Skip to main content
SpringerLink
Log in

Epi-Fluorescence Microscopy

  • Protocol
  • First Online:
Cell Imaging Techniques

Part of the book series: Methods in Molecular Biology ((MIMB,volume 931))

Abstract

Epi-fluorescence microscopy is available in most life sciences research laboratories, and when optimized can be a central laboratory tool. In this chapter, the epi-fluorescence light path is introduced and the various components are discussed in detail. Recommendations are made for incident lamp light sources, excitation and emission filters, dichroic mirrors, objective lenses, and charge-coupled device (CCD) cameras in order to obtain the most sensitive epi-fluorescence microscope. The even illumination of metal-halide lamps combined with new “hard” coated filters and mirrors, a high resolution monochrome CCD camera, and a high NA objective lens are all recommended for high resolution and high sensitivity fluorescence imaging. Recommendations are also made for multicolor imaging with the use of monochrome cameras, motorized filter turrets, individual filter cubes, and corresponding dyes being the best choice for sensitive, high resolution multicolor imaging. Images should be collected using Nyquist sampling and images should be corrected for background intensity contributions and nonuniform illumination across the field of view. Photostable fluorescent probes and proteins that absorb a lot of light (i.e., high extinction co-efficients) and generate a lot of fluorescence signal (i.e., high quantum yields) are optimal. A neuronal immune-fluorescence labeling protocol is also presented. Finally, in order to maximize the utility of sensitive wide-field microscopes and generate the highest resolution images with high signal-to-noise, advice for combining wide-field epi-fluorescence imaging with restorative image deconvolution is presented.

Note: Company names and brands are mentioned throughout the chapter. This information is meant to facilitate scientists who are new to the field and wish to find out more information about the various components of the epi-fluorescence microscope. The authors in no way endorse specific products and apologize for any company who may have inadvertently been left out.

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 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
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. Blaydes JP, Vojtesek B, Bloomberg GB, Hupp TR (2000) The development and use of phospho-specific antibodies to study protein phosphorylation. Methods Mol Biol 99:177–189

    PubMed  CAS  Google Scholar 

  2. DiGiovanna MP, Roussel RR, Stern DF (2002) Production of antibodies that recognize specific tyrosine-phosphorylated peptides. Curr Protoc Cell Biol Chapter 16:Unit 16 16

    Google Scholar 

  3. Xu N, Xu M, Zhang YY (2005) Optical detection of single molecules in living cells. Sheng Li Xue Bao 57:271–277

    PubMed  Google Scholar 

  4. Lang E, Baier J, Kohler J (2006) Epifluorescence, confocal and total internal reflection microscopy for single-molecule experiments: a quantitative comparison. J Microsc 222:118–123

    Article  PubMed  CAS  Google Scholar 

  5. Triller A, Choquet D (2008) New concepts in synaptic biology derived from single-molecule imaging. Neuron 59:359–374

    Article  PubMed  CAS  Google Scholar 

  6. Day RN, Schaufele F (2008) Fluorescent protein tools for studying protein dynamics in living cells: a review. J Biomed Opt 13:031202

    Article  PubMed  Google Scholar 

  7. Giepmans BN, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312:217–224

    Article  PubMed  CAS  Google Scholar 

  8. Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909

    Article  PubMed  CAS  Google Scholar 

  9. Allen GJ, Kwak JM, Chu SP, Llopis J, Tsien RY, Harper JF, Schroeder JI (1999) Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells. Plant J 19:735–747

    Article  PubMed  CAS  Google Scholar 

  10. Dailey ME, Khodjakov A, Rieder CL, Swedlow JR, Andrews PD, Waters JC, Claxton NS, Olenych SG, Griffin JD, Davidson MW (2007) The automated microscope

    Google Scholar 

  11. Wolf DE (2007) Fundamentals of fluorescence and fluorescence microscopy. Methods Cell Biol 81:63–91

    Article  PubMed  Google Scholar 

  12. Webb DJ, Donais K, Whitmore LA, Thomas SM, Turner CE, Parsons JT, Horwitz AF (2004) FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat Cell Biol 6:154–161

