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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 21 — Oct. 21, 2013
  • pp: 25291–25306

Bleaching-corrected fluorescence microspectroscopy with nanometer peak position resolution

Iztok Urbančič, Zoran Arsov, Ajasja Ljubetič, Daniele Biglino, and Janez Štrancar  »View Author Affiliations

Optics Express, Vol. 21, Issue 21, pp. 25291-25306 (2013)

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Fluorescence microspectroscopy (FMS) with environmentally sensitive dyes provides information about local molecular surroundings at microscopic spatial resolution. Until recently, only probes exhibiting large spectral shifts due to local changes have been used. For filter-based experimental systems, where signal at different wavelengths is acquired sequentially, photostability has been required in addition. Herein, we systematically analyzed our spectral fitting models and bleaching correction algorithms which mitigate both limitations. We showed that careful analysis of data acquired by stochastic wavelength sampling enables nanometer spectral peak position resolution even for highly photosensitive fluorophores. To demonstrate how small spectral shifts and changes in bleaching rates can be exploited, we analyzed vesicles in different lipid phases. Our findings suggest that a wide range of dyes, commonly used in bulk spectrofluorimetry but largely avoided in microspectroscopy due to the above-mentioned restrictions, can be efficiently applied also in FMS.

© 2013 Optical Society of America

OCIS Codes
(000.2170) General : Equipment and techniques
(000.4430) General : Numerical approximation and analysis
(070.4790) Fourier optics and signal processing : Spectrum analysis
(180.2520) Microscopy : Fluorescence microscopy
(300.6280) Spectroscopy : Spectroscopy, fluorescence and luminescence
(110.4234) Imaging systems : Multispectral and hyperspectral imaging

ToC Category:

Original Manuscript: August 12, 2013
Revised Manuscript: October 7, 2013
Manuscript Accepted: October 8, 2013
Published: October 16, 2013

Iztok Urbančič, Zoran Arsov, Ajasja Ljubetič, Daniele Biglino, and Janez Štrancar, "Bleaching-corrected fluorescence microspectroscopy with nanometer peak position resolution," Opt. Express 21, 25291-25306 (2013)

