Skip to main content
SpringerLink
Log in

Multi-parametric O2 Imaging in Three-Dimensional Neural Cell Models with the Phosphorescent Probes

  • Protocol
  • First Online:
Neuronal Cell Death

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1254))

Abstract

Recent progress in bio-imaging has allowed detailed mechanistic studies of neural cell function in complex 3D tissue models including multicellular aggregates , neurospheres , excised brain slices , ganglia , and organoids . Molecular oxygen (O2 ) is an important metabolite and an environmental parameter which determines the viability and physiological status of neural cells within tissue. Here we describe standard method for monitoring O2 in 3D tissue models using phosphorescence lifetime imaging microscopy (PLIM ) and cell-penetrating O2-sensing probes . The O2 probes can be multiplexed with many conventional fluorescence based live cell biomarkers and also end-point immunofluorescence staining . The multi-parametric O2 imaging method is particularly useful for areas such as stem cell development and differentiation , hypoxia research, neurodegenerative disorders , regeneration of brain tissue, evaluation of new drugs , and development of novel tissue models.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Northington FJ, Chavez-Valdez R, Martin LJ (2011) Neuronal cell death in neonatal hypoxia-ischemia. Ann Neurol 69:743–758

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Green DR, Galluzzi L, Kroemer G (2011) Mitochondria and the autophagy–inflammation–cell death axis in organismal aging. Science 333:1109–1112

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Sims NR, Muyderman H (2010) Mitochondria, oxidative metabolism and cell death in stroke. Biochim Biophys Acta 1802:80–91

    Article  CAS  PubMed  Google Scholar 

  4. Kalyanaraman B, Darley-Usmar V, Davies KJA et al (2012) Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radic Biol Med 52:1–6

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Tait SW, Green DR (2010) Mitochondria and cell death: outer membrane permeabilization and beyond. Nat Rev Mol Cell Biol 11:621–632

    Article  CAS  PubMed  Google Scholar 

  6. Jespersen SN, Østergaard L (2011) The roles of cerebral blood flow, capillary transit time heterogeneity, and oxygen tension in brain oxygenation and metabolism. JCBFM 32:264–277

    Google Scholar 

  7. Devor A, Sakadžić S, Srinivasan VJ et al (2012) Frontiers in optical imaging of cerebral blood flow and metabolism. JCBFM 32:1259–1276

    CAS  Google Scholar 

  8. Papkovsky DB, Dmitriev RI (2013) Biological detection by optical oxygen sensing. Chem Soc Rev 42:8700–8732

    Article  CAS  PubMed  Google Scholar 

  9. Lecoq J, Roussakis A, Parpaleix E et al (2011) Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels. Nat Med 17:893–898

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Quaranta M, Borisov SM, Klimant I (2012) Indicators for optical oxygen sensors. Bioanal Rev 4:115–157

    Article  PubMed Central  PubMed  Google Scholar 

  11. Dmitriev RI, Kondrashina AV, Koren K et al (2014) Small molecule phosphorescent probes for O2 imaging in 3D tissue models. Biomater Sci 2:853–866

    Article  CAS  Google Scholar 

  12. Dmitriev RI, Zhdanov AV, Nolan YM et al (2013) Imaging of neurosphere oxygenation with phosphorescent probes. Biomaterials 34:9307–9317

    Article  CAS  PubMed  Google Scholar 

  13. Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707–1710

    Article  CAS  PubMed  Google Scholar 

  14. Monni E, Congiu T, Massa D et al (2011) Human neurospheres: from stained sections to three-dimensional assembly. Translational Neurosci 2:43–48

    Article  Google Scholar 

  15. Pampaloni F, Reynaud EG, Stelzer EH (2007) The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8:839–845

    Article  CAS  PubMed  Google Scholar 

  16. Gil‐Perotín S, Duran‐Moreno M, Cebrián‐Silla A et al (2013) Adult neural stem cells from the subventricular zone: a review of the neurosphere assay. Anat Rec 296:1435–1452

    Article  Google Scholar 

  17. Gähwiler BH, Capogna M, Debanne D et al (1997) Organotypic slice cultures: a technique has come of age. Trends Neurosci 20:471–477

    Article  PubMed  Google Scholar 

  18. Ohnishi T, Matsumura H, Izumoto S et al (1998) A novel model of glioma cell invasion using organotypic brain slice culture. Cancer Res 58:2935–2940

    CAS  PubMed  Google Scholar 

  19. Zimmer J, Kristensen BW, Jakobsen B et al (2000) Excitatory amino acid neurotoxicity and modulation of glutamate receptor expression in organotypic brain slice cultures. Amino Acids 19:7–21

    Article  CAS  PubMed  Google Scholar 

  20. Dionne KR, Smith Leser J, Lorenzen KA et al (2011) A brain slice culture model of viral encephalitis reveals an innate CNS cytokine response profile and the therapeutic potential of caspase inhibition. Exp Neurol 228:222–231

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Su T, Paradiso B, Long Y-S et al (2011) Evaluation of cell damage in organotypic hippocampal slice culture from adult mouse: a potential model system to study neuroprotection. Brain Res 1385:68–76