    Article  PubMed  CAS  Google Scholar 

  13. Abramowitz M, Davidson MW (2003) Light sources for optical microscopy

    Google Scholar 

  14. Hohman B (2007) LED light source: major advance in fluorescent microscopy. Biomed Instrum Technol 41:461–464

    Article  PubMed  Google Scholar 

  15. Albeanu DF, Soucy E, Sato TF, Meister M, Murthy VN (2008) LED arrays as cost effective and efficient light sources for widefield microscopy. PLoS One 3:e2146

    Article  PubMed  Google Scholar 

  16. Cole RW, Turner JN (2008) Light-emitting diodes are better illumination sources for biological microscopy than conventional sources. Microsc Microanal 14:243–250

    PubMed  CAS  Google Scholar 

  17. Frigault MM, Lacoste J, Swift JL, Brown CM (2009) Live-cell microscopy—tips and tools. J Cell Sci 122:753–767

    Article  PubMed  CAS  Google Scholar 

  18. Abramowitz M, Spring KR, Keller HE, Davidson MW (2002) Basic principles of microscope objectives. Biotechniques 33:772–774; 776–778; 780–771

    Google Scholar 

  19. Keller HE (2006) Objective lenses for confocl microscopy. In: Pawley J (ed) Handbook of biological confocal microscopy, 3rd edn. Springer, New York, pp 145–161

    Chapter  Google Scholar 

  20. North AJ (2006) Seeing is believing? A beginners’ guide to practical pitfalls in image acquisition. J Cell Biol 172:9–18

    Article  PubMed  CAS  Google Scholar 

  21. Standish B (2008) High performance with fluorescence optical filters. BioOptics World 35–37

    Google Scholar 

  22. Erdogan T, Pradhan A, Mizrahi V (2003) Optical filters impact fluorescence fidelity. Biophotonics Int 10:34–38

    Google Scholar 

  23. Spring KR, Fellers TJ, Davidson MW. Introduction to charged-coupled devices (CCDs).

    Google Scholar 

  24. Spring KR (2007) Cameras for digital microscopy. Methods Cell Biol 81:171–186

    Article  PubMed  Google Scholar 

  25. Tran P (2005) CCD cameras for fluorescence imaging of living cells. In: Goldman RD, Spector DL (eds) Live cell imaging: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, pp 87–100

    Google Scholar 

  26. Tsien RY, Ernst L, Waggoner A (2006) Fluorophores for confocal microscopy: photophysics and photochemistry. In: Pawley J (ed) Handbook of biological confocal microscopy, 3rd edn. Springer, New York, pp 338–352

    Chapter  Google Scholar 

  27. Lichtman JW, Conchello JA (2005) Fluorescence microscopy. Nat Methods 2:910–919

    Article  PubMed  CAS  Google Scholar 

  28. Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239

    Article  PubMed  CAS  Google Scholar 

  29. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805

    Article  PubMed  CAS  Google Scholar 

  30. Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572

    Article  PubMed  CAS  Google Scholar 

  31. Day RN, Davidson MW (2009) The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38:2887–2921

    Article  PubMed  CAS  Google Scholar 

  32. Tsien RY (2005) Building and breeding molecules to spy on cells and tumors. FEBS Lett 579:927–932

    Article  PubMed  CAS  Google Scholar 

  33. Goldman RD, Spector DL (2005) Live cell imaging: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  34. Zacharias DA, Violin JD, Newton AC, Tsien RY (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296:913–916

    Article  PubMed  CAS  Google Scholar 

  35. Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY (2001) Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem 276:29188–29194

    Article  PubMed  CAS  Google Scholar 

  36. Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20:87–90

    Article  PubMed  CAS  Google Scholar 

  37. Karasawa S, Araki T, Nagai T, Mizuno H, Miyawaki A (2004) Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem J 381:307–312

    Article  PubMed  CAS  Google Scholar 

  38. Ai HW, Henderson JN, Remington SJ, Campbell RE (2006) Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochem J 400:531–540

