Location via proxy:   [ UP ]  
[Report a bug]   [Manage cookies]                
Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Fluorescence microscopy

Nature Methods volume 2, pages 910–919 (2005)Cite this article

Abstract

Although fluorescence microscopy permeates all of cell and molecular biology, most biologists have little experience with the underlying photophysical phenomena. Understanding the principles underlying fluorescence microscopy is useful when attempting to solve imaging problems. Additionally, fluorescence microscopy is in a state of rapid evolution, with new techniques, probes and equipment appearing almost daily. Familiarity with fluorescence is a prerequisite for taking advantage of many of these developments. This review attempts to provide a framework for understanding excitation of and emission by fluorophores, the way fluorescence microscopes work, and some of the ways fluorescence can be optimized.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Fluorescence fundamentals.
Figure 2: The spectral and structural properties of fluorophores.
Figure 3: The fluorescence microscope.

Similar content being viewed by others

References

  1. Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  Google Scholar 

  2. Valeur, B. Molecular Fluorescence: Principles and Applications. (Wiley-VCH, Weinheim, 2002).

    Google Scholar 

  3. Lakowicz, J.R., ed. Principles of Fluorescence Spectroscopy. 2nd ed. (Plenum Press, New York, 1999).

    Book  Google Scholar 

  4. Turro, N.J. Modern Molecular Photochemistry. (University Science Books, Sausalito, California, 1991).

    Google Scholar 

  5. Irvine, D.J., Purbhoo, M.A., Krogsgaard, M. & Davis, M.M. Direct observation of ligand recognition by T cells. Nature 419, 845–849 (2002).

    Article  CAS  Google Scholar 

  6. Wang, L., Jackson, W.C., Steinbach, P.A. & Tsien, R.Y. Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc. Natl. Acad. Sci. USA 101, 16745–16749 (2004).

    Article  CAS  Google Scholar 

  7. Deerinck, T.J. et al. Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy. J. Cell Biol. 126, 901–910 (1994).

    Article  CAS  Google Scholar 

  8. Suhling, K., French, P.M. & Phillips, D. Time-resolved fluorescence microscopy. Photochem. Photobiol. Sci. 4, 13–22 (2005).

    Article  CAS  Google Scholar 

  9. Elson, D. et al. Time-domain fluorescence lifetime imaging applied to biological tissue. Photochem. Photobiol. Sci. 3, 795–801 (2004).

    Article  CAS  Google Scholar 

  10. Jares-Erijman, E.A. & Jovin, T.M. FRET imaging. Nat. Biotechnol. 21, 1387–1395 (2003).

    Article  CAS  Google Scholar 

  11. Mizuno, H. et al. Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. Mol. Cell 12, 1051–1058 (2003).

    Article  CAS  Google Scholar 

  12. Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. & Miyawaki, A. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 12651–12656 (2002).

    Article  CAS  Google Scholar 

  13. Mason, W.T., ed. Fluorescent and Luminescent Probes for Biological Activity: A Practical Guide for Quantitative Real-Time Analysis. (Academic Press Harcourt Brace and Co., Boston, 1993).

    Google Scholar 

  14. Periasamy, A., ed. Methods in Cellular Imaging (Oxford University Press, Oxford, 2001).

    Book  Google Scholar 

  15. Rudolf, R., Mongillo, M., Rizzuto, R. & Pozzan, T. Looking forward to seeing calcium. Nat. Rev. Mol. Cell Biol. 4, 579–586 (2003).

    Article  CAS  Google Scholar 

  16. Tsien, R.Y. Fluorescent indicators of ion concentrations. Methods Cell Biol. 30, 127–156 (1989).

    Article  CAS  Google Scholar 

  17. Bassnett, S., Reinisch, L. & Beebe, D.C. Intracellular pH measurement using single excitation-dual emission fluorescence ratios. Am. J. Physiol. 258, 171–178 (1990).

    Article  Google Scholar 

  18. Yuste, R. & Konnerth, A., eds. Imaging in Neuroscience and Development: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, New York, 2005).

