Abstract

The use of spectrally distinguishable fluorescent dyes enables imaging of multiple targets. However, in two-photon microscopy, the number of fluorescent labels with distinct emission spectra that can be effectively excited and resolved is constrained by the confined tuning range of the excitation laser and the broad and overlapping nature of fluorophore two-photon absorption spectra. This limitation effectively reduces the number of available imaging channels. Here, we demonstrate that two-photon steady state anisotropy imaging (2PSSA) offers the capability to resolve otherwise unresolvable fluorescent tracers both in live cells and in mouse tumor models. This approach expands the number of biological targets that can be imaged simultaneously, increasing the total amount of information that can be obtained through imaging.

© 2014 Optical Society of America

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  1. N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
    [CrossRef]
  2. M. J. Pittet and R. Weissleder, Cell 147, 983 (2011).
    [CrossRef]
  3. D. Entenberg, J. Wyckoff, B. Gligorijevic, E. T. Roussos, V. V. Verkhusha, J. W. Pollard, and J. Condeelis, Nat. Protoc. 6, 1500 (2011).
    [CrossRef]
  4. M. H. Brenner, D. Cai, J. A. Swanson, and J. P. Ogilvie, Opt. Express 21, 17256 (2013).
    [CrossRef]
  5. A. J. Radosevich, M. B. Bouchard, S. A. Burgess, B. R. Chen, and E. M. Hillman, Opt. Lett. 33, 2164 (2008).
    [CrossRef]
  6. P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Debarre, and E. Beaurepaire, Nat. Methods 9, 815 (2012).
    [CrossRef]
  7. H. Brismar and B. Ulfhake, Nat. Biotechnol. 15, 373 (1997).
    [CrossRef]
  8. K. Carlsson and A. Liljeborg, J. Microsc. 191, 119 (1998).
    [CrossRef]
  9. J. D. Driscoll, A. Y. Shih, S. Iyengar, J. J. Field, G. A. White, J. A. Squier, G. Cauwenberghs, and D. Kleinfeld, J. Neurophysiol. 105, 3106 (2011).
    [CrossRef]
  10. F. Perrin, J. Phys. Radium 7, 390 (1926).
  11. C. E. Bigelow, D. L. Conover, and T. H. Foster, Opt. Lett. 28, 695 (2003).
    [CrossRef]
  12. J. Lazar, A. Bonder, S. Timr, and S. J. Firestein, Nat. Methods 8, 684 (2011).
    [CrossRef]
  13. J. M. Dubach, C. Vinegoni, R. Mazitschek, P. F. Feruglio, L. Cameron, and R. Weissleder, Nat. Commun. 5, 3946 (2014).
    [CrossRef]
  14. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer, 2006).

2014 (1)

J. M. Dubach, C. Vinegoni, R. Mazitschek, P. F. Feruglio, L. Cameron, and R. Weissleder, Nat. Commun. 5, 3946 (2014).
[CrossRef]

2013 (1)

2012 (1)

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Debarre, and E. Beaurepaire, Nat. Methods 9, 815 (2012).
[CrossRef]

2011 (4)

M. J. Pittet and R. Weissleder, Cell 147, 983 (2011).
[CrossRef]

D. Entenberg, J. Wyckoff, B. Gligorijevic, E. T. Roussos, V. V. Verkhusha, J. W. Pollard, and J. Condeelis, Nat. Protoc. 6, 1500 (2011).
[CrossRef]

J. D. Driscoll, A. Y. Shih, S. Iyengar, J. J. Field, G. A. White, J. A. Squier, G. Cauwenberghs, and D. Kleinfeld, J. Neurophysiol. 105, 3106 (2011).
[CrossRef]

J. Lazar, A. Bonder, S. Timr, and S. J. Firestein, Nat. Methods 8, 684 (2011).
[CrossRef]

2008 (1)

2005 (1)

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef]

2003 (1)

1998 (1)

K. Carlsson and A. Liljeborg, J. Microsc. 191, 119 (1998).
[CrossRef]

1997 (1)

H. Brismar and B. Ulfhake, Nat. Biotechnol. 15, 373 (1997).
[CrossRef]

1926 (1)

F. Perrin, J. Phys. Radium 7, 390 (1926).

Beaurepaire, E.

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Debarre, and E. Beaurepaire, Nat. Methods 9, 815 (2012).
[CrossRef]

Bigelow, C. E.

Bonder, A.

J. Lazar, A. Bonder, S. Timr, and S. J. Firestein, Nat. Methods 8, 684 (2011).
[CrossRef]

Bouchard, M. B.

Brenner, M. H.

Brismar, H.

H. Brismar and B. Ulfhake, Nat. Biotechnol. 15, 373 (1997).
[CrossRef]

Burgess, S. A.

Cai, D.

Cameron, L.

J. M. Dubach, C. Vinegoni, R. Mazitschek, P. F. Feruglio, L. Cameron, and R. Weissleder, Nat. Commun. 5, 3946 (2014).
[CrossRef]

Carlsson, K.

K. Carlsson and A. Liljeborg, J. Microsc. 191, 119 (1998).
[CrossRef]

Cauwenberghs, G.

J. D. Driscoll, A. Y. Shih, S. Iyengar, J. J. Field, G. A. White, J. A. Squier, G. Cauwenberghs, and D. Kleinfeld, J. Neurophysiol. 105, 3106 (2011).
[CrossRef]

Chen, B. R.

Condeelis, J.

D. Entenberg, J. Wyckoff, B. Gligorijevic, E. T. Roussos, V. V. Verkhusha, J. W. Pollard, and J. Condeelis, Nat. Protoc. 6, 1500 (2011).
[CrossRef]

Conover, D. L.

Debarre, D.

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Debarre, and E. Beaurepaire, Nat. Methods 9, 815 (2012).
[CrossRef]

Driscoll, J. D.

J. D. Driscoll, A. Y. Shih, S. Iyengar, J. J. Field, G. A. White, J. A. Squier, G. Cauwenberghs, and D. Kleinfeld, J. Neurophysiol. 105, 3106 (2011).
[CrossRef]

Dubach, J. M.

J. M. Dubach, C. Vinegoni, R. Mazitschek, P. F. Feruglio, L. Cameron, and R. Weissleder, Nat. Commun. 5, 3946 (2014).
[CrossRef]

Entenberg, D.

D. Entenberg, J. Wyckoff, B. Gligorijevic, E. T. Roussos, V. V. Verkhusha, J. W. Pollard, and J. Condeelis, Nat. Protoc. 6, 1500 (2011).
[CrossRef]

Feruglio, P. F.

J. M. Dubach, C. Vinegoni, R. Mazitschek, P. F. Feruglio, L. Cameron, and R. Weissleder, Nat. Commun. 5, 3946 (2014).
[CrossRef]

Field, J. J.

J. D. Driscoll, A. Y. Shih, S. Iyengar, J. J. Field, G. A. White, J. A. Squier, G. Cauwenberghs, and D. Kleinfeld, J. Neurophysiol. 105, 3106 (2011).
[CrossRef]

Firestein, S. J.

J. Lazar, A. Bonder, S. Timr, and S. J. Firestein, Nat. Methods 8, 684 (2011).
[CrossRef]

Foster, T. H.

Gligorijevic, B.

D. Entenberg, J. Wyckoff, B. Gligorijevic, E. T. Roussos, V. V. Verkhusha, J. W. Pollard, and J. Condeelis, Nat. Protoc. 6, 1500 (2011).
[CrossRef]

Hillman, E. M.

Iyengar, S.

J. D. Driscoll, A. Y. Shih, S. Iyengar, J. J. Field, G. A. White, J. A. Squier, G. Cauwenberghs, and D. Kleinfeld, J. Neurophysiol. 105, 3106 (2011).
[CrossRef]

Kleinfeld, D.

J. D. Driscoll, A. Y. Shih, S. Iyengar, J. J. Field, G. A. White, J. A. Squier, G. Cauwenberghs, and D. Kleinfeld, J. Neurophysiol. 105, 3106 (2011).
[CrossRef]

Labroille, G.

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Debarre, and E. Beaurepaire, Nat. Methods 9, 815 (2012).
[CrossRef]

Lakowicz, J. R.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer, 2006).

Lazar, J.

J. Lazar, A. Bonder, S. Timr, and S. J. Firestein, Nat. Methods 8, 684 (2011).
[CrossRef]

Liljeborg, A.

K. Carlsson and A. Liljeborg, J. Microsc. 191, 119 (1998).
[CrossRef]

Livet, J.

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Debarre, and E. Beaurepaire, Nat. Methods 9, 815 (2012).
[CrossRef]

Loulier, K.

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Debarre, and E. Beaurepaire, Nat. Methods 9, 815 (2012).
[CrossRef]

Mahou, P.

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Debarre, and E. Beaurepaire, Nat. Methods 9, 815 (2012).
[CrossRef]

Matho, K. S.

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Debarre, and E. Beaurepaire, Nat. Methods 9, 815 (2012).
[CrossRef]

Mazitschek, R.

J. M. Dubach, C. Vinegoni, R. Mazitschek, P. F. Feruglio, L. Cameron, and R. Weissleder, Nat. Commun. 5, 3946 (2014).
[CrossRef]

Morin, X.

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Debarre, and E. Beaurepaire, Nat. Methods 9, 815 (2012).
[CrossRef]

Ogilvie, J. P.

Perrin, F.

F. Perrin, J. Phys. Radium 7, 390 (1926).

Pittet, M. J.

M. J. Pittet and R. Weissleder, Cell 147, 983 (2011).
[CrossRef]

Pollard, J. W.

D. Entenberg, J. Wyckoff, B. Gligorijevic, E. T. Roussos, V. V. Verkhusha, J. W. Pollard, and J. Condeelis, Nat. Protoc. 6, 1500 (2011).
[CrossRef]

Radosevich, A. J.

Roussos, E. T.

D. Entenberg, J. Wyckoff, B. Gligorijevic, E. T. Roussos, V. V. Verkhusha, J. W. Pollard, and J. Condeelis, Nat. Protoc. 6, 1500 (2011).
[CrossRef]

Shaner, N. C.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef]

Shih, A. Y.

J. D. Driscoll, A. Y. Shih, S. Iyengar, J. J. Field, G. A. White, J. A. Squier, G. Cauwenberghs, and D. Kleinfeld, J. Neurophysiol. 105, 3106 (2011).
[CrossRef]

Squier, J. A.

J. D. Driscoll, A. Y. Shih, S. Iyengar, J. J. Field, G. A. White, J. A. Squier, G. Cauwenberghs, and D. Kleinfeld, J. Neurophysiol. 105, 3106 (2011).
[CrossRef]

Steinbach, P. A.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef]

Supatto, W.

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Debarre, and E. Beaurepaire, Nat. Methods 9, 815 (2012).
[CrossRef]

Swanson, J. A.

Timr, S.

J. Lazar, A. Bonder, S. Timr, and S. J. Firestein, Nat. Methods 8, 684 (2011).
[CrossRef]

Tsien, R. Y.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef]

Ulfhake, B.

H. Brismar and B. Ulfhake, Nat. Biotechnol. 15, 373 (1997).
[CrossRef]

Verkhusha, V. V.

D. Entenberg, J. Wyckoff, B. Gligorijevic, E. T. Roussos, V. V. Verkhusha, J. W. Pollard, and J. Condeelis, Nat. Protoc. 6, 1500 (2011).
[CrossRef]

Vinegoni, C.

J. M. Dubach, C. Vinegoni, R. Mazitschek, P. F. Feruglio, L. Cameron, and R. Weissleder, Nat. Commun. 5, 3946 (2014).
[CrossRef]

Weissleder, R.

J. M. Dubach, C. Vinegoni, R. Mazitschek, P. F. Feruglio, L. Cameron, and R. Weissleder, Nat. Commun. 5, 3946 (2014).
[CrossRef]

M. J. Pittet and R. Weissleder, Cell 147, 983 (2011).
[CrossRef]

White, G. A.

J. D. Driscoll, A. Y. Shih, S. Iyengar, J. J. Field, G. A. White, J. A. Squier, G. Cauwenberghs, and D. Kleinfeld, J. Neurophysiol. 105, 3106 (2011).
[CrossRef]

Wyckoff, J.

D. Entenberg, J. Wyckoff, B. Gligorijevic, E. T. Roussos, V. V. Verkhusha, J. W. Pollard, and J. Condeelis, Nat. Protoc. 6, 1500 (2011).
[CrossRef]

Zimmerley, M.

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Debarre, and E. Beaurepaire, Nat. Methods 9, 815 (2012).
[CrossRef]

Cell (1)

M. J. Pittet and R. Weissleder, Cell 147, 983 (2011).
[CrossRef]

J. Microsc. (1)

K. Carlsson and A. Liljeborg, J. Microsc. 191, 119 (1998).
[CrossRef]

J. Neurophysiol. (1)

J. D. Driscoll, A. Y. Shih, S. Iyengar, J. J. Field, G. A. White, J. A. Squier, G. Cauwenberghs, and D. Kleinfeld, J. Neurophysiol. 105, 3106 (2011).
[CrossRef]

J. Phys. Radium (1)

F. Perrin, J. Phys. Radium 7, 390 (1926).

Nat. Biotechnol. (1)

H. Brismar and B. Ulfhake, Nat. Biotechnol. 15, 373 (1997).
[CrossRef]

Nat. Commun. (1)

J. M. Dubach, C. Vinegoni, R. Mazitschek, P. F. Feruglio, L. Cameron, and R. Weissleder, Nat. Commun. 5, 3946 (2014).
[CrossRef]

Nat. Methods (3)

J. Lazar, A. Bonder, S. Timr, and S. J. Firestein, Nat. Methods 8, 684 (2011).
[CrossRef]

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef]

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Debarre, and E. Beaurepaire, Nat. Methods 9, 815 (2012).
[CrossRef]

Nat. Protoc. (1)

D. Entenberg, J. Wyckoff, B. Gligorijevic, E. T. Roussos, V. V. Verkhusha, J. W. Pollard, and J. Condeelis, Nat. Protoc. 6, 1500 (2011).
[CrossRef]

Opt. Express (1)

Opt. Lett. (2)

Other (1)

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer, 2006).

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Figures (3)

Fig. 1.
Fig. 1.

Two-photon fluorescence anisotropy imaging. A, Schematic illustration of the imaging setup, F:spectral and polarization filters; PBS: polarizing beam splitter; HWP: half wave-plate; GT: Glan-Thompson. B, Emission spectrum for fluorescein and FITC-dextran. Half the wavelength was utilized for two-photon imaging (arrow) and optical emission filter bandwidth (green box). C, Images of fluorescence (top) and anisotropy (bottom) collected in two-photon excitation for a water-based solution of fluorescein and FITC-dextran. Lookup table for color anisotropy assignment is shown on right.

Fig. 2.
Fig. 2.

Two-photon in vivo and in vitro fluorescence (left) and anisotropy (right) images of A, MitoTracker Green in HT1080 cells; B, FITC-dextran in mouse ear vasculature; C, FITC-dextran in tumor associated macrophages; and D, GFP expressing HT1080 cells in a tumor. Lookup tables for anisotropy color assignments are shown on right.

Fig. 3.
Fig. 3.

Simultaneous multilabel imaging by 2PSSA microscopy (left, fluorescence; right, anisotropy). A, In vitro imaging of HT1080 GFP cells loaded with MitoTracker Green. B, Maximum intensity projection image stack of an HT1080 GFP tumor implanted in a skinfold dorsal window chamber with FITC-dextran vascular perfusion. Red, GFP; blue, FITC-dextran. C, Maximum intensity projection image stack at the periphery of an HT1080 GFP tumor in a mouse vascularly injected with FITC-dextran 24 h before and during imaging. Red, GFP; blue, vascular FITC-dextran; yellow, tumor associated macrophages FITC-dextran. Lookup tables for anisotropy color assignments are shown on right.

Equations (2)

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r0r=1+τfτr,
r=IGII+2GI,

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