Abstract

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

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2012 (3)

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. Methods 9(8), 815–818 (2012).
[CrossRef] [PubMed]

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. Express 3(9), 1972–1977 (2012).
[CrossRef] [PubMed]

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]

2011 (4)

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,” Development 138(13), 2705–2715 (2011).
[CrossRef] [PubMed]

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]

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[CrossRef] [PubMed]

E. R. Tkaczyk and A. H. Tkaczyk, “Multiphoton flow cytometry strategies and applications,” Cytometry A 79(10), 775–788 (2011).
[CrossRef] [PubMed]

2010 (5)

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,” Cell 143(1), 134–144 (2010).
[CrossRef] [PubMed]

K. Red-Horse, H. Ueno, I. L. Weissman, and M. A. Krasnow, “Coronary arteries form by developmental reprogramming of venous cells,” Nature 464(7288), 549–553 (2010).
[CrossRef] [PubMed]

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]

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]

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]

2009 (8)

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 ONE 4(2), e4418 (2009).
[CrossRef] [PubMed]

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]

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]

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]

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]

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. Express 17(15), 12741–12752 (2009).
[CrossRef] [PubMed]

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. Express 17(16), 13737–13746 (2009).
[CrossRef] [PubMed]

D. S. Moore, “Optimal coherent control of sensitivity and selectivity in spectrochemical analysis,” Anal. Bioanal. Chem. 393(1), 51–56 (2009).
[CrossRef] [PubMed]

2008 (4)

H. Kawano, T. Kogure, Y. Abe, H. Mizuno, and A. Miyawaki, “Two-photon dual-color imaging using fluorescent proteins,” Nat. Methods 5(5), 373–374 (2008).
[CrossRef] [PubMed]

H. W. Ai, K. L. Hazelwood, M. W. Davidson, and R. E. Campbell, “Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors,” Nat. Methods 5(5), 401–403 (2008).
[CrossRef] [PubMed]

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. B 25(6), A140–A150 (2008).
[CrossRef]

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. Methods 5(6), 545–551 (2008).
[CrossRef] [PubMed]

2007 (6)

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. Methods 4(9), 741–746 (2007).
[CrossRef] [PubMed]

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. Methods 4(7), 555–557 (2007).
[CrossRef] [PubMed]

L. M. Davis and G. Shen, “Extension of multidimensional microscopy to ultrasensitive applications with maximum-likelihood analysis,” Proc. SPIE 6443, 64430N, 64430N-12 (2007).
[CrossRef]

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]

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,” Nature 450(7166), 56–62 (2007).
[CrossRef] [PubMed]

T. Misgeld, M. Kerschensteiner, F. M. Bareyre, R. W. Burgess, and J. W. Lichtman, “Imaging axonal transport of mitochondria in vivo,” Nat. Methods 4(7), 559–561 (2007).
[CrossRef] [PubMed]

2006 (6)

B. N. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, “The fluorescent toolbox for assessing protein location and function,” Science 312(5771), 217–224 (2006).
[CrossRef] [PubMed]

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]

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. Express 14(2), 759–766 (2006).
[CrossRef] [PubMed]

L. T. Schelhas, J. C. Shane, and M. Dantus, “Advantages of ultrashort phase-shaped pulses for selective two-photon activation and biomedical imaging,” Nanomedicine 2(3), 177–181 (2006).
[CrossRef] [PubMed]

Y. Garini, I. T. Young, and G. McNamara, “Spectral imaging: principles and applications,” Cytometry A 69(8), 735–747 (2006).
[CrossRef] [PubMed]

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. B 23(4), 750–759 (2006).
[CrossRef]

2005 (4)

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]

V. V. Lozovoy and M. Dantus, “Systematic control of nonlinear optical processes using optimally shaped femtosecond pulses,” ChemPhysChem 6(10), 1970–2000 (2005).
[CrossRef] [PubMed]

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, “A guide to choosing fluorescent proteins,” Nat. Methods 2(12), 905–909 (2005).
[CrossRef] [PubMed]

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]

2004 (2)

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. A 108(1), 53–58 (2004).
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2003 (3)

I. Pastirk, J. Dela Cruz, K. Walowicz, V. Lozovoy, and M. Dantus, “Selective two-photon microscopy with shaped femtosecond pulses,” Opt. Express 11(14), 1695–1701 (2003).
[CrossRef] [PubMed]

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).
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2002 (2)

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
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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. A 106(41), 9369–9373 (2002).
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2001 (1)

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).
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2000 (3)

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,” Neuron 28(1), 41–51 (2000).
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1999 (1)

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).
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1998 (2)

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1996 (1)

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1990 (1)

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B. N. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, “The fluorescent toolbox for assessing protein location and function,” Science 312(5771), 217–224 (2006).
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H. W. Ai, K. L. Hazelwood, M. W. Davidson, and R. E. Campbell, “Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors,” Nat. Methods 5(5), 401–403 (2008).
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Bearman, G.

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).
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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,” Nature 450(7166), 56–62 (2007).
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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).
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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 ONE 4(2), e4418 (2009).
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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. Methods 4(7), 555–557 (2007).
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Burgess, R. W.

T. Misgeld, M. Kerschensteiner, F. M. Bareyre, R. W. Burgess, and J. W. Lichtman, “Imaging axonal transport of mitochondria in vivo,” Nat. Methods 4(7), 559–561 (2007).
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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).
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H. W. Ai, K. L. Hazelwood, M. W. Davidson, and R. E. Campbell, “Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors,” Nat. Methods 5(5), 401–403 (2008).
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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).
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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 ONE 4(2), e4418 (2009).
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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. Methods 4(9), 741–746 (2007).
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Chudakov, D. M.

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. Methods 4(9), 741–746 (2007).
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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,” Development 138(13), 2705–2715 (2011).
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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,” Cell 143(1), 134–144 (2010).
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Comstock, M.

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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).
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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).
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Dantus, M.

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).
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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).
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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. B 25(6), A140–A150 (2008).
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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. B 23(4), 750–759 (2006).
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L. T. Schelhas, J. C. Shane, and M. Dantus, “Advantages of ultrashort phase-shaped pulses for selective two-photon activation and biomedical imaging,” Nanomedicine 2(3), 177–181 (2006).
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V. V. Lozovoy and M. Dantus, “Systematic control of nonlinear optical processes using optimally shaped femtosecond pulses,” ChemPhysChem 6(10), 1970–2000 (2005).
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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. A 108(1), 53–58 (2004).
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M. Comstock, V. Lozovoy, I. Pastirk, and M. Dantus, “Multiphoton intrapulse interference 6; binary phase shaping,” Opt. Express 12(6), 1061–1066 (2004).
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I. Pastirk, J. Dela Cruz, K. Walowicz, V. Lozovoy, and M. Dantus, “Selective two-photon microscopy with shaped femtosecond pulses,” Opt. Express 11(14), 1695–1701 (2003).
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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).
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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. A 106(41), 9369–9373 (2002).
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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. Methods 5(6), 545–551 (2008).
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H. W. Ai, K. L. Hazelwood, M. W. Davidson, and R. E. Campbell, “Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors,” Nat. Methods 5(5), 401–403 (2008).
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Dela Cruz, J. M.

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. A 108(1), 53–58 (2004).
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W. Denk, “Two-photon excitation in functional biological imaging,” J. Biomed. Opt. 1(3), 296–304 (1996).
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W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
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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,” Nature 450(7166), 56–62 (2007).
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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 ONE 4(2), e4418 (2009).
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B. N. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, “The fluorescent toolbox for assessing protein location and function,” Science 312(5771), 217–224 (2006).
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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. Methods 4(9), 741–746 (2007).
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Feng, G.

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,” Neuron 28(1), 41–51 (2000).
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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. Methods 4(9), 741–746 (2007).
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Fraser, S. E.

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).
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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. Methods 4(7), 555–557 (2007).
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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. Methods 4(7), 555–557 (2007).
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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).
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B. N. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, “The fluorescent toolbox for assessing protein location and function,” Science 312(5771), 217–224 (2006).
[CrossRef] [PubMed]

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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).
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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. Methods 4(7), 555–557 (2007).
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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 ONE 4(2), e4418 (2009).
[CrossRef] [PubMed]

Gunaratne, T. C.

Gunn, J. M.

Haraguchi, T.

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
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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).
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Hazelwood, K. L.

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Figures (4)

Fig. 1
Fig. 1

Experimental setup. M1-M5: mirrors, G = grating, CM = curved mirror, SLM = spatial light modulator, DM1 = 660DCXR dichroic mirror, DM2 = 595DCXR dichroic mirror, OBJ = 60x NA1.2 water immersion objective; SP = 650nm short pass filter.

Fig. 2
Fig. 2

Fluorescent protein spectra and selective excitations. (a) Two-photon brightness (dotted curves) and fluorescence emission spectra (solid curves) of mAmetrine, TagRFPt and mKate2 are plotted in green, orange, and red, respectively (adapted from ref [30]). We note that TagRFPt, which was used in this study, is known to have a very similar spectral response to TagRFP shown here [43, 45]. Normalized spectrum of the broadband Ti:Sa laser is plotted in black (long dashed curve). The 595DCXR dichroic mirror transform function is plotted in blue. (b) SHG signal from a β-BaBO4 crystal for a TL pulse (dashed line) and two selective two-photon excitations with SHG centered at 390 nm (blue phase-shaped pulse, blue solid line) and 410 nm (red phase-shaped pulse, red solid line).

Fig. 3
Fig. 3

Images of two-photon excitation of a mixed sample, in which each mammalian COS-7 cell only expresses one of either mAmetrine, TagRFPt or mKate2 fluorescent proteins. Cells were excited by the TL (left column), blue phase-shaped (middle column) and red phase-shaped (right column) pulses. Solid arrows, putative mAmetrine-expressing cells; open arrows, putative TagRFPt-expressing cells; solid arrowhead, putative mKate2-expressing cell. The TL pulses create higher image intensities than the shaped pulses do. To avoid saturation, lower PMT voltages have been applied to lower the fluorescence intensity of ~7 fold. Additionally, brightness and contrast of panels (a) and (b) have been scaled down ~10 fold to avoid saturation in display. Scale bar is 20 µm.

Fig. 4
Fig. 4

Linear unmixing. (a) Signal of mAmetrine, TagRFPt and mKate2 from Fig. 3 (middle and right columns) is restored in three separate images. (b) Pseudo-colored merge image of a sample co-expressing all three FPs after linear unmixing. In (a) and (b), mAmetrine, TagRFPt and mKate2 are pseudo-colored in blue, green and red in the merge image, respectively. Gamma correction (0.5) was applied to both panels in order to better present the dimly labeled cells. Scale bars are 20 µm.

Tables (1)

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Table 1 Constants quantifying the fractions of fluorophores intensity in observed images

Equations (3)

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Sg(ω) | E (2) (ω) | 2
E (2) (ω)= dω'| E(ω') || E(ωω') |exp{ i[ φ(ω')+φ(ωω') ] }
[ I blue,GREEN I blue,RED I red,GREEN ]= [ X A ^ X R ^ X K ^ Y A ^ Y R ^ Y K ^ Z A ^ Z R ^ Z K ^ ][ I A I R I K ]

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