Advertisement

European Biophysics Journal

, Volume 39, Issue 6, pp 947–957 | Cite as

Understanding biological dynamics: following cells and molecules to track functions and mechanisms

  • A. Palamidessi
  • I. Testa
  • E. Frittoli
  • S. Barozzi
  • M. Garrè
  • D. Mazza
  • P. P. Di Fiore
  • A. Diaspro
  • G. Scita
  • Mario Faretta
Original Paper

Abstract

The dissection of the molecular circuitries at the base of cell life and the identification of their abnormal transformation during carcinogenesis rely on the characterization of biological phenotypes generated by targeted overexpression or deletion of gene products through genetic manipulation. Fluorescence microscopy provides a wide variety of tools to monitor cell life with minimal perturbations. The observation of living cells requires the selection of a correct balance between temporal, spatial and “statistical” resolution according to the process to be analyzed. In the following paper ad hoc developed optical tools for dynamical tracking from cellular to molecular resolution will be presented. Particular emphasis will be devoted to discuss how to exploit light–matter interaction to selectively target specific molecular species, understanding the relationships between their intracellular compartmentalization and function.

Keywords

Fluorescence microscopy Cell tracking Fluorescence recovery after photobleaching Photoactivation Two-photon microscopy 

Notes

Acknowledgments

This work was supported by IIT (Italian Institute of Technology, Genoa, Italy) and by IFOM (FIRC Institute of Molecular Oncology, Milan, Italy). The authors are grateful to Dr. Francesca Ballarini for her help in manuscript preparation.