    Article  CAS  PubMed  Google Scholar 

  22. McCord AM, Jamal M, Shankavarum UT et al (2009) Physiologic oxygen concentration enhances the stem-like properties of CD133+ human glioblastoma cells in vitro. Mol Cancer Res 7:489–497

    Article  CAS  PubMed  Google Scholar 

  23. Lancaster MA, Renner M, Martin C-A et al (2013) Cerebral organoids model human brain development and microcephaly. Nature 501:373–379

    Article  CAS  PubMed  Google Scholar 

  24. Finikova OS, Lebedev AY, Aprelev A et al (2008) Oxygen microscopy by two‐photon‐excited phosphorescence. Chem Phys Chem 9:1673–1679

    CAS  PubMed Central  PubMed  Google Scholar 

  25. Kazmi SMS, Salvaggio AJ, Estrada AD et al (2013) Three-dimensional mapping of oxygen tension in cortical arterioles before and after occlusion. Biomed Opt Express 4:1061–1073

    Article  PubMed Central  PubMed  Google Scholar 

  26. Becker W (2012) Fluorescence lifetime imaging – techniques and applications. J Microsc 247:119–136

    Article  CAS  PubMed  Google Scholar 

  27. Bershteyn M, Kriegstein AR (2013) Cerebral organoids in a dish: progress and prospects. Cell 155:19–20

    Article  CAS  PubMed  Google Scholar 

  28. Stoppini L, Buchs P-A, Muller D (1991) A simple method for organotypic cultures of nervous tissue. J Neurosci Meth 37:173–182

    Article  CAS  Google Scholar 

  29. Barry DS, Pakan JMP, O’Keeffe GW et al (2013) The spatial and temporal arrangement of the radial glial scaffold suggests a role in axon tract formation in the developing spinal cord. J Anat 222:203–213

    Article  CAS  PubMed  Google Scholar 

  30. Pampaloni F, Ansari N, Stelzer EH (2013) High-resolution deep imaging of live cellular spheroids with light-sheet-based fluorescence microscopy. Cell Tiss Res 352:161–167

    Article  Google Scholar 

  31. Wilt BA, Burns LD, Wei Ho ET et al (2009) Advances in light microscopy for neuroscience. Annu Rev Neurosci 32:435–506

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Graf BW, Boppart SA (2010) Imaging and analysis of three-dimensional cell culture models. In: Live cell imaging, vol 591, Methods in molecular biology. Springer, New York, pp 211–227

    Google Scholar 

  33. Dmitriev RI, Papkovsky DB (2012) Optical probes and techniques for O2 measurement in live cells and tissue. Cell Mol Life Sci 69:2025–2039

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Mehta G, Hsiao AY, Takayama S (2012) Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. J Control Release 164:192–204

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Frigault MM, Lacoste J, Swift JL et al (2009) Live-cell microscopy–tips and tools. J Cell Sci 122:753–767

    Article  CAS  PubMed  Google Scholar 

  36. Kondrashina AV, Dmitriev R, Borisov SM et al (2012) A phosphorescent nanoparticle-based probe for sensing and imaging of (intra)cellular oxygen in multiple detection modalities. Adv Funct Mater 22:4931–4939

    Article  CAS  Google Scholar 

  37. Fercher A, Borisov SM, Zhdanov A et al (2011) Intracellular O2 sensing probe based on cell-penetrating phosphorescent nanoparticles. ACS Nano 5:5499–5508

    Article  CAS  PubMed  Google Scholar 

  38. Tsytsarev V, Arakawa H, Borisov SM et al (2013) In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe. J Neurosci Meth 216:146–151

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Science Foundation Ireland, grants 12/RC/2276 and 12/TIDA/B2413, and the EC FP7 Program, grant NanoBio4Trans (No. 304842-2). We thank Dr. J. M.P. Pakan and Dr. Y.M. Nolan (Department of Anatomy and Neuroscience, University College Cork) for the help in preparation of brain slices and neurosphere cultures.

Author information

Authors and Affiliations

  1. School of Biochemistry and Cell Biology, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Ireland

    Ruslan I. Dmitriev & Dmitri B. Papkovsky

Authors
  1. Ruslan I. Dmitriev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dmitri B. Papkovsky
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ruslan I. Dmitriev .

Editor information

Editors and Affiliations

  1. Dept. of Veterinary Sciences, University of Torino, Grugliasco, Torino, Italy

    Laura Lossi

  2. Dept. of Veterinary Sciences, University of Torino, Grugliasco, Torino, Italy

    Adalberto Merighi

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Dmitriev, R.I., Papkovsky, D.B. (2015). Multi-parametric O2 Imaging in Three-Dimensional Neural Cell Models with the Phosphorescent Probes. In: Lossi, L., Merighi, A. (eds) Neuronal Cell Death. Methods in Molecular Biology, vol 1254. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2152-2_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2152-2_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2151-5

  • Online ISBN: 978-1-4939-2152-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation