Skip to main content

Application of Fluorescence Lifetime Imaging (FLIM) to Measure Intracellular Environments in a Single Cell

  • Chapter
  • First Online:

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 1035))

Abstract

Fluorescence lifetime imaging (FLIM) has now been used in many bioscience fields, which comes from the quantification of fluorescence lifetime. The procedure for obtaining lifetime images is very similar to that used in fluorescence microscopy. However, obtaining reliable lifetime images requires an understanding of the theory of fluorescence lifetime, principle of FLIM systems, and evaluation procedure of intracellular environments. In this chapter, the materials, methods, and notes on FLIM measurements have been described, in conjunction with a brief explanation of the background of FLIM.

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

Buying options

Chapter
USD   29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   159.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   159.99
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

Learn about institutional subscriptions

References

  1. Chorvat D Jr, Chorvatova A (2009) Multi-wavelength fluorescence lifetime spectroscopy: a new approach to the study of endogenous fluorescence in living cells and tissues. Laser Phys Lett 6:175–193

    Article  CAS  Google Scholar 

  2. Berezin MY, Achilefu S (2010) Fluorescence lifetime measurements and biological imaging. Chem Rev 110:2641–2684

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  4. Marcu L (2012) Fluorescence lifetime techniques in medical applications. Ann Biomed Eng 40:304–331

    Article  PubMed  PubMed Central  Google Scholar 

  5. Ohta N, Nakabayashi T (2014) Intracellular autofluorescent species: structure, spectroscopy, and photophysics. In: Ghukasyan VV, Heikal AA (eds) Natural biomarkers for cellular metabolism, biology, techniques, and applications. CRC Press, Boca Raton, pp 41–64

    Google Scholar 

  6. Nakabayashi T, Ohta N (2015) Sensing of intracellular environments by fluorescence lifetime imaging of exogenous fluorophores. Anal Chem 31:275–285

    CAS  Google Scholar 

  7. Sarder P, Maji D, Achilefu S (2015) Molecular probes for fluorescence lifetime imaging. Bioconjug Chem 26:963–974

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hirvonen LM, Suhling K (2017) Wide-field TCSPC: methods and applications. Meas Sci Technol 28:012003

    Google Scholar 

  9. Nakabayashi T, Okamoto H, Tasumi M (1998) Vibrational relaxation dynamics of trans-stilbene in the lowest excited singlet state. Pump and probe wavelength dependencies of the picosecond time-resolved anti-Stokes Raman spectrum. J Phys Chem A 102:9686–9695

    Article  CAS  Google Scholar 

  10. Nakabayashi T, Islam MS, Li L et al (2014) Studies on external electric field effects on absorption and fluorescence spectra of NADH. Chem Phys Lett 595-596:25–30

    Article  CAS  Google Scholar 

  11. Suhling K, Siegel J, Phillips D et al (2002) Imaging the environment of green fluorescent protein. Biophys J 83:3589–3595

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. van Manen HJ, Verkuijlen P, Wittendorp P et al (2008) Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy. Biophys J 94:L67–L69

    Article  PubMed  PubMed Central  Google Scholar 

  13. Ogikubo S, Nakabayashi T, Adachi T et al (2011) Intracellular pH sensing using autofluorescence lifetime microscopy. J Phys Chem B 115:10385–10390

    Article  CAS  PubMed  Google Scholar 

  14. Awasthi K, Yamamoto K, Furuya K et al (2015) Fluorescence characteristics and lifetime images of photosensitizers of talaporfin sodium and sodium pheophorbide a in normal and cancer cells. Sensors 15:11417–11430

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Thomas JA, Buchsbaum RN, Zimniak A et al (1979) Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18:2210–2218

    Article  CAS  PubMed  Google Scholar 

  16. Nakabayashi T, Hino K, Ohta Y et al (2011) Electric-field-induced changes in absorption and fluorescence of the green fluorescent protein chromophore in a PMMA film. J Phys Chem B 115:8622–8626

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Takakazu Nakabayashi or Nobuhiro Ohta .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer International Publishing AG

About this chapter

Cite this chapter

Nakabayashi, T., Awasthi, K., Ohta, N. (2017). Application of Fluorescence Lifetime Imaging (FLIM) to Measure Intracellular Environments in a Single Cell. In: Dmitriev, R. (eds) Multi-Parametric Live Cell Microscopy of 3D Tissue Models. Advances in Experimental Medicine and Biology, vol 1035. Springer, Cham. https://doi.org/10.1007/978-3-319-67358-5_8

Download citation

Publish with us

Policies and ethics