Journal of Fluorescence

, 19:1037 | Cite as

Two-Color Two-Photon Fluorescence Laser Scanning Microscopy

  • S. Quentmeier
  • S. Denicke
  • K.-H. Gericke
Original Paper


We present the first realization of a Two-Color Two-Photon Laser-Scanning Microscope (2c2pLSM) and UV fluorescence images of cells acquired with this technique. Fluorescence is induced by two-color two-photon absorption using the fundamental and the second harmonic of a Ti:Sa femtosecond laser. Simultaneous absorption of an 800 nm photon and a 400 nm photon energetically corresponds to one-photon absorption at 266 nm. This technique for Laser-Scanning Microscopy extends the excitation wavelength range of a Ti:Sa powered fluorescence microscope to the UV. In addition to the known advantages of multi-photon microscopy like intrinsic 3D resolution, reduced photo damage and high penetration depth 2c2pLSM offers the possibility of using standard high numeric aperture objectives for UV fluorescence imaging. The effective excitation wavelength of 266 nm corresponds especially well to the excitation spectrum of tryptophan. Hence, it is an ideal tool for label free fluorescence studies and imaging of intrinsic protein fluorescence which originates mainly from tryptophan. Thus a very sensitive natural lifetime probe can be used for monitoring protein reactions or changes in conformation. First measurements of living MIN-6 cells reveal differences between the UV fluorescence lifetimes of the nucleus and cytoplasm. The significance of this method was further demonstrated by monitoring the binding of biotin to avidin.


2c2p Two-color two-photon Tryptophan Fluorescence Fluorescence lifetime 


  1. 1.
    Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:933. doi: 10.1038/nmeth818 CrossRefGoogle Scholar
  2. 2.
    Bird D, Gu M (2003) Two-photon fluorescence endoscopy with a micro-optic scanning head. Opt Lett 28(17):1552. doi: 10.1364/OL.28.001552 CrossRefPubMedGoogle Scholar
  3. 3.
    Flusberg BA, Jung JC, Cocker ED, Anderson EP, Schnitzer MJ (2005) In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope. Opt Lett 30(17):2272. doi: 10.1364/OL.30.002272 CrossRefPubMedGoogle Scholar
  4. 4.
    König K, Riemann I, Ehlers A, Buckle R, Dimitrow E, Kaatz M, Fluhr J, Elsner P (2006) In vivo multiphoton tomography of skin cancer. Proc. SPIE 6089Google Scholar
  5. 5.
    Zipfel WR, Wiliams RM, Christie R, Nikitin AY, Hyman B, Webb WW (2003) Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc Natl Acad Sci USA 100(12):7075. doi: 10.1073/pnas.0832308100 CrossRefPubMedGoogle Scholar
  6. 6.
    Quentmeier S, Denicke S, Ehlers J-E, Niesner RA, Gericke K-H (2008) Two-color two-photon excitation using femtosecond laser pulses. J Phys Chem B 112:5768. doi: 10.1021/jp7113994 CrossRefPubMedGoogle Scholar
  7. 7.
    Chen J, Midorikawa K (2004) Two-color two-photon 4Pi fluorescence microscopy. Opt Lett 29(12):1354. doi: 10.1364/OL.29.001354 CrossRefPubMedGoogle Scholar
  8. 8.
    Blanca CM, Saloma C (2001) Two-color excitation fluorescence microscopy through highly scattering media. Appl Opt 40(16):2722. doi: 10.1364/AO.40.002722 CrossRefPubMedGoogle Scholar
  9. 9.
    Lakowicz JR, Gryczynsiki I, Malak H, Gryczynski Z (1996) Two-color two-photon excitation of fluorescence. Photochem Photobiol 64(4):632. doi: 10.1111/j.1751-1097.1996.tb03116.x CrossRefPubMedGoogle Scholar
  10. 10.
    Dobrydnev B, Havey M (1995) Theoretical investigation of two-color, two-photon, 6s 2S1/2-->5d 2Dj-->11p 2P3/2 excitation and depolarization spectra in atomic Cs. Phys Rev 52(5):4010. doi: 10.1103/PhysRevA.52.4010 CrossRefGoogle Scholar
  11. 11.
    Cambaliza MO, Saloma C (2000) Advantages of two-color excitation fluorescence microscopy with two confocal excitation beams. Opt Commun 184:25. doi: 10.1016/S0030-4018(00)00929-9 CrossRefGoogle Scholar
  12. 12.
    Gryczynski I, Malak H, Lakowicz JR (1997) Two-color two-photon excitation of Indole. Biospec 3(2):97. doi: 10.1002/(SICI)1520-6343(1997)3:2<97::AID-BSPY2>3.0.CO;2-P CrossRefGoogle Scholar
  13. 13.
    Lim M, Saloma C (2003) Primary spherical aberration in two-color two-photon excitation fluorescence microscopy with two confocal excitation beams. Appl Opt 42(17):3398. doi: 10.1364/AO.42.003398 CrossRefPubMedGoogle Scholar
  14. 14.
    Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Kluwer Academic/Plenum, New YorkGoogle Scholar
  15. 15.
    Engelborghs Y (2001) The analysis of time resolved protein fluorescence in multi-tryptophan proteins. Spectrochimica Acta 57:2255. doi: 10.1016/S1386-1425(01)00485-1 CrossRefPubMedGoogle Scholar
  16. 16.
    Kurzban GP, Gitlin G, Bayer EA, Wilchek M, Horowitz PM (1990) Biotin binding changes the conformation and decreases tryptophan accessibility of streptavidin. J Protein Chem 9(6):673. doi: 10.1007/BF01024762 CrossRefPubMedGoogle Scholar
  17. 17.
    Sinha RP, Häder D-P (2002) UV-induced DNA damage and repair: a review. Photochem Photobiol Sci 1:225. doi: 10.1039/b201230h CrossRefPubMedGoogle Scholar
  18. 18.
    Gan XS, Gu M (2000) Fluorescence microscopic imaging through tissue-like turbid media. J Appl Phys 87(7):3214. doi: 10.1063/1.372326 CrossRefGoogle Scholar
  19. 19.
    Centonze VE, White JG (1998) Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J 75:2015. doi: 10.1016/S0006-3495(98)77643-X CrossRefPubMedGoogle Scholar
  20. 20.
    Kenworthy AK (2007) Fluorescence recovery after photobleaching studies of lipid rafts. Methods Mol Biol 398:179. doi: 10.1007/978-1-59745-513-8_13 CrossRefPubMedGoogle Scholar
  21. 21.
    Fleming GR, Morris JM, Robbins RJ, Woolfe GJ, Thistlethwaite PJ, Robinson GW (1978) Nonexponential fluorescence decay of aqueous tryptophan and two related peptides by picosecond spectroscopy. Proc Natl Acad Sci USA 75(10):4652. doi: 10.1073/pnas.75.10.4652 CrossRefPubMedGoogle Scholar
  22. 22.
    Siemiarczuk A, Petersen CE, Ha C-E, Yang J, Bhagavan NV (2004) Analysis of tryptophan fluorescence lifetimes on a series of human serum albumin mutants with substitutions in subdomain A. Cell Biochem Biophys 40:115. doi: 10.1385/CBB:40:2:115 CrossRefPubMedGoogle Scholar
  23. 23.
    Niesner R, Peker B, 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 5:1141. doi: 10.1002/cphc.200400066 CrossRefPubMedGoogle Scholar
  24. 24.
    Turconi S, Bingham RP, Haupts U, Pope AJ (2001) Developments in fluorescence lifetime-based analysis for ultra-HTS. Drug Discov Today 6(12):SS27Google Scholar
  25. 25.
    Pugliese L, Malcovati M, Coda A, Bolognesi M (1994) Crystal structure of apo-avidin from hen egg-white. J Mol Biol 235(1):42. doi: 10.1016/S0022-2836(05)80010-5 CrossRefPubMedGoogle Scholar
  26. 26.
    Mei G, Pugliese L, Rosato N, Toma L, Bolognesi M, Finazzi-Agrò A (1994) Biotin and biotin analogues specifically modify the fluorescence decay of avidin. J Mol Biol 242:559CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Institute for Physical und Theoretical ChemistryUniversity of BraunschweigBraunschweigGermany

Personalised recommendations