OSA's Digital Library

Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 14 — Jul. 15, 2013
  • pp: 17256–17264

Two-photon imaging of multiple fluorescent proteins by phase-shaping and linear unmixing with a single broadband laser

Meredith H. Brenner, Dawen Cai, Joel A. Swanson, and Jennifer P. Ogilvie  »View Author Affiliations

Optics Express, Vol. 21, Issue 14, pp. 17256-17264 (2013)

View Full Text Article

Enhanced HTML    Acrobat PDF (1211 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



Imaging multiple fluorescent proteins (FPs) by two-photon microscopy has numerous applications for studying biological processes in thick and live samples. Here we demonstrate a setup utilizing a single broadband laser and a phase-only pulse-shaper to achieve imaging of three FPs (mAmetrine, TagRFPt, and mKate2) in live mammalian cells. Phase-shaping to achieve selective excitation of the FPs in combination with post-imaging linear unmixing enables clean separation of the fluorescence signal of each FP. This setup also benefits from low overall cost and simple optical alignment, enabling easy adaptation in a regular biomedical research laboratory.

© 2013 OSA

OCIS Codes
(180.2520) Microscopy : Fluorescence microscopy
(190.4180) Nonlinear optics : Multiphoton processes
(320.5540) Ultrafast optics : Pulse shaping
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:

Original Manuscript: May 28, 2013
Revised Manuscript: July 3, 2013
Manuscript Accepted: July 5, 2013
Published: July 11, 2013