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  1. J. Pawley, Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, 2006).
  2. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, 2006).
  3. T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett.546(1), 87–92 (2003). [CrossRef] [PubMed]
  4. Y. Garini and E. Tauber, “Spectral imaging: methods, design, and applications,” in Biomedical Optical Imaging Technologies, R. Liang, ed. (Springer, 2013), pp. 111–161.
  5. R. A. Neher, M. Mitkovski, F. Kirchhoff, E. Neher, F. J. Theis, and A. Zeug, “Blind source separation techniques for the decomposition of multiply labeled fluorescence images,” Biophys. J.96(9), 3791–3800 (2009). [CrossRef] [PubMed]
  6. H. Xu and B. W. Rice, “In-vivo fluorescence imaging with a multivariate curve resolution spectral unmixing technique,” J. Biomed. Opt.14(6), 064011 (2009). [CrossRef] [PubMed]
  7. A. M. Valm, J. L. Mark Welch, C. W. Rieken, Y. Hasegawa, M. L. Sogin, R. Oldenbourg, F. E. Dewhirst, and G. G. Borisy, “Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging,” Proc. Natl. Acad. Sci. U.S.A.108(10), 4152–4157 (2011). [CrossRef] [PubMed]
  8. F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express20(12), 12729–12741 (2012). [CrossRef] [PubMed]
  9. A. P. Demchenko, Y. Mély, G. Duportail, and A. S. Klymchenko, “Monitoring biophysical properties of lipid membranes by environment-sensitive fluorescent probes,” Biophys. J.96(9), 3461–3470 (2009). [CrossRef] [PubMed]
  10. L. A. Bagatolli and E. Gratton, “Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures,” Biophys. J.78(1), 290–305 (2000). [CrossRef] [PubMed]
  11. A. S. Klymchenko, S. Oncul, P. Didier, E. Schaub, L. Bagatolli, G. Duportail, and Y. Mély, “Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone,” Biochim. Biophys. Acta1788(2), 495–499 (2009). [CrossRef] [PubMed]
  12. S. Haldar and A. Chattopadhyay, “Application of NBD-labeled lipids in membrane and cell biology,” in Fluorescent Methods to Study Biological Membranes, Y. Mély and G. Duportail, eds., Springer Series on Fluorescence No. 13 (Springer, 2013), pp. 37–50.
  13. S. Pajk, M. Garvas, J. Štrancar, and S. Pečar, “Nitroxide-fluorophore double probes: a potential tool for studying membrane heterogeneity by ESR and fluorescence,” Org. Biomol. Chem.9(11), 4150–4159 (2011). [CrossRef] [PubMed]
  14. Z. Arsov, I. Urbančič, M. Garvas, D. Biglino, A. Ljubetič, T. Koklič, and J. Štrancar, “Fluorescence microspectroscopy as a tool to study mechanism of nanoparticles delivery into living cancer cells,” Biomed. Opt. Express2(8), 2083–2095 (2011). [CrossRef] [PubMed]
  15. I. Urbančič, A. Ljubetič, Z. Arsov, and J. Štrancar, “Coexistence of probe conformations in lipid phases-a polarized fluorescence microspectroscopy study,” Biophys. J.105(4), 919–927 (2013). [CrossRef] [PubMed]
  16. T. Schmidt, G. J. Schütz, W. Baumgartner, H. J. Gruber, and H. Schindler, “Imaging of single molecule diffusion,” Proc. Natl. Acad. Sci. U.S.A.93(7), 2926–2929 (1996). [CrossRef] [PubMed]
  17. N. Bobroff, “Position measurement with a resolution and noise‐limited instrument,” Rev. Sci. Instrum.57(6), 1152–1157 (1986). [CrossRef]
  18. A. Bullen and P. Saggau, “High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes,” Biophys. J.76(4), 2272–2287 (1999). [CrossRef] [PubMed]
  19. S. Duhr, S. Arduini, and D. Braun, “Thermophoresis of DNA determined by microfluidic fluorescence,” Eur. Phys,” J. E Soft Matter15(3), 277–286 (2004). [CrossRef]
  20. J. P. Rigaut and J. Vassy, “High-resolution three-dimensional images from confocal scanning laser microscopy. Quantitative study and mathematical correction of the effects from bleaching and fluorescence attenuation in depth,” Anal. Quant. Cytol. Histol.13(4), 223–232 (1991). [PubMed]
  21. J. Markham and J.-A. Conchello, “Artefacts in restored images due to intensity loss in three-dimensional fluorescence microscopy,” J. Microsc.204(2), 93–98 (2001). [CrossRef] [PubMed]
  22. T. Zal and N. R. J. Gascoigne, “Photobleaching-corrected FRET efficiency imaging of live cells,” Biophys. J.86(6), 3923–3939 (2004). [CrossRef] [PubMed]
  23. R. Lansford, G. Bearman, and S. E. Fraser, “Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy,” J. Biomed. Opt.6(3), 311–318 (2001). [CrossRef] [PubMed]
