics. The authors declare that there are no competing interests. Data availability: The repositories of the open-source Instant-FLIM-Control [67] and Instant-FLIM-Analysis [68] software are hosted on GitHub and they are freely available for academic use. Detailed protocols describing how to use the two software packages are included within the repositories. All data needed to evaluate the conclusions in this paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.Attached Files
Published - optica-8-6-885.pdf
Submitted - 2020.02.05.936039v1.full.pdf
Supplemental Material - 5243711.pdf
Supplemental Material - MovieS1.mp4
Supplemental Material - MovieS2.mp4
Supplemental Material - MovieS3.mp4
Supplemental Material - MovieS4.mp4
Supplemental Material - MovieS5.mp4
Supplemental Material - MovieS6.mp4
Supplemental Material - MovieS7.mp4
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Additional details
- Alternative title
- Instant FLIM enables 4D in vivo lifetime imaging of intact brains
- Alternative title
- High-speed, long-term, 4D in vivo lifetime imaging in intact and injured zebrafish and mouse brains by instant FLIM
- Eprint ID
- 101180
- Resolver ID
- CaltechAUTHORS:20200207-102104833
- R01 CA194697
- NIH
- R01 CA222405
- NIH
- R01 NS107553
- NIH
- CBET-1554516
- NSF
- University of Notre Dame
- Elizabeth and Michael Gallagher Family
- Alfred P. Sloan Foundation
- Berry Family Foundation
- Created
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2020-02-10Created from EPrint's datestamp field
- Updated
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2021-07-12Created from EPrint's last_modified field