Virtual Issues
Vol. 8, Iss. 8 Virtual Journal for Biomedical Optics

Meredith H. Brenner, Dawen Cai, Joel A. Swanson, and Jennifer P. Ogilvie, "Two-photon imaging of multiple fluorescent proteins by phase-shaping and linear unmixing with a single broadband laser," Opt. Express 21, 17256-17264 (2013)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. R. Y. Tsien, “The green fluorescent protein,” Annu. Rev. Biochem.67(1), 509–544 (1998). [CrossRef] [PubMed]
  2. N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, “A guide to choosing fluorescent proteins,” Nat. Methods2(12), 905–909 (2005). [CrossRef] [PubMed]
  3. E. R. Tkaczyk and A. H. Tkaczyk, “Multiphoton flow cytometry strategies and applications,” Cytometry A79(10), 775–788 (2011). [CrossRef] [PubMed]
  4. M. Qian, D. Cai, K. J. Verhey, and B. Tsai, “A lipid receptor sorts polyomavirus from the endolysosome to the endoplasmic reticulum to cause infection,” PLoS Pathog.5(6), e1000465 (2009). [CrossRef] [PubMed]
  5. D. Cai, D. P. McEwen, J. R. Martens, E. Meyhofer, and K. J. Verhey, “Single molecule imaging reveals differences in microtubule track selection between Kinesin motors,” PLoS Biol.7(10), e1000216 (2009). [CrossRef] [PubMed]
  6. J. Lippincott-Schwartz and G. H. Patterson, “Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging,” Trends Cell Biol.19(11), 555–565 (2009). [CrossRef] [PubMed]
  7. D. Cai, A. D. Hoppe, J. A. Swanson, and K. J. Verhey, “Kinesin-1 structural organization and conformational changes revealed by FRET stoichiometry in live cells,” J. Cell Biol.176(1), 51–63 (2007). [CrossRef] [PubMed]
  8. H. W. Ai, K. L. Hazelwood, M. W. Davidson, and R. E. Campbell, “Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors,” Nat. Methods5(5), 401–403 (2008). [CrossRef] [PubMed]
  9. B. N. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, “The fluorescent toolbox for assessing protein location and function,” Science312(5771), 217–224 (2006). [CrossRef] [PubMed]
  10. T. Kogure, S. Karasawa, T. Araki, K. Saito, M. Kinjo, and A. Miyawaki, “A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy,” Nat. Biotechnol.24(5), 577–581 (2006). [CrossRef] [PubMed]
  11. J. Livet, T. A. Weissman, H. Kang, R. W. Draft, J. Lu, R. A. Bennis, J. R. Sanes, and J. W. Lichtman, “Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system,” Nature450(7166), 56–62 (2007). [CrossRef] [PubMed]
  12. H. J. Snippert, L. G. van der Flier, T. Sato, J. H. van Es, M. van den Born, C. Kroon-Veenboer, N. Barker, A. M. Klein, J. van Rheenen, B. D. Simons, and H. Clevers, “Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells,” Cell143(1), 134–144 (2010). [CrossRef] [PubMed]
  13. K. Red-Horse, H. Ueno, I. L. Weissman, and M. A. Krasnow, “Coronary arteries form by developmental reprogramming of venous cells,” Nature464(7288), 549–553 (2010). [CrossRef] [PubMed]
  14. G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, “Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP,” Neuron28(1), 41–51 (2000). [CrossRef] [PubMed]
  15. T. Misgeld, M. Kerschensteiner, F. M. Bareyre, R. W. Burgess, and J. W. Lichtman, “Imaging axonal transport of mitochondria in vivo,” Nat. Methods4(7), 559–561 (2007). [CrossRef] [PubMed]
  16. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990). [CrossRef] [PubMed]
  17. W. Denk, “Two-photon excitation in functional biological imaging,” J. Biomed. Opt.1(3), 296–304 (1996). [CrossRef] [PubMed]
  18. H. Kawano, T. Kogure, Y. Abe, H. Mizuno, and A. Miyawaki, “Two-photon dual-color imaging using fluorescent proteins,” Nat. Methods5(5), 373–374 (2008). [CrossRef] [PubMed]
  19. S. E. Tillo, T. E. Hughes, N. S. Makarov, A. Rebane, and M. Drobizhev, “A new approach to dual-color two-photon microscopy with fluorescent proteins,” BMC Biotechnol.10(1), 6 (2010). [CrossRef] [PubMed]
  20. E. Sahai, J. Wyckoff, U. Philippar, J. E. Segall, F. Gertler, and J. Condeelis, “Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy,” BMC Biotechnol.5(1), 14 (2005). [CrossRef] [PubMed]
  21. 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]
  22. D. Entenberg, J. Wyckoff, B. Gligorijevic, E. T. Roussos, V. V. Verkhusha, J. W. Pollard, and J. Condeelis, “Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging,” Nat. Protoc.6(10), 1500–1520 (2011). [CrossRef] [PubMed]
  23. P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Débarre, and E. Beaurepaire, “Multicolor two-photon tissue imaging by wavelength mixing,” Nat. Methods9(8), 815–818 (2012). [CrossRef] [PubMed]
  24. K. Wang, T. M. Liu, J. Wu, N. G. Horton, C. P. Lin, and C. Xu, “Three-color femtosecond source for simultaneous excitation of three fluorescent proteins in two-photon fluorescence microscopy,” Biomed. Opt. Express3(9), 1972–1977 (2012). [CrossRef] [PubMed]
  25. J. P. Ogilvie, D. Débarre, X. Solinas, J. L. Martin, E. Beaurepaire, and M. Joffre, “Use of coherent control for selective two-photon fluorescence microscopy in live organisms,” Opt. Express14(2), 759–766 (2006). [CrossRef] [PubMed]
  26. L. T. Schelhas, J. C. Shane, and M. Dantus, “Advantages of ultrashort phase-shaped pulses for selective two-photon activation and biomedical imaging,” Nanomedicine2(3), 177–181 (2006). [CrossRef] [PubMed]
  27. R. S. Pillai, C. Boudoux, G. Labroille, N. Olivier, I. Veilleux, E. Farge, M. Joffre, and E. Beaurepaire, “Multiplexed two-photon microscopy of dynamic biological samples with shaped broadband pulses,” Opt. Express17(15), 12741–12752 (2009). [CrossRef] [PubMed]
  28. K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Multifarious control of two-photon excitation of multiple fluorophores achieved by phase modulation of ultra-broadband laser pulses,” Opt. Express17(16), 13737–13746 (2009). [CrossRef] [PubMed]
  29. D. S. Moore, “Optimal coherent control of sensitivity and selectivity in spectrochemical analysis,” Anal. Bioanal. Chem.393(1), 51–56 (2009). [CrossRef] [PubMed]
  30. M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods8(5), 393–399 (2011). [CrossRef] [PubMed]
  31. I. Pastirk, J. Dela Cruz, K. Walowicz, V. Lozovoy, and M. Dantus, “Selective two-photon microscopy with shaped femtosecond pulses,” Opt. Express11(14), 1695–1701 (2003). [CrossRef] [PubMed]
  32. E. R. Tkaczyk, A. H. Tkaczyk, K. Mauring, J. Y. Ye, J. R. Baker, and T. B. Norris, “Control of two-photon fluorescence of common dyes and conjugated dyes,” J. Fluoresc.19(3), 517–532 (2009). [CrossRef] [PubMed]
  33. M. Comstock, V. Lozovoy, I. Pastirk, and M. Dantus, “Multiphoton intrapulse interference 6; binary phase shaping,” Opt. Express12(6), 1061–1066 (2004). [CrossRef] [PubMed]
  34. D. Meshulach and Y. Silberberg, “Coherent quantum control of two-photon transitions by a femtosecond laser pulse,” Nature396(6708), 239–242 (1998). [CrossRef]
  35. V. V. Lozovoy, I. Pastirk, K. A. Walowicz, and M. Dantus, “Multiphoton intrapulse interference. II. Control of two- and three-photon laser induced fluorescence with shaped pulses,” J. Chem. Phys.118(7), 3187–3196 (2003). [CrossRef]
  36. K. A. Walowicz, I. Pastirk, V. V. Lozovoy, and M. Dantus, “Multiphoton Intrapulse Interference. 1. Control of Multiphoton Processes in Condensed Phases,” J. Phys. Chem. A106(41), 9369–9373 (2002). [CrossRef]
  37. A. M. Weiner, “Femtosecond pulse shaping using spatial light modulators,” Rev. Sci. Instrum.71(5), 1929–1960 (2000). [CrossRef]
  38. V. V. Lozovoy and M. Dantus, “Systematic control of nonlinear optical processes using optimally shaped femtosecond pulses,” ChemPhysChem6(10), 1970–2000 (2005). [CrossRef] [PubMed]
  39. J. M. Dela Cruz, I. Pastirk, V. V. Lozovoy, K. A. Walowicz, and M. Dantus, “Multiphoton Intrapulse Interference 3: Probing Microscopic Chemical Environments,” J. Phys. Chem. A108(1), 53–58 (2004). [CrossRef]
  40. Y. Coello, V. V. Lozovoy, T. C. Gunaratne, B. Xu, I. Borukhovich, C.- Tseng, T. Weinacht, and M. Dantus, “Interference without an interferometer: a different approach to measuring, compressing, and shaping ultrashort laser pulses,” J. Opt. Soc. Am. B25(6), A140–A150 (2008). [CrossRef]
  41. B. Xu, J. M. Gunn, J. M. D. Cruz, V. V. Lozovoy, and M. Dantus, “Quantitative investigation of the multiphoton intrapulse interference phase scan method for simultaneous phase measurement and compensation of femtosecond laser pulses,” J. Opt. Soc. Am. B23(4), 750–759 (2006). [CrossRef]
  42. E. M. Merzlyak, J. Goedhart, D. Shcherbo, M. E. Bulina, A. S. Shcheglov, A. F. Fradkov, A. Gaintzeva, K. A. Lukyanov, S. Lukyanov, T. W. Gadella, and D. M. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods4(7), 555–557 (2007). [CrossRef] [PubMed]
  43. N. C. Shaner, M. Z. Lin, M. R. McKeown, P. A. Steinbach, K. L. Hazelwood, M. W. Davidson, and R. Y. Tsien, “Improving the photostability of bright monomeric orange and red fluorescent proteins,” Nat. Methods5(6), 545–551 (2008). [CrossRef] [PubMed]
  44. D. Shcherbo, E. M. Merzlyak, T. V. Chepurnykh, A. F. Fradkov, G. V. Ermakova, E. A. Solovieva, K. A. Lukyanov, E. A. Bogdanova, A. G. Zaraisky, S. Lukyanov, and D. M. Chudakov, “Bright far-red fluorescent protein for whole-body imaging,” Nat. Methods4(9), 741–746 (2007). [CrossRef] [PubMed]
  45. E. Salomonnson, L. A. Mihalko, V. V. Verkhusha, K. E. Luker, and G. D. Luker, “Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy,” J. Biomed. Opt.17(9), 096001 (2012). [CrossRef] [PubMed]
  46. H. Tsurui, H. Nishimura, S. Hattori, S. Hirose, K. Okumura, and T. Shirai, “Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition,” J. Histochem. Cytochem.48(5), 653–662 (2000). [CrossRef] [PubMed]
  47. 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]
  48. I. B. Clark, V. Muha, A. Klingseisen, M. Leptin, and H. A. Müller, “Fibroblast growth factor signalling controls successive cell behaviours during mesoderm layer formation in Drosophila,” Development138(13), 2705–2715 (2011). [CrossRef] [PubMed]
  49. J. W. Boardman, “Inversion Of Imaging Spectrometry Data Using Singular Value Decomposition,” in Geoscience and Remote Sensing Symposium, 1989. IGARSS'89. 12th Canadian Symposium on Remote Sensing., 1989 International (1989), 2069–2072. [CrossRef]
  50. Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct.27(5), 367–374 (2002). [CrossRef] [PubMed]
  51. B. Kraus, M. Ziegler, and H. Wolff, Linear Fluorescence Unmixing in Cell Biological Research (2007).
  52. S. Kramer-Hämmerle, F. Ceccherini-Silberstein, C. Bickel, H. Wolff, M. Vincendeau, T. Werner, V. Erfle, and R. Brack-Werner, “Identification of a novel Rev-interacting cellular protein,” BMC Cell Biol.6(1), 20 (2005). [CrossRef] [PubMed]
  53. Y. Garini, I. T. Young, and G. McNamara, “Spectral imaging: principles and applications,” Cytometry A69(8), 735–747 (2006). [CrossRef] [PubMed]
  54. L. M. Davis and G. Shen, “Extension of multidimensional microscopy to ultrasensitive applications with maximum-likelihood analysis,” Proc. SPIE6443, 64430N, 64430N-12 (2007). [CrossRef]
  55. M. Ducros, L. Moreaux, J. Bradley, P. Tiret, O. Griesbeck, and S. Charpak, “Spectral unmixing: analysis of performance in the olfactory bulb in vivo,” PLoS ONE4(2), e4418 (2009). [CrossRef] [PubMed]
  56. B. Xu, Y. Coello, V. V. Lozovoy, and M. Dantus, “Two-photon fluorescence excitation spectroscopy by pulse shaping ultrabroad-bandwidth femtosecond laser pulses,” Appl. Opt.49(32), 6348–6353 (2010). [CrossRef] [PubMed]
  57. H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J.77(4), 2226–2236 (1999). [CrossRef] [PubMed]
  58. D. Pestov, Y. Andegeko, V. V. Lozovoy, and M. Dantus, “Photobleaching and photoenhancement of endogenous fluorescence observed in two-photon microscopy with broadband laser sources,” J. Opt.12(8), 084006 (2010). [CrossRef]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.


Fig. 1 Fig. 2 Fig. 3
Fig. 4

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited