Abstract

The need for optical sectioning in bio-imaging has amongst others led to the development of the two-photon scanning microscopy. However, this comes with some intrinsic fundamental limitations in the temporal domain as the focused spot has to be scanned mechanically in the sample plane. Hence for a large number of biological applications where imaging speed is a limiting factor, it would be significantly advantageous to generate widefield excitations with an optical sectioning comparable to the two-photon scanning microscopy. Recently by using the technique of temporal focusing it was shown that high axial resolution widefield excitation can be generated in picosecond time scales without any mechanical moving parts. However the achievable axial resolution is still well above that of a two-photon scanning microscope. Here we demonstrate a new ultrafast widefield two-photon imaging technique termed Multifocal Temporal Focusing (MUTEF) which relies on the generation of a set of diffraction limited beams produced by an Echelle grating that scan across a second tilted diffraction grating in picosecond time scale, generating a widefield excitation area with an axial resolution comparable to a two-photon scanning microscope. Using this method we have shown widefield two-photon imaging on fixed biological samples with an axial sectioning with a FWHM of ~0.85 μm.

© 2010 OSA

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [PubMed]

2010 (1)

B. K. Andrasfalvy, B. V. Zemelman, J. Tang, and A. Vaziri, “Two-photon single-cell optogenetic control of neuronal activity by sculpted light,” Proc. Natl. Acad. Sci. U.S.A. 107(26), 11981–11986 (2010).
[CrossRef] [PubMed]

2009 (1)

2008 (2)

M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing in nonlinear microscopy,” Opt. Commun. 281(7), 1796–1805 (2008).
[CrossRef] [PubMed]

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

2007 (1)

S. A. Diddams, L. Hollberg, and V. Mbele, “Molecular fingerprinting with the resolved modes of a femtosecond laser frequency comb,” Nature 445(7128), 627–630 (2007).
[CrossRef] [PubMed]

2005 (3)

1998 (1)

1994 (1)

R. M. Williams, D. W. Piston, and W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8(11), 804–813 (1994).
[PubMed]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Andrasfalvy, B. K.

B. K. Andrasfalvy, B. V. Zemelman, J. Tang, and A. Vaziri, “Two-photon single-cell optogenetic control of neuronal activity by sculpted light,” Proc. Natl. Acad. Sci. U.S.A. 107(26), 11981–11986 (2010).
[CrossRef] [PubMed]

Bewersdorf, J.

de Sars, V.

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Diddams, S. A.

S. A. Diddams, L. Hollberg, and V. Mbele, “Molecular fingerprinting with the resolved modes of a femtosecond laser frequency comb,” Nature 445(7128), 627–630 (2007).
[CrossRef] [PubMed]

Durst, M.

Durst, M. E.

M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing in nonlinear microscopy,” Opt. Commun. 281(7), 1796–1805 (2008).
[CrossRef] [PubMed]

Emiliani, V.

Hell, S. W.

Hollberg, L.

S. A. Diddams, L. Hollberg, and V. Mbele, “Molecular fingerprinting with the resolved modes of a femtosecond laser frequency comb,” Nature 445(7128), 627–630 (2007).
[CrossRef] [PubMed]

Mbele, V.

S. A. Diddams, L. Hollberg, and V. Mbele, “Molecular fingerprinting with the resolved modes of a femtosecond laser frequency comb,” Nature 445(7128), 627–630 (2007).
[CrossRef] [PubMed]

Oron, D.

Papagiakoumou, E.

Pick, R.

Piston, D. W.

R. M. Williams, D. W. Piston, and W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8(11), 804–813 (1994).
[PubMed]

Shank, C. V.

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

Shroff, H.

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

Silberberg, Y.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Tal, E.

Tang, J.

B. K. Andrasfalvy, B. V. Zemelman, J. Tang, and A. Vaziri, “Two-photon single-cell optogenetic control of neuronal activity by sculpted light,” Proc. Natl. Acad. Sci. U.S.A. 107(26), 11981–11986 (2010).
[CrossRef] [PubMed]

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

van Howe, J.

Vaziri, A.

B. K. Andrasfalvy, B. V. Zemelman, J. Tang, and A. Vaziri, “Two-photon single-cell optogenetic control of neuronal activity by sculpted light,” Proc. Natl. Acad. Sci. U.S.A. 107(26), 11981–11986 (2010).
[CrossRef] [PubMed]

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

Webb, W. W.

R. M. Williams, D. W. Piston, and W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8(11), 804–813 (1994).
[PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Williams, R. M.

R. M. Williams, D. W. Piston, and W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8(11), 804–813 (1994).
[PubMed]

Xu, C.

M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing in nonlinear microscopy,” Opt. Commun. 281(7), 1796–1805 (2008).
[CrossRef] [PubMed]

G. H. Zhu, J. van Howe, M. Durst, W. Zipfel, and C. Xu, “Simultaneous spatial and temporal focusing of femtosecond pulses,” Opt. Express 13(6), 2153–2159 (2005).
[CrossRef] [PubMed]

Zemelman, B. V.

B. K. Andrasfalvy, B. V. Zemelman, J. Tang, and A. Vaziri, “Two-photon single-cell optogenetic control of neuronal activity by sculpted light,” Proc. Natl. Acad. Sci. U.S.A. 107(26), 11981–11986 (2010).
[CrossRef] [PubMed]

Zhu, G.