References

  1. Bansal V, Patel S, Saggau P (2006) High-speed addressable confocal microscopy for functional imaging of cellular activity. J Biomed Opt 11:34003. doi: 10.1117/1.2209562 CrossRefPubMedGoogle Scholar
  2. Diaspro A, Testa I, Faretta M, Magrassi R, Barozzi S, Parazzoli D, Vicidomini G (2006) 3D localized photoactivation of pa-GFP in living cells using two-photon interactions. Conf Proc IEEE Eng Med Biol Soc 1:389–391CrossRefPubMedGoogle Scholar
  3. Graf R, Rietdorf J, Zimmermann T (2005) Live cell spinning disk microscopy. Adv Biochem Eng Biotechnol 95:57–75PubMedGoogle Scholar
  4. Hell SW (2007) Far-field optical nanoscopy. Science 316:1153–1158. doi: 10.1126/science.1137395 CrossRefPubMedGoogle Scholar
  5. Hell SW (2009) Microscopy and its focal switch. Nat Methods 6:24–32. doi: 10.1038/nmeth.1291 CrossRefPubMedGoogle Scholar
  6. Keller PJ, Schmidt AD, Wittbrodt J, Stelzer EH (2008) Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322:1065–1069. doi: 10.1126/science.1162493 CrossRefPubMedGoogle Scholar
  7. Kim SY, Gitai Z, Kinkhabwala A, Shapiro L, Moerner WE (2006) Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus. Proc Natl Acad Sci USA 103:10929–10934. doi: 10.1073/pnas.0604503103 CrossRefPubMedGoogle Scholar
  8. Kurtz R, Fricke M, Kalb J, Tinnefeld P, Sauer M (2006) Application of multiline two-photon microscopy to functional in vivo imaging. J Neurosci Methods 151:276–286. doi: 10.1016/j.jneumeth.2005.12.003 CrossRefPubMedGoogle Scholar
  9. Lanzetti L, Palamidessi A, Areces L, Scita G, Di Fiore PP (2004) Rab5 is a signalling GTPase involved in actin remodelling by receptor tyrosine kinases. Nature 429:309–314. doi: 10.1038/nature02542 CrossRefPubMedGoogle Scholar
  10. Leake MC, Chandler JH, Wadhams GH, Bai F, Berry RM, Armitage JP (2006) Stoichiometry and turnover in single, functioning membrane protein complexes. Nature 443:355–358. doi: 10.1038/nature05135 CrossRefPubMedGoogle Scholar
  11. Leake MC, Greene NP, Godun RM, Granjon T, Buchanan G, Chen S, Berry RM, Palmer T, Berks BC (2008) Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging. Proc Natl Acad Sci USA 105:15376–15381. doi: 10.1073/pnas.0806338105 CrossRefPubMedGoogle Scholar
  12. Lippincott-Schwartz J, Manley S (2009) Putting super-resolution fluorescence microscopy to work. Nat Methods 6:21–23. doi: 10.1038/nmeth.f.233 CrossRefPubMedGoogle Scholar
  13. Maddox PS, Moree B, Canman JC, Salmon ED (2003) Spinning disk confocal microscope system for rapid high-resolution, multimode, fluorescence speckle microscopy and green fluorescent protein imaging in living cells. Methods Enzymol 360:597–617. doi: 10.1016/S0076-6879(03)60130-8 CrossRefPubMedGoogle Scholar
  14. Palamidessi A, Frittoli E, Garre M, Faretta M, Mione M, Testa I, Diaspro A, Lanzetti L, Scita G, Di Fiore PP (2008) Endocytic trafficking of Rac is required for the spatial restriction of signaling in cell migration. Cell 134:135–147. doi: 10.1016/j.cell.2008.05.034 CrossRefPubMedGoogle Scholar
  15. Reddy GD, Saggau P (2005) Fast three-dimensional laser scanning scheme using acousto-optic deflectors. J Biomed Opt 10:064038. doi: 10.1117/1.2141504 CrossRefPubMedGoogle Scholar
  16. Saggau P (2006) New methods and uses for fast optical scanning. Curr Opin Neurobiol 16:543–550. doi: 10.1016/j.conb.2006.08.011 CrossRefPubMedGoogle Scholar
  17. Sahai E, Marshall CJ (2002) RHO-GTPases and cancer. Nat Rev Cancer 2:133–142. doi: 10.1038/nrc725 CrossRefPubMedGoogle Scholar
  18. Sahai E, Marshall CJ (2003) Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol 5:711–719. doi: 10.1038/ncb1019 CrossRefPubMedGoogle Scholar
  19. Testa I, Garre M, Parazzoli D, Barozzi S, Ponzanelli I, Mazza D, Faretta M, Diaspro A (2008a) Photoactivation of pa-GFP in 3D: optical tools for spatial confinement. Eur Biophys J 37:1219–1227. doi: 10.1007/s00249-008-0317-9 CrossRefPubMedGoogle Scholar
  20. Testa I, Parazzoli D, Barozzi S, Garre M, Faretta M, Diaspro A (2008b) Spatial control of pa-GFP photoactivation in living cells. J Microsc 230:48–60. doi: 10.1111/j.1365-2818.2008.01951.x CrossRefPubMedGoogle Scholar
  21. Ulbrich MH, Isacoff EY (2007) Subunit counting in membrane-bound proteins. Nat Methods 4:319–321PubMedGoogle Scholar
  22. Wolleschensky R, Zimmermann B, Kempe M (2006) High-speed confocal fluorescence imaging with a novel line scanning microscope. J Biomed Opt 11:064011. doi: 10.1117/1.2402110 CrossRefPubMedGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2009

Authors and Affiliations

  • A. Palamidessi
    • 3
  • I. Testa
    • 2
  • E. Frittoli
    • 3
  • S. Barozzi
    • 1
    • 4
  • M. Garrè
    • 3
    • 4
  • D. Mazza
    • 2
  • P. P. Di Fiore
    • 3
    • 5
  • A. Diaspro
    • 2
    • 3
  • G. Scita
    • 3
    • 5
  • Mario Faretta
    • 1
    • 6
  1. 1.Dipartimento di Oncologia SperimentaleIEO, Istituto Europeo di OncologiaMilanItaly
  2. 2.LAMBS-MicroScoBio, Department of PhysicsUniversity of GenoaGenoaItaly
  3. 3.IFOM, Fondazione Istituto FIRC di Oncologia MolecolareMilanItaly
  4. 4.Consortium for Genomic Technologies, COGENTECHMilanItaly
  5. 5.Dipartimento di Medicina, Chirurgia ed OdontoiatriaUniversita′ degli Studi di MilanoMilanItaly
  6. 6.Department of Experimental OncologyIEOMilanItaly

Personalised recommendations