  24. D. M. Benson, J. Bryan, A. L. Plant, A. M. Gotto, and L. C. Smith, “Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells,” J. Cell Biol.100(4), 1309–1323 (1985). [CrossRef] [PubMed]
  25. G. J. Brakenhoff, K. Visscher, and E. J. Gijsbers, “Fluorescence bleach rate imaging,” J. Microsc.175(2), 154–161 (1994). [CrossRef]
  26. D. Wüstner, A. Landt Larsen, N. J. Faergeman, J. R. Brewer, and D. Sage, “Selective visualization of fluorescent sterols in Caenorhabditis elegans by bleach-rate-based image segmentation,” Traffic11(4), 440–454 (2010). [CrossRef] [PubMed]
  27. I. Kusters, N. Mukherjee, M. R. de Jong, S. Tans, A. Koçer, and A. J. M. Driessen, “Taming membranes: functional immobilization of biological membranes in hydrogels,” PLoS ONE6(5), e20435 (2011). [CrossRef] [PubMed]
  28. K. Akashi, H. Miyata, H. Itoh, and K. Kinosita., “Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope,” Biophys. J.71(6), 3242–3250 (1996). [CrossRef] [PubMed]
  29. S. Waldenstrøm and K. R. Naqvi, “The overlap integrals of two harmonic-oscillator wavefunctions: some remarks on originals and reproductions,” Chem. Phys. Lett.85(5-6), 581–584 (1982). [CrossRef]
  30. M. Abramowitz and I. A. Stegun, Handbook of Mathematical Functions: With Formulas, Graphs and Mathematical Tables (Dover, 1972).
  31. D. B. Siano and D. E. Metzler, “Band shapes of the electronic spectra of complex molecules,” J. Chem. Phys.51(5), 1856–1861 (1969). [CrossRef]
  32. A. Kalauzi, D. Mutavdzić, D. Djikanović, K. Radotić, and M. Jeremić, “Application of asymmetric model in analysis of fluorescence spectra of biologically important molecules,” J. Fluoresc.17(3), 319–329 (2007). [CrossRef] [PubMed]
  33. E. A. Burstein and V. I. Emelyanenko, “Log-normal description of fluorescence spectra of organic fluorophores,” Photochem. Photobiol.64(2), 316–320 (1996). [CrossRef]
  34. T. Hirschfeld, “Quantum efficiency independence of the time integrated emission from a fluorescent molecule,” Appl. Opt.15(12), 3135–3139 (1976). [CrossRef] [PubMed]
  35. O. R., “Shaving the last 50 ms off NMinimize Web. 29 April 2012 http://mathematica.stackexchange.com/a/4877/5443 ,” (2012).
  36. F. Iachello and M. Ibrahim, “Analytic and algebraic evaluation of Franck−Condon overlap integrals,” J. Phys. Chem. A102(47), 9427–9432 (1998). [CrossRef]
  37. M. Lampton, B. Margon, and S. Bowyer, “Parameter estimation in X-ray astronomy,” Astrophys. J.208, 177 (1976). [CrossRef]
  38. R. Neher and E. Neher, “Optimizing imaging parameters for the separation of multiple labels in a fluorescence image,” J. Microsc.213(1), 46–62 (2004). [CrossRef] [PubMed]
  39. R. Koynova and M. Caffrey, “Phases and phase transitions of the phosphatidylcholines,” Biochim. Biophys. Acta1376(1), 91–145 (1998). [CrossRef] [PubMed]
  40. W. K. Subczynski, A. Wisniewska, J.-J. Yin, J. S. Hyde, and A. Kusumi, “Hydrophobic barriers of lipid bilayer membranes formed by reduction of water penetration by alkyl chain unsaturation and cholesterol,” Biochemistry33(24), 7670–7681 (1994). [CrossRef] [PubMed]
  41. S. Fery-Forgues, J.-P. Fayet, and A. Lopez, “Drastic changes in the fluorescence properties of NBD probes with the polarity of the medium: involvement of a TICT state?” J. Photochem. Photobiol. Chem.70(3), 229–243 (1993). [CrossRef]
  42. Z. Arsov and L. Quaroni, “Direct interaction between cholesterol and phosphatidylcholines in hydrated membranes revealed by ATR-FTIR spectroscopy,” Chem. Phys. Lipids150(1), 35–48 (2007). [CrossRef] [PubMed]
  43. T. Parasassi, M. Di Stefano, M. Loiero, G. Ravagnan, and E. Gratton, “Influence of cholesterol on phospholipid bilayers phase domains as detected by Laurdan fluorescence,” Biophys. J.66(1), 120–132 (1994). [CrossRef] [PubMed]
  44. J. Löbau, M. Sass, W. Pohle, C. Selle, M. H. J. Koch, and K. Wolfrum, “Chain fluidity and phase behaviour of phospholipids as revealed by FTIR and sum-frequency spectroscopy,” J. Mol. Struct.480–481, 407–411 (1999). [CrossRef]
  45. L. Li, H. Wang, and J.-X. Cheng, “Quantitative coherent anti-Stokes Raman scattering imaging of lipid distribution in coexisting domains,” Biophys. J.89(5), 3480–3490 (2005). [CrossRef] [PubMed]
  46. L. Opilik, T. Bauer, T. Schmid, J. Stadler, and R. Zenobi, “Nanoscale chemical imaging of segregated lipid domains using tip-enhanced Raman spectroscopy,” Phys. Chem. Chem. Phys.13(21), 9978–9981 (2011). [CrossRef] [PubMed]

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