    Article  PubMed  CAS  Google Scholar 

  39. Kremers GJ, Gilbert SG, Cranfill PJ, Davidson MW, Piston DW (2011) Fluorescent proteins at a glance. J Cell Sci 124:157–160

    Article  PubMed  CAS  Google Scholar 

  40. Rizzo MA, Davidson MW, Piston DW (2009) Fluorescent protein tracking and detection: applications using fluorescent proteins in living cells. Cold Spring Harb Protoc 2009:pdb top64

    Google Scholar 

  41. Rizzo MA, Davidson MW, Piston DW (2009) Fluorescent protein tracking and detection: fluorescent protein structure and color variants. Cold Spring Harb Protoc 2009:pdb top63

    Google Scholar 

  42. Johnson ID (2006) Practical considerations in the selection and application of fluorescent probes. In: Pawley J (ed) Handbook of biological confocal microscopy, 3rd edn. Springer, New York, pp 353–367

    Chapter  Google Scholar 

  43. The Handbook—A Guide to Fluorescent Probes and Labeling Technologies

    Google Scholar 

  44. Patterson G, Day RN, Piston D (2001) Fluorescent protein spectra. J Cell Sci 114:837–838

    PubMed  CAS  Google Scholar 

  45. Jayaraman S, Haggie P, Wachter RM, Remington SJ, Verkman AS (2000) Mechanism and cellular applications of a green fluorescent protein-based halide sensor. J Biol Chem 275:6047–6050

    Article  PubMed  CAS  Google Scholar 

  46. Nalbant P, Hodgson L, Kraynov V, Toutchkine A, Hahn KM (2004) Activation of endogenous Cdc42 visualized in living cells. Science 305:1615–1619

    Article  PubMed  CAS  Google Scholar 

  47. Kraynov VS, Chamberlain C, Bokoch GM, Schwartz MA, Slabaugh S, Hahn KM (2000) Localized Rac activation dynamics visualized in living cells. Science 290:333–337

    Article  PubMed  CAS  Google Scholar 

  48. Pertz O, Hahn KM (2004) Designing biosensors for Rho family proteins—deciphering the dynamics of Rho family GTPase activation in living cells. J Cell Sci 117:1313–1318

    Article  PubMed  CAS  Google Scholar 

  49. Jaiswal JK, Goldman ER, Mattoussi H, Simon SM (2004) Use of quantum dots for live cell imaging. Nat Methods 1:73–78

    Article  PubMed  Google Scholar 

  50. Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307:538–544

    Article  PubMed  CAS  Google Scholar 

  51. Allison DW, Gelfand VI, Spector I, Craig AM (1998) Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors. J Neurosci 18:2423–2436

    PubMed  CAS  Google Scholar 

  52. Fischer M, Kaech S, Knutti D, Matus A (1998) Rapid actin-based plasticity in dendritic spines. Neuron 20:847–854

    Article  PubMed  CAS  Google Scholar 

  53. Liu Y, Fisher DA, Storm DR (1993) Analysis of the palmitoylation and membrane targeting domain of neuromodulin (GAP-43) by site-specific mutagenesis. Biochemistry 32:10714–10719

    Article  PubMed  CAS  Google Scholar 

  54. Brown CM (2007) Fluorescence microscopy—avoiding the pitfalls. J Cell Sci 120:1703–1705

    Article  PubMed  CAS  Google Scholar 

  55. Stack RF, Bayles CJ, Girard AM, Martin K, Opansky C, Schulz K, Cole RW (2011) Quality assurance testing for modern optical imaging systems. Microsc Microanal 17:598–606

    Article  PubMed  CAS  Google Scholar 

  56. Kraus B, Zeigler M, Wolff H (2007) Linear fluorescence unmixing in cell biological research. In: Modern research and educational topics in microscopy. Formatex Microscopy Book Series, Badajoz, Spain, pp 863–873