M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing in nonlinear microscopy,” Opt. Commun. 281(7), 1796–1805 (2008).
[CrossRef] [PubMed]

Zhu, G. H.

Zipfel, W.

FASEB J. (1)

R. M. Williams, D. W. Piston, and W. W. Webb, “Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry,” FASEB J. 8(11), 804–813 (1994).
[PubMed]

Nature (1)

S. A. Diddams, L. Hollberg, and V. Mbele, “Molecular fingerprinting with the resolved modes of a femtosecond laser frequency comb,” Nature 445(7128), 627–630 (2007).
[CrossRef] [PubMed]

Opt. Commun. (1)

M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing in nonlinear microscopy,” Opt. Commun. 281(7), 1796–1805 (2008).
[CrossRef] [PubMed]

Opt. Express (3)

Opt. Lett. (2)

Proc. Natl. Acad. Sci. U.S.A. (2)

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

B. K. Andrasfalvy, B. V. Zemelman, J. Tang, and A. Vaziri, “Two-photon single-cell optogenetic control of neuronal activity by sculpted light,” Proc. Natl. Acad. Sci. U.S.A. 107(26), 11981–11986 (2010).
[CrossRef] [PubMed]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

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Figures (5)

Fig. 1
Fig. 1

Overview of multifocal temporal focusing (MUTEF) for ultrafast high axial resolution widefield imaging. An Echelle grating is imaged via a telescope onto a diffraction grating with lines orthogonal to the former. In this fashion multiple beamlets each of which filling the backfocal aperture of an objective via dispersion in one dimension and via diffraction in the other dimension scan in picosecond time scale on the diffraction grating and the sample leading to a widefield excitation area.

Fig. 2
Fig. 2

Details of the pulse propagation and the geometry in MUTEF at the diffraction and the Echelle grating.

Fig. 3
Fig. 3

a) Comparison of the axial localization of excitation in different two-photon imaging schemes for a spot size of ~6µm. While regular two-photon excitation (green trace) with such a beam size only leads to a poor axial confinement (FWHM~7µm) and the widefield temporal focusing (blue line) to a confinement of ~1.6µm, both the line scan temporal focusing (black trace) and the MUTEF imaging (red) curve yield the same axial confinement (~0.85µm) as a two-photon scanning microscope. However only in MUTEF imaging a wide field of view can be achieved without any mechanical scanning. b) Lateral intensity distribution of the MUTEF excitation

Fig. 4
Fig. 4

Demonstration of the axial confinement of MUTEF imaging and its comparison to temporal focusing and widefield 2-photon excitation for a spot size of ~6µm on fluorescently labeled pollen grains. a) Overview fluorescence image of a pollen grain using a fluorescence lamp. b) Comparison between MUTEF, and wide-field 2-photon imaging at two axial planes separated by 50nm. While the MUTEF imaging clearly leads to the excitation of either the upper or the lower spike, the temporal focusing imaging leads to the excitation of both spike at x = 0nm and the widefield temporal focusing in addition to significant amount of out of focus florescence. c) Excitation of a spike with a directional component in the axial direction when the sample is moved by steps of 100nm. One can clearly see that the excitation area on the spike moves from the bole to the tip of the spike for the MUTEF imaging, whereas in the temporal focusing the spike is more homogenously excited over the entire range of the axial range and the widefield two-photon imaging almost does not give any sectioning. d) The three-dimensional representation of an image stack over a range of ~20µm shows clearly the higher optical section capability of the MUTEF imaging compared to the two other alternative methods. The integration time for all two-photon images was 100ms.

Fig. 5
Fig. 5

Demonstration of axial confinement of MUTEF imaging and its comparison to temporal focusing and widefield 2-photon excitation for a spot size of ~6µm on fixed mouse kidney cells labeled with Alexa flour conjugated to WGA labeling apical surface. a) Overview fluorescence image of the fixed kidney cells b) Comparison between MUTEF, temporal focusing and wide-field 2-photon imaging at three axial planes separated by 50nm. In the MUTEF imaging an inverted V-shape structure is excited at x = 0nm for which the excitation moves to the tip of the structure at x = 50nm and is significantly reduced on the right hand side tip at x = 100nm. Imaging the same region using temporal focusing leads to a more unspecific excitation of a region which remains almost unchanged over the axial range. Using just 2-photon excitation without any temporal focusing leads in addition to a significant amount of out of focus background. c) The three-dimensional representation of an image stack over a range of ~20µm shows clearly the higher optical section capability of the MUTEF imaging compared to the two other alternative methods. The integration time for all two-photon images was 100ms.

Equations (8)

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T P E ( Δ z ) = [ 1 + ( Δ z z R ) 2 ] 1 2 ,
T P E ( Δ z ) = [ 1 + ( Δ z z R ) 2 ] 1 ,
Δ λ ε sin α 2 = 2 λ π ω 0 z ,
σ sin α 1 M 2 s sin α 2 ,
a M 1 M 2 s ,
2 b / sin α 1 20 M 2 s sin α 2 ,
Δ λ f 2 ε sin α 2 = D .
d 3 b s M 1 2 s 2 M 2 2 π 4 λ ,

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