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Sample Preparation and Choice of Fluorophores for Single and Dual Color Photo-Activated Localization Microscopy (PALM) with Bacterial Cells

  • Juri N. Bach
  • Giacomo Giacomelli
  • Marc BramkampEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1563)

Abstract

Photo-activated localization microscopy (PALM) is one of the light microscopy techniques providing highest resolution. Single photo-activatable or photo-switchable fluorescent molecules are stochastically excited. The point spread function of this event is recorded and the exact fluorophore position is calculated. This chapter describes how bacterial samples can be prepared for PALM to achieve routinely a resolution of ≤30 nm using fluorophores such as mNeonGreen, Dendra2, and PAmCherry. It is also explained how to perform multicolor PALM and combine it with total internal reflection (TIRF) microscopy to increase resolution.

Key words

Localization Microscopy PALM STORM Dendra2 PAmCherry mNeonGreen Super resolution Dual color PALM 

References

  1. 1.
    Abbe E (1883) XV.—The relation of aperture and power in the microscope (continued)*. J R Microsc Soc 3(6):790–812Google Scholar
  2. 2.
    Rayleigh L (1879) XXXI. Investigations in optics, with special reference to the spectroscope. Philos Mag 8(49):261–274. Series 5Google Scholar
  3. 3.
    Rudner DZ, Losick R (2010) Protein subcellular localization in bacteria. Cold Spring Harb Perspect Biol 2(4):a000307CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Shapiro L, McAdams HH, Losick R (2009) Why and how bacteria localize proteins. Science 326(5957):1225–1228CrossRefPubMedGoogle Scholar
  5. 5.
    Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10):793–795CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Betzig E et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793):1642–1645CrossRefPubMedGoogle Scholar
  7. 7.
    Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91(11):4258–4272CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Adam V, Nienhaus K, Bourgeois D, Nienhaus GU (2009) Structural basis of enhanced photoconversion yield in green fluorescent protein-like protein Dendra2. Biochemistry 48(22):4905–4915CrossRefPubMedGoogle Scholar
  9. 9.
    Bach JN, Bramkamp M (2013) Flotillins functionally organize the bacterial membrane. Mol Microbiol 88(6):1205–1217CrossRefPubMedGoogle Scholar
  10. 10.
    Bach JN, Bramkamp M (2015) Dissecting the molecular properties of prokaryotic flotillins. PLoS One 10(1):e0116750CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bramkamp M, Lopez D (2015) Exploring the existence of lipid rafts in bacteria. Microbiol Mol Biol Rev 79(1):81–100CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Donovan C, Schwaiger A, Kramer R, Bramkamp M (2010) Subcellular localization and characterization of the ParAB system from Corynebacterium glutamicum. J Bacteriol 192(13):3441–3451CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Donovan C, Sieger B, Kramer R, Bramkamp M (2012) A synthetic Escherichia coli system identifies a conserved origin tethering factor in Actinobacteria. Mol Microbiol 84(1):105–116CrossRefPubMedGoogle Scholar
  14. 14.
    Chozinski TJ, Gagnon LA, Vaughan JC (2014) Twinkle, twinkle little star: photoswitchable fluorophores for super-resolution imaging. FEBS Lett 588(19):3603–3612CrossRefPubMedGoogle Scholar
  15. 15.
    Annibale P, Vanni S, Scarselli M, Rothlisberger U, Radenovic A (2011) Quantitative photo activated localization microscopy: unraveling the effects of photoblinking. PLoS One 6(7):e22678CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Jacq M et al (2015) Remodeling of the Z-ring nanostructure during the Streptococcus pneumoniae cell cycle revealed by photoactivated localization microscopy. MBio 6(4)Google Scholar
  17. 17.
    Buss J et al (2013) In vivo organization of the FtsZ-ring by ZapA and ZapB revealed by quantitative super-resolution microscopy. Mol Microbiol 89(6):1099–1120CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Haas BL, Matson JS, DiRita VJ, Biteen JS (2014) Imaging live cells at the nanometer-scale with single-molecule microscopy: obstacles and achievements in experiment optimization for microbiology. Molecules 19(8):12116–12149CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Lee SH, Shin JY, Lee A, Bustamante C (2012) Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM). Proc Natl Acad Sci U S A 109(43):17436–17441CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Shaner NC et al (2013) A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 10(5):407–409CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Wang S, Moffitt JR, Dempsey GT, Xie XS, Zhuang X (2014) Characterization and development of photoactivatable fluorescent proteins for single-molecule-based superresolution imaging. Proc Natl Acad Sci U S A 111(23):8452–8457CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Shcherbakova DM, Sengupta P, Lippincott-Schwartz J, Verkhusha VV (2014) Photocontrollable fluorescent proteins for superresolution imaging. Annu Rev Biophys 43:303–329CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Ong WQ, Citron YR, Schnitzbauer J, Kamiyama D, Huang B (2015) Heavy water: a simple solution to increasing the brightness of fluorescent proteins in super-resolution imaging. Chem Commun 51(70):13451–13453CrossRefGoogle Scholar
  24. 24.
    Andresen M et al (2008) Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy. Nat Biotechnol 26(9):1035–1040CrossRefPubMedGoogle Scholar
  25. 25.
    Subach OM, Entenberg D, Condeelis JS, Verkhusha VV (2012) A FRET-facilitated photoswitching using an orange fluorescent protein with the fast photoconversion kinetics. J Am Chem Soc 134(36):14789–14799CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Subach FV et al (2009) Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat Methods 6(2):153–159CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Juri N. Bach
    • 1
  • Giacomo Giacomelli
    • 1
  • Marc Bramkamp
    • 1
    Email author
  1. 1.Faculty of BiologyLudwig-Maximilians-University MunichMunichGermany

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