    Google Scholar 

  57. Swedlow JR, Hu K, Andrews PD, Roos DS, Murray JM (2002) Measuring tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and wide-field fluorescence microscopy. Proc Natl Acad Sci U S A 99:2014–2019

    Article  PubMed  CAS  Google Scholar 

  58. Swedlow JR, Platani M (2002) Live cell imaging using wide-field microscopy and deconvolution. Cell Struct Funct 27:335–341

    Article  PubMed  Google Scholar 

  59. McNally JG, Karpova T, Cooper J, Conchello JA (1999) Three-dimensional imaging by deconvolution microscopy. Methods 19:373–385

    Article  PubMed  CAS  Google Scholar 

  60. Swedlow JR (2007) Quantitative fluorescence microscopy and image deconvolution. Methods Cell Biol 81:447–465

    Article  PubMed  CAS  Google Scholar 

  61. Wallace W, Schaefer LH, Swedlow JR (2001) A workingperson’s guide to deconvolution in light microscopy. Biotechniques 31:1076–1078; 1080; 1082 passim

    Google Scholar 

  62. Murray JM (2005) Confocal microscopy, deconvolution, and structured illumination methods. In: Goldman RD, Spector DL (eds) Live cell imaging: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, pp 239–279

    Google Scholar 

  63. Shaw PJ (2006) Comparison of widefield/deconvolution and confocal microscopy for three-dimensional imaging. In: Pawley J (ed) Handbook of biological confocal microscopy, 3rd edn. Springer, New York, pp 453–467

    Chapter  Google Scholar 

  64. Holmes TJ, Biggs D, Abu-Tarif A (2006) Blind deconvolution. In: Pawley J (ed) Handbook of biological confocal microscopy, 3rd edn. Springer, New York, pp 468–487

    Chapter  Google Scholar 

  65. Wallace W, Schaefer LH, Swedlow JR, Fellers TJ, Davidson MW. Deconvolution in optical microscopy

    Google Scholar 

  66. Nyquist H (1928) Certain topics in telegraph transmission theory. Trans AIEE 47:617–644

    Google Scholar 

  67. Cole RW, Jinadasa T, Brown CM (2011) Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control. Nat Protoc 6(12):1929–1941

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the McGill Life Sciences Complex Imaging Facility (CMB) and by grant MH071674 from NIH to DJW. Thank you goes out to the following people: Loralei Dewe formerly of Media Cybernetics and David Hitrys formerly of QImaging Corporation for their work performing the deconvolution of raw widefield images using AutoQuant X software for Fig. 7 and for generating the images used in Fig. 8a, b; Media Cybernetics for providing funding to print Fig. 8 in color; Cory Glowinski from Bitplane Inc. for generating the 3D iso-surfaces for Fig. 8c, d using Imaris Software; Adam Wegner for collecting the neuronal images in Fig. 5; Michael Davidson for providing the microscope and fluorescence illuminator figures in Fig. 1a, b; and Olympus Canada for providing the equipment in order to generate the images for Figs. 2 and 4; Brady Eason for critically reading the chapter.

Author information

Authors and Affiliations

  1. Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA

    Donna J. Webb

  2. Department of Physiology, Life Sciences Complex Imaging Facility, McGill University, Montreal, Canada

    Claire M. Brown

Authors
  1. Donna J. Webb
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Claire M. Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Claire M. Brown .

Editor information

Editors and Affiliations

  1. College of Medicine, Professor, Department of Pathology, University of Vermont, Beaumont Ave 89, Burlington, 05405-1742, Vermont, USA

    Douglas J. Taatjes

  2. , Abt. Zell- und Molekularpathologie, Universität Zürich, Schmelzbergstr. 12, Zürich, 8091, Switzerland

    Jürgen Roth

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Webb, D.J., Brown, C.M. (2012). Epi-Fluorescence Microscopy. In: Taatjes, D., Roth, J. (eds) Cell Imaging Techniques. Methods in Molecular Biology, vol 931. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-056-4_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-056-4_2

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-055-7

  • Online ISBN: 978-1-62703-056-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation