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Label-Free Spatial Analysis of Free and Enzyme-Bound NAD(P)H in the Presence of High Concentrations of Melanin

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Abstract

The analysis of autofluorescence, often regarded as undesired noise during the imaging of biological samples, allows label free, unbiased detection of NAD(P)H and melanin in native samples. Because both the emission and absorption spectra of these fluorophores overlap and they can hence not be differentiated using emission filters or with different excitation wavelengths, fluorescence lifetime imaging microscopy (FLIM) is used to differentiate between them. In the present paper the application of two-photon excitation microscopy is presented to investigate the autofluorescence of fungal spores. The model organism which was examined is Aspergillus ochraceus. Furthermore a strategy is developed which allows to quantitatively analyze the fluorescence lifetimes of melanin, free NAD(P)H and protein-bound NAD(P)H using forward convolution of a multiexponential decay function with the instrument response function (IRF) and subsequent fitting to the experimental fluorescence data. As a consequence proteins, which are able to bind NAD(P)H, are located with sub-cellular resolution. Furthermore a spatial differentiation of the fluorophores NAD(P)H and melanin inside the spores, is revealed.

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References

  1. Lakowicz JR (2006) Principles of fluorescence spectroscopy. Vol, 3rd edn. Springer, New York

    Book  Google Scholar 

  2. Baker NR (2008) Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annu Rev Plant Biol 59(1):89–113

    Article  PubMed  CAS  Google Scholar 

  3. Imanishi Y, Lodowski KH, Koutalos Y (2007) Two-photon microscopy: shedding light on the chemistry of vision†. Biochemistry 46(34):9674–9684

    Article  PubMed  CAS  Google Scholar 

  4. Meredith P, Sarna T (2006) The physical and chemical properties of eumelanin. Pigment Cell Res 19(6):572–594

    Article  PubMed  CAS  Google Scholar 

  5. Durrell LW (1964) The composition and structure of walls of dark fungus spores. Mycopathologia 23(4):339–345

    Article  Google Scholar 

  6. Bell AA, Wheeler MH (1986) Biosynthesis and functions of fungal melanins. Annu Rev Phytopathol 24(1):411–451

    Article  CAS  Google Scholar 

  7. Quentmeier S, Quentmeier CC, Walla PJ, Gericke K-H (2009) Two-color two-photon excitation of intrinsic protein fluorescence: label-free observation of proteolytic digestion of bovine serum albumin. Chemphyschem 10(9–10):1607–1613

    Article  PubMed  CAS  Google Scholar 

  8. Tran ML, Powell BJ, Meredith P (2006) Chemical and structural disorder in eumelanins: a possible explanation for broadband absorbance. Biophys J 90(3):743–752

    Article  PubMed  CAS  Google Scholar 

  9. Lakowicz JR, Szmacinski H, Nowaczyk K, Johnson ML (1992) Fluorescence lifetime imaging of free and protein-bound NADH. Proc Natl Acad Sci USA 89(4):1271–1275

    Article  PubMed  CAS  Google Scholar 

  10. Niesner R, Narang P, Spiecker H, Andresen V, Gericke K-H, Gunzer M (2008) selective detection of NADPH oxidase in polymorphonuclear cells by means of NAD(P)H-based fluorescence lifetime imaging. J Biophys, 2008

  11. Niesner R, Bülent P, Schlüsche P, Gericke K-H (2004) Noniterative biexponential fluorescence lifetime imaging in the investigation of cellular metabolism by means of NAD(P)H autofluorescence. Chemphyschem 5(8):1141–1149

    Article  PubMed  CAS  Google Scholar 

  12. Elson D et al (2004) Time-domain fluorescence lifetime imaging applied to biological tissue. Photochem Photobiol Sci 3(8):795–801

    Article  PubMed  CAS  Google Scholar 

  13. Gerritsen HC, Asselbergs MAH, Agronskaia AV, Van Sark WGJHM (2002) Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution. J Microsc 206(3):218–224

    Article  PubMed  CAS  Google Scholar 

  14. Niesner R, Gericke K-H (2008) Fluorescence lifetime imaging in biosciences: technologies and applications. Front Phys 3(1)

  15. Denicke S, Ehlers J-E, Niesner R, Quentmeier S, Gericke K-H (2007) Steady-state and time-resolved two-photon fluorescence microscopy: a versatile tool for probing cellular environment and function. Phys Scripta 76(3):C115

    Article  CAS  Google Scholar 

  16. Carriles R, Schafer DN, Sheetz KE, Field JJ, Cisek R, Barzda V, Sylvester AW, Squier JA (2009) Invited review article: imaging techniques for harmonic and multiphoton absorption fluorescence microscopy. Rev Sci Instrum 80(8):081101

    Article  PubMed  Google Scholar 

  17. Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21(11):1369–1377

    Article  PubMed  CAS  Google Scholar 

  18. Ehlers A, Riemann I, Stark M, König K (2007) Multiphoton fluorescence lifetime imaging of human hair. Microsc Res Tech 70(2):154–161

    Article  PubMed  Google Scholar 

  19. Habenicht A, Hjelm J, Mukhtar E, Bergström F, Johansson LBÅ (2002) Two-photon excitation and time-resolved fluorescence: I. The proper response function for analysing single-photon counting experiments. Chem Phys Lett 354(5–6):367–375

    Article  CAS  Google Scholar 

  20. Zheltikov AM (2006) Let there be white light: supercontinuum generation by ultrashort laser pulses. Phys Usp 49(6):605

    Article  CAS  Google Scholar 

  21. Available from: http://www.picoquant.com/datasheets/photon_counting/PDM_Series.pdf.

  22. Forest SE, Lam WC, Millar DP, Nofsinger JB, Simon JD (2000) A model for the activated energy transfer within eumelanin aggregates. J Phys Chem B 104(4):811–814

    Article  CAS  Google Scholar 

  23. Zhang Q, Piston DW, Goodman RH (2002) Regulation of corepressor function by nuclear NADH. Science 295(5561):1895–1897

    PubMed  CAS  Google Scholar 

  24. Arcangeli C, Yu W, Cannistraro S, Gratton E (2000) Two-photon autofluorescence microscopy and spectroscopy of Antarctic fungus: new approach for studying effects of UV-B irradiation. Biopolymers 57(4):218–225

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

Financial support by the Deutsche Forschungsgemeinschaft (SH) and the German National Merit Foundation (MG) are kindly acknowledged. The authors also thank PicoQuant GmbH for material support. Support of the IGSM Braunschweig is also gratefully acknowledged.

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Authors and Affiliations

  1. Institute of Physical and Theoretical Chemistry, Technische Universität Braunschweig, Hans-Sommerstraße 10, 38106, Braunschweig, Germany

    Sebastian Herbrich & Karl-Heinz Gericke

  2. Institute of Biochemical Engineering, Technische Universität Braunschweig, Gaußstraße 17, 38106, Braunschweig, Germany

    Matthias Gehder & Rainer Krull

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  1. Sebastian Herbrich
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  2. Matthias Gehder
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  3. Rainer Krull
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  4. Karl-Heinz Gericke
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Correspondence to Karl-Heinz Gericke.

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Herbrich, S., Gehder, M., Krull, R. et al. Label-Free Spatial Analysis of Free and Enzyme-Bound NAD(P)H in the Presence of High Concentrations of Melanin. J Fluoresc 22, 349–355 (2012). https://doi.org/10.1007/s10895-011-0965-5

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  • DOI: https://doi.org/10.1007/s10895-011-0965-5

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