    Google Scholar 

  19. Zhang, J., Campbell, R.E., Ting, A.Y. & Tsien, R.Y. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3, 906–918 (2002).

    Article  CAS  Google Scholar 

  20. Griesbeck, O. Fluorescent proteins as sensors for cellular functions. Curr. Opin. Neurobiol. 14, 636–641 (2004).

    Article  CAS  Google Scholar 

  21. Ohmichi, T. et al. DNA-based biosensor for monitoring pH in vitro and in living cells. Biochemistry 44, 7125–7130 (2005).

    Article  CAS  Google Scholar 

  22. Grinvald, A. & Hildesheim, R. VSDI: a new era in functional imaging of cortical dynamics. Nat. Rev. Neurosci. 5, 874–885 (2004).

    Article  CAS  Google Scholar 

  23. Kuhn, B., Fromherz, P. & Denk, W. High sensitivity of Stark-shift voltage-sensing dyes by one- or two-photon excitation near the red spectral edge. Biophys. J. 87, 631–639 (2004).

    Article  CAS  Google Scholar 

  24. Reiff, D.F. et al. In vivo performance of genetically encoded indicators of neural activity in flies. J. Neurosci. 25, 4766–4778 (2005).

    Article  CAS  Google Scholar 

  25. Goldman, R.D. & Spector, D.L., eds. Live Cell Imaging: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, New York, 2004).

    Google Scholar 

  26. Inoué, S. & Spring, K.R. Video Microscopy. 2nd edn. (Plenum Publishing, New York, 1997).

    Book  Google Scholar 

  27. Abramowitz, M., Spring, K.R., Keller, H.E. & Davidson, M.W. Basic principles of microscope objectives. Biotechniques 33, 772–781 (2002).

    Article  CAS  Google Scholar 

  28. Panchuk-Voloshina, N. et al. Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright photostable conjugates. J. Histochem. Cytochem. 47, 1179–1188 (1999).

    Article  CAS  Google Scholar 

  29. Ono, M. et al. Quantitative comparison of anti-fading mounting media for confocal laser scanning microscopy. J. Histochem. Cytochem. 49, 305–331 (2001).

    Article  CAS  Google Scholar 

  30. Johnson, G.D. & Nogueira Araujo, G.M. A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Methods 43, 349–350 (1981).

    Article  CAS  Google Scholar 

  31. Johnson, G.D. et al. Fading of immunofluorescence during microscopy: a study of the phenomenon and its remdedy. J. Immunol. Methods 55, 231–242 (1982).

    Article  CAS  Google Scholar 

  32. Giloh, H. & Sedat, J.W. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217, 1252–1255 (1982).

    Article  CAS  Google Scholar 

  33. Alivisatos, A.P., Gu, W. & Larabell, C. Quantum dots as cellular probes. Annu. Rev. Biomed. Eng. 7, 55–76 (2005).

    Article  CAS  Google Scholar 

  34. van Gijlswijk, R.P. et al. Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization. J. Histochem. Cytochem. 45, 375–382 (1997).

    Article  CAS  Google Scholar 

  35. Cullander, C. Imaging in the far-red with electronic light microscopy: requirements and limitations. J. Microsc. 176, 281–286 (1994).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular and Cell Biology, Harvard University, 7 Divinity Avenue, Cambridge, 02138, Massachusetts, USA

    Jeff W Lichtman

  2. Molecular, Cell and Developmental Biology, Oklahoma Medical Research Foundation, 825 N.E., 13th Street, Oklahoma City, 73104, Oklahoma, USA

    José-Angel Conchello

  3. Program in Biomedical Engineering, University of Oklahoma, 100 East Boyd Street, Norman, 73019, Oklahoma, USA

    José-Angel Conchello

Authors
  1. Jeff W Lichtman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. José-Angel Conchello
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jeff W Lichtman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lichtman, J., Conchello, JA. Fluorescence microscopy. Nat Methods 2, 910–919 (2005). https://doi.org/10.1038/nmeth817

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth817

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing