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Spectrally resolved time-correlated single photon counting: a novel approach for characterization of endogenous fluorescence in isolated cardiac myocytes

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Abstract

A new setup for time-resolved fluorescence micro-spectroscopy of cells, based on multi-dimensional time-correlated single photon counting, was designed and tested. Here we demonstrate that the spectrometer allows fast and reproducible measurements of endogenous flavin fluorescence measured directly in living cardiac cells after excitation with visible picosecond laser diodes. Two complementary approaches for the analysis of spectrally- and time-resolved autofluorescence data are presented, comprising the fluorescence decay fitting by exponential series and the time-resolved emission spectroscopy analysis. In isolated cardiac myocytes, we observed three distinct lifetime pools with characteristic lifetime values spanning from picosecond to nanosecond range and the time-dependent red shift of the autofluorescence emission spectra. We compared obtained results to in vitro recordings of free flavin adenine dinucleotide (FAD) and FAD in lipoamide dehydrogenase (LipDH). The developed setup combines the strength of both spectral and fluorescence lifetime analysis and provides a solid base for the study of complex systems with intrinsic fluorescence, such as identification of the individual flavinoprotein components in living cardiac cells. This approach therefore constitutes an important instrumental advancement towards redox fluorimetry of living cardiomyocytes, with the perspective of its applications in the investigation of oxidative metabolic state under pathophysiological conditions, such as ischemia and/or metabolic disorders.

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References

  • Bastiaens PI, van Hoek A, Wolkers WF, Brochon JC, Visser AJ (1992) Comparison of the dynamical structures of lipoamide dehydrogenase and glutathione reductase by time-resolved polarized flavin fluorescence. Biochemistry 31(31):7050–7060

    Article  Google Scholar 

  • Becker W (2005) Advanced time-correlated single photon counting techniques. Springer, Berlin Heidelberg New York

    Google Scholar 

  • Becker W, Bergmann A, Biskup C, Zimmer T, Klöcker N, Benndorf K (2002) Multi-wavelength TCSPC lifetime imaging. In: Ammasi Periasamy, Peter T So; (eds) Proc. SPIE - Multiphoton Microscopy in the Biomedical Sciences II, 4620:79–84

  • Bird DK, Eliceiri KW, Fan Ch-H, White JG (2004) Simultaneous two-photon spectral and lifetime fluorescence microscopy. Appl Opt 43(27):5173–5182

    Article  ADS  Google Scholar 

  • Bugar I, Chorvat D Jr, Palszegi T, Mach P, Urban J, Szöcs V (2002) Femto- and picosecond fluorescence dynamics of merocyanine 540: experiments and modeling. Femtochemistry and Femtobiology, Proceedings of 7th femtochemistry conference in Toledo, Word Scientific, Singapore, 298

  • Chosrowjan H, Mataga N, Taniguchi S, Tanaka F, Visser AJWG (2003) The stacked flavin adenine dinucleotide conformation in water is fluorescent on picosecond time scale. Chem Phys Lett 378:354–358

    Article  ADS  Google Scholar 

  • Chorvat D Jr, Bassien-Capsa V, Cagalinec M, Kirchnerova J, Mateasik A, Comte B, Chorvatova A (2004) Mitochondrial autofluorescence induced by visible light in single cardiac myocytes studied by spectrally resolved confocal microscopy. Laser Phys (14): 220–230

  • Chorvat D Jr, Kirchnerova J, Cagalinec M, Smolka J, Mateasik A, Chorvatova A (2005) Spectral unmixing of flavin autofluorescence components in cardiac myocytes. Biophys J 89(6): L55–57

    Article  Google Scholar 

  • Chung YG, Schwartz JA, Gardner CM, Sawaya RE, Jacques SL (1997) Diagnostic potential of laser-induced autofluorescence emission in brain tissue. J Korean Med Sci 12(2):135–142

    Google Scholar 

  • Dickinson ME, Bearman G, Tilie S, Lansford R, Fraser SE (2001) Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy. Biotechniques 31:1272–1278

    Google Scholar 

  • Gadella TWJ Jr, Jovin TM, Clegg RM (1993) Fluorescence lifetime imaging microscopy (FLIM)—spatial resolution of microstructures on the nanosecond time-scale. Biophys Chem 48:221–239

    Article  Google Scholar 

  • Huang S, Heikal AA, Webb WW (2002) Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. Biophys J 82:2811–2825

    Article  Google Scholar 

  • Koti ASR, Krishna MMG, Periasamy N (2001) Time-resolved area-normalized emission spectroscopy (TRANES): a novel method for confirming emission from two excited states. J Phys Chem A 105:1767–1771

    Article  Google Scholar 

  • Kunz WS, Gellerich FN (1993) Quantification of the content of fluorescent flavoproteins in mitochondria from liver, kidney cortex, skeletal muscle, and brain. Biochem Med Metab Biol 50:103–110

    Article  Google Scholar 

  • O’Connor DV, Phillips D (1984) Time-correlated single photon counting. Academic, London

    Google Scholar 

  • Petushkov VN, van Stokkum IHM, Gobets B, van Mourik F, Lee J, van Grondelle R, Visser AJWG (2003) Ultrafast fluorescence relaxation spectroscopy of 6,7-dimethyl-(8-ribityl)-lumazine and riboflavin, free and bound to antenna proteins from bioluminescent bacteria. J Phys Chem B 107:10934–10939

    Article  Google Scholar 

  • Poguet J, Mugnier J, Valeur B (1989) Correction of systematic phase errors in frequency-domain fluorometry. J Phys [E] 22:855–862

    ADS  Google Scholar 

  • Rocheleau JV, Head WS, Piston DW (2004) Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response. J Biol Chem 279:31780–31787

    Article  Google Scholar 

  • Romashko DN, Marban E, O’Rourke B (1998) Subcellular metabolic transients and mitochondrial redox waves in heart cells. Proc Natl Acad Sci USA 95:1618–1623

    Article  ADS  Google Scholar 

  • Schweitzer D, Hammer M, Schweitzer F, Anders R, Doebbecke T, Schenke S, Gaillard ER (2004) In vivo measurement of time-resolved autofluorescence at the human fundus. J Biomed Opt 9(6):1214–1222

    Article  Google Scholar 

  • van den Berg PAW, Feenstra KA, van Hoek A, Mark AE, Berendsen HJC, Visser AJWG (2002) Dynamic conformations of flavin adenine dinucleotide: simulated molecular dynamics of the flavin cofactor related to the time-resolved fluorescence characteristics. J Phys Chem B 106:8858–8869

    Article  Google Scholar 

  • Vetrova EV, Kudryasheva NS, Visser AJWG, van Hoek A (2005) Characteristics of endogenous flavin fluorescence of Photobacterium leiognathi luciferase and Vibrio fischeri NAD(P)H:FMN-oxidoreductase. Luminescence 20(3):205–209

    Article  Google Scholar 

  • Visser AJWG (1984) Kinetics of stacking interactions in flavin adenine dinucleotide from time-resolved flavin fluorescence. Photochem Photobiol (40):703–706

  • Wahl P, Auchet JC, Visser AJ, Veeger C (1975) A pulse fluorometry study of lipoamide dehydrogenase. Evidence for non-equivalent FAD centers. Eur J Biochem 50(2):413–418

    Article  Google Scholar 

  • Wang XF, Periasamy A, Herman B, Coleman DM (1992) Fluorescence lifetime imaging microscopy (FLIM): instrumentation and applications. Crit Rev Anal Chem 23:369–395

    Article  Google Scholar 

  • Zhong D, Zewail AH (2001) Femtosecond dynamics of flavoproteins: charge separation and recombination in riboflavine (vitamin B2)-binding protein and in glucose oxidase enzyme. Proc Natl Acad Sci USA 98(21):11867–11872

    Article  ADS  Google Scholar 

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Acknowledgments

This work was supported by Collaborative Linkage Grant LST.CLG.979836 from NATO to DC and AC; FRSQ (N 2948), CFI (N 9684) and CIHR (MOP 74600) grants to AC. We would like to thank V. Bassien-Capsa for cell isolation and A. Mateasik for programming of custom routines for data analysis.

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Authors and Affiliations

  1. International Laser Centre, Bratislava, Slovak Republic

    D. Chorvat Jr

  2. Research Center, CHU Sainte-Justine, 3175 Cote Sainte Catherine, Montreal, H3T 1C5, Canada

    A. Chorvatova

  3. Department of Pediatrics, University of Montreal, Montreal, Canada

    A. Chorvatova

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  1. D. Chorvat Jr
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  2. A. Chorvatova
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Correspondence to A. Chorvatova.

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Chorvat, D., Chorvatova, A. Spectrally resolved time-correlated single photon counting: a novel approach for characterization of endogenous fluorescence in isolated cardiac myocytes. Eur Biophys J 36, 73–83 (2006). https://doi.org/10.1007/s00249-006-0104-4

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  • DOI: https://doi.org/10.1007/s00249-006-0104-4

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