Skip to main content
SpringerLink
Log in

Optimizing Long-Term Live Cell Imaging

  • Protocol
  • First Online:
Fluorescent Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2440))

Abstract

Live cell microscopy has become a common technique for exploring dynamic biological processes. When combined with fluorescent markers of cellular structures of interest, or fluorescent reporters of a biological activity of interest, live cell microscopy enables precise temporally and spatially resolved quantitation of the biological processes under investigation. However, because living cells are not normally exposed to light, live cell fluorescence imaging is significantly hindered by the effects of photodamage, which encompasses photobleaching of fluorophores and phototoxicity of the cells under observation. In this chapter, we outline several methods for optimizing and maintaining long-term imaging of live cells while simultaneously minimizing photodamage. This protocol demonstrates the intracellular trafficking of early and late endosomes following phagocytosis using both two and three dimensional imaging, but this protocol can easily be modified to image any biological process of interest in nearly any cell type.

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

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
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

Similar content being viewed by others

References

  1. Cole R (2014) Live-cell imaging. Cell Adh Migr 8:452–459

    Article  PubMed  PubMed Central  Google Scholar 

  2. Magidson V, Khodjakov A (2013) Circumventing photodamage in live-cell microscopy. Methods Cell Biol 114:545–560

    Article  PubMed  Google Scholar 

  3. Icha J, Weber M, Waters JC et al (2017) Phototoxicity in live fluorescence microscopy, and how to avoid it. Bioessays 39:1700003

    Article  Google Scholar 

  4. Stennett EMS, Ciuba MA, Levitus M (2014) Photophysical processes in single molecule organic fluorescent probes. Chem Soc Rev 43:1057–1075

    Article  CAS  PubMed  Google Scholar 

  5. Laloi C, Havaux M (2015) Key players of singlet oxygen-induced cell death in plants. Front Plant Sci 6:39

    Article  PubMed  PubMed Central  Google Scholar 

  6. Thorpe GW, Fong CS, Alic N et al (2004) Cells have distinct mechanisms to maintain protection against different reactive oxygen species: oxidative-stress-response genes. Proc Natl Acad Sci U S A 101:6564–6569

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682

    Article  CAS  PubMed  Google Scholar 

  8. Flannagan RS, Jaumouillé V, Grinstein S (2011) The cell biology of phagocytosis. Annu Rev Pathol 7:61–98

    Article  CAS  PubMed  Google Scholar 

  9. Hennies CM, Lehn MA, Janssen EM (2015) Quantitating MHC class II trafficking in primary dendritic cells using imaging flow cytometry. J Immunol Methods 423:18–28

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bohdanowicz M, Grinstein S (2010) Vesicular traffic: a Rab SANDwich. Curr Biol 20:R311–R314

    Article  CAS  PubMed  Google Scholar 

  11. Hwang J-R, Byeon Y, Kim D et al (2020) Recent insights of T cell receptor-mediated signaling pathways for T cell activation and development. Exp Mol Med 52:750–761

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kagan JC, Iwasaki A (2012) Phagosome as the organelle linking innate and adaptive immunity. Traffic 13:1053–1061

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hampton MB, Vissers MC, Winterbourn CC (1994) A single assay for measuring the rates of phagocytosis and bacterial killing by neutrophils. J Leukoc Biol 55:147–152

    Article  CAS  PubMed  Google Scholar 

  14. Paul D, Achouri S, Yoon Y-Z et al (2013) Phagocytosis dynamics depends on target shape. Biophys J 105:1143–1150

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Vieira OV, Bucci C, Harrison RE et al (2003) Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase. Mol Cell Biol 23:2501–2514

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Yin C, Kim Y, Argintaru D et al (2016) Rab17 mediates differential antigen sorting following efferocytosis and phagocytosis. Cell Death Dis 7:e2529

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wong C-O, Gregory S, Hu H et al (2017) Lysosomal degradation is required for sustained phagocytosis of bacteria by macrophages. Cell Host Microbe 21:719–730.e6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kleijmeer MJ, Ossevoort MA, van Veen CJ et al (1995) MHC class II compartments and the kinetics of antigen presentation in activated mouse spleen dendritic cells. J Immunol 154:5715–5724

    CAS  PubMed  Google Scholar 

  19. Banaz N, Mäkelä J, Uphoff S (2019) Choosing the right label for single-molecule tracking in live bacteria: side-by-side comparison of photoactivatable fluorescent protein and halo tag dyes. J Phys D Appl Phys 52:064002

    Article  CAS  PubMed  Google Scholar 

  20. Lambert TJ (2019) FPbase: a community-editable fluorescent protein database. Nat Methods 16:277–278

    Article  CAS  PubMed  Google Scholar 

  21. Perskvist N, Roberg K, Kulyté A et al (2002) Rab5a GTPase regulates fusion between pathogen-containing phagosomes and cytoplasmic organelles in human neutrophils. J Cell Sci 115:1321–1330

    Article  CAS  PubMed  Google Scholar 

  22. Rupper A, Grove B, Cardelli J (2001) Rab7 regulates phagosome maturation in. Dictyostelium 114:2449–2460

    CAS  Google Scholar 

  23. Bogdanov AM, Kudryavtseva EI, Lukyanov KA (2012) Anti-fading media for live cell GFP imaging. PLoS One 7:e53004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Comes MC, Casti P, Mencattini A et al (2019) The influence of spatial and temporal resolutions on the analysis of cell-cell interaction: a systematic study for time-lapse microscopy applications. Sci Rep 9:6789

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kreft M, Stenovec M, Zorec R (2005) Focus-drift correction in time-lapse confocal imaging. Ann N Y Acad Sci 1048:321–330

    Article  PubMed  Google Scholar 

  26. Zhao Q, Young IT, de Jong JGS (2011) Photon budget analysis for fluorescence lifetime imaging microscopy. J Biomed Opt 16:086007

    Article  PubMed  Google Scholar 

  27. Mubaid F, Brown CM (2017) Less is more: longer exposure times with low light intensity is less photo-toxic. Microsc Today 25:26–35

    Article  CAS  Google Scholar 

  28. Wu P-H, Nelson N, Tseng Y (2010) A general method for improving spatial resolution by optimization of electron multiplication in CCD imaging. Opt Express 18:5199–5212

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jin X, Hirakawa K (2012) Analysis and processing of pixel binning for color image sensor. EURASIP J Adv Signal Process 2012:125

    Article  Google Scholar 

  30. Hu X, Jalal S, Sheetz M et al (2020) Micro-stepping extended focus reduces photobleaching and preserves structured illumination super-resolution features. J Cell Sci 133:jcs240796

    Article  CAS  PubMed  Google Scholar 

  31. Shihavuddin A, Basu S, Rexhepaj E et al (2017) Smooth 2D manifold extraction from 3D image stack. Nat Commun 8:15554

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 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 

  33. Carlton PM, Boulanger J, Kervrann C et al (2010) Fast live simultaneous multiwavelength four-dimensional optical microscopy. Proc Natl Acad Sci U S A 107:16016–16022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was funded by a Canadian Institutes of Health Research Project Grant (PJT-162203) to BH.

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, The University of Western Ontario, London, ON, Canada

    Alex Lac, Austin Le Lam & Bryan Heit

  2. Robarts Research Institute, London, ON, Canada

    Bryan Heit

Authors
  1. Alex Lac
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Austin Le Lam
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bryan Heit
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bryan Heit .

Editor information

Editors and Affiliations

  1. Department of Microbiology and Immunology, Center for Human Immunology, The University of Western Ontario, London, ON, Canada

    Bryan Heit

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Lac, A., Le Lam, A., Heit, B. (2022). Optimizing Long-Term Live Cell Imaging. In: Heit, B. (eds) Fluorescent Microscopy. Methods in Molecular Biology, vol 2440. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2051-9_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2051-9_3

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2050-2

  • Online ISBN: 978-1-0716-2051-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation