Abstract
Reactive molecular oxygen (O2) plays important roles in bioenergetics and metabolism and is implicated in biochemical pathways underlying angiogenesis, fertilization, wound healing and regeneration. Here we describe how to use the scanning micro-optrode technique (SMOT) to measure extracellular fluxes of dissolved O2. The self-referencing O2-specific micro-optrode (also termed micro-optode and optical fiber microsensor) is a tapered optical fiber with an O2-sensitive fluorophore coated onto the tip. The O2 concentration is quantified by fluorescence quenching of the fluorophore emission upon excitation with blue–green light. The micro-optrode presents high spatial and temporal resolutions with improved signal-to-noise ratio (in the picomole range). In this protocol, we provide step-by-step instructions for micro-optrode calibration, validation, example applications and data analysis. We describe how to use the technique for cells (Xenopus oocyte), tissues (Xenopus epithelium and rat cornea), organs (Xenopus gills and mouse skin) and appendages (Xenopus tail), and provide recommendations on how to adapt the approach to different model systems. The basic, user-friendly system presented here can be readily installed to reliably and accurately measure physiological O2 fluxes in a wide spectrum of biological models and physiological responses. The full protocol can be performed in ~4 h.
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Data availability
The representative data that support and use the approach are included in this protocol. Extended data are available in the support research paper24. In addition, all primary data underlying the figures shown in this protocol are available from the corresponding authors upon reasonable request.
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Acknowledgements
This work was supported by two NIH grants (EY019101 and R21EB015737), an AFOSR grant (FA9550-16-1-0052) and by an unrestricted grant from Research to Prevent Blindness, Inc., to the University of California (UC), Davis Ophthalmology Department. F.F. was supported by Fundação para a Ciência e Tecnologia (FCT) grant SFRH/BD/87256/2012. We thank A. Gomes (Departamento de Biologia, CBMA, Universidade do Minho, Portugal) for support. We are grateful to A. L. Miller (Department of Biology, Hong Kong University of Science and Technology, China), M. Horb (director of the NXR, MBL) and E. Pearl (NXR, MBL), as well as A. M. Shipley (Applicable Electronics) for generously hosting F.F.’s visit to MBL (summer 2014), for help in providing access to local research facilities and materials (SMOT and tadpoles (NXR RRID: SCR_013731) included), and for helpful comments. We are also grateful to C. A. Shipley, Applicable Electronics, for lending a SMOT system to the Zhao lab. We offer special thanks to E. Karplus (Science Wares, Inc.), designer and programmer of the SMOT, for providing software, technical assistance and support for its efficient installation and operation. We appreciate the input given by A. M. Shipley and E. Karplus to establish an efficient protocol in the Zhao lab. We are further thankful to E. Karplus for helping with the mathematics and interpretation behind the equations used to calculate O2 flux. We are grateful to Y. Yu (Department of Molecular & Cell Biology, UC Berkeley) for generously providing glass beads and for helpful discussion. We appreciate the technical information about micro-optrodes provided by S. Kerschensteiner and C. Huber (PreSens, GmbH). We thank Y. Shen (Department of Dermatology, UC Davis; Department of Occupational and Environmental Health, Zhejiang University, China) for kindly providing the euthanized mice. We thank the Zhao lab members for helpful discussions.
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F.F. designed and optimized the protocol. G.L. provided training in oocyte wounding. B.R. provided the intact and wounded rat eyes. L.M. executed some experiments. F.F. performed the physiological measurements and analyzed the data. M.Z. provided the funds for experiments and manuscript. All authors contributed to the scientific discussions and study design as appropriate. F.F. wrote the manuscript. B.R., VK.R. and M.Z. edited the manuscript. All authors read, commented and accepted the final manuscript.
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F.F. and M.Z. declare the existence of a non-financial competing interest. The measurements were performed using turnkey systems provided free for use at MBL and lent to the Zhao lab at no cost by Applicable Electronics, LLC, and Science Wares, Inc. The companies had no influence over the research (design, execution or interpretation), or its reporting; no restrictions on data sharing have been imposed. The other authors declare no competing interests.
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Ferreira, F., Raghunathan, V., Luxardi, G., Zhu, K. & Zhao, M. Nat. Commun. 9, 4296 (2018): https://www.nature.com/articles/s41467-018-06614-2
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Supplementary Figure 1 Further potentialities of the micro-optrode under testing.
(a) Invasive measurement of endogenous intravenous partial pressure of oxygen (pO2) in the heated rat tail. After manually impaling the housing needle, micro-optrode is pushed (injected) into the blood circulation. Right side schemes are a zoom in of the transverse section of the rat tail (dotted line in left scheme). Rat scheme (top view) is displayed in the same orientation as the whole organism anteroposterior (A–P), dorsoventral (D–V) and left-right (L–R) axes (middle scheme). (b) Invasive measurement of endogenous intraocular pO2 in the enucleated non-human primate eye (gifted by the California National Primate Research Center). After manually impaling the housing needle, micro-optrode is injected into the eye chambers. Eye scheme (lateral view) is displayed in the temporal (T) to nasal (N) axis orientation. (c) Invasive measurement of endogenous intratissue pO2 in the regenerating bud of tadpole tails. Using a broken pulled capillary mounted on a manual micropositioner, a temporary hole is made in the regenerating bud, facilitating the entry of the micro-optrode. In this case and in other soft or fragile tissues, housing needle cannot be impaled. Tadpole tail scheme (lateral view) is displayed in the same orientation as the whole organism A–P, D–V and left-right L–R axes (bottom scheme). Furthermore, we expect that the optical fiber of the micro-optrode is sufficiently break-resistant and sharp to directly impale target samples. All procedures involving animals were approved by the relevant Institutional and National regulatory boards.
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Supplementary Fig. 1 Further potentialities of the micro-optrode under testing
Supplementary Data
Template Excel worksheet for a quick and stepwise flux calculation and visualization (see Box 4). Data of template is from the gill of a tadpole used in Fig. 5d
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Ferreira, F., Luxardi, G., Reid, B. et al. Real-time physiological measurements of oxygen using a non-invasive self-referencing optical fiber microsensor. Nat Protoc 15, 207–235 (2020). https://doi.org/10.1038/s41596-019-0231-x
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DOI: https://doi.org/10.1038/s41596-019-0231-x
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