Advertisement

Total Internal Reflection Fluorescence (TIRF) Microscopy for Real-Time Imaging of Nanoparticle-Cell Plasma Membrane Interaction

  • Ladan Parhamifar
  • S. Moein MoghimiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 906)

Abstract

Nanoparticulate systems are widely used for site-specific drug and gene delivery as well as for medical imaging. The mode of nanoparticle-cell interaction may have a significant effect on the pathway of nanoparticle internalization and subsequent intracellular trafficking. Total internal reflection fluorescence (TIRF) microscopy allows for real-time monitoring of nanoparticle-membrane interaction events, which can provide vital information in relation to design and surface engineering of therapeutic nanoparticles for cell-specific targeting. In contrast to other microscopy techniques, the bleaching effect by lasers in TIRF microscopy is considerably less when using fluorescent nanoparticles and it reduces photo-induced cytotoxicity during visualization of live-cell events since it only illuminates the specific area near or at the plasma membrane.

Key words

TIRFM Live-cell imaging Cell surface Widefield microscopy Trafficking Nano-particles 

Notes

Acknowledgements

Financial support from the Danish Agency for Science, Technology and Innovation (Det frie forskningsråd for teknologi og production), reference 274-08-0534, and Leica Microsystems (Ballerup, Denmark) are greatly acknowledged.

References

  1. 1.
    Toomre D, Bewersdorf J (2010) A new wave of cellular imaging. Annu Rev Cell Dev Biol 26:285–314PubMedCrossRefGoogle Scholar
  2. 2.
    Axelrod D (1989) Total internal reflection fluorescence microscopy. Methods Cell Biol 30:245–270PubMedCrossRefGoogle Scholar
  3. 3.
    Mattheyses AL, Simon SM, Rappoport JZ (2010) Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci 123:3621–3628PubMedCrossRefGoogle Scholar
  4. 4.
    Stout AL, Axelrod D (1989) Evanescent field excitation of fluorescence by epi-illumination microscopy. Appl Opt 28:5237–5242PubMedCrossRefGoogle Scholar
  5. 5.
    Weiger MC, Wang CC, Krajcovic M, Melvin AT, Rhoden JJ, Haugh JM (2009) Spontaneous phosphoinositide 3-kinase signaling dynamics drive spreading and random migration of fibroblasts. J Cell Sci 122:313–323PubMedCrossRefGoogle Scholar
  6. 6.
    Murray TA, Liu Q, Whiteaker P, Wu J, Lukas RJ (2009) Nicotinic acetylcholine receptor alpha7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors. Acta Pharmacol Sin 30:828–841PubMedCrossRefGoogle Scholar
  7. 7.
    Aaron JS, Greene AC, Kotula PG, Bachand GD, Timlin JA (2010) Advanced optical imaging reveals the dependence of particle geometry on interactions between CdSe quantum dots and immune cells. Small 7:334–341PubMedCrossRefGoogle Scholar
  8. 8.
    Ji W, Xu P, Li Z, Lu J, Liu L, Zhan Y, Chen Y, Hille B, Xu T, Chen L (2008) Functional stoichiometry of the unitary calcium-release-activated calcium channel. Proc Natl Acad Sci U S A 105:13668–13673PubMedCrossRefGoogle Scholar
  9. 9.
    Jones JT, Myers JW, Ferrell JE, Meyer T (2004) Probing the precision of the mitotic clock with a live-cell fluorescent biosensor. Nat Biotechnol 22:306–312PubMedCrossRefGoogle Scholar
  10. 10.

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Centre for Pharmaceutical Nanotechnology and NanotoxicologyUniversity of CopenhagenCopenhagen ØDenmark

Personalised recommendations