Abstract

We present a simple and robust way to reject out-of-focus background when performing deep two-photon excited fluorescence (TPEF) imaging in thick tissue. The technique is based on the use of a deformable mirror (DM) to introduce illumination aberrations that preferentially degrade TPEF signal while leaving TPEF background relatively unchanged. A subtraction of aberrated from unaberrated images leads to background rejection. We present a heuristic description of our technique, which we corroborate with experiment. An added benefit of our technique is that it leads to somewhat improved image resolution.

© 2006 Optical Society of America

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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
  4. M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth," J. Neurosci. Meth. 111, 29-37 (2001).
    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
  20. R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, T. M. Jovin, "Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes," Micron 34, 293-300 (2003).
    [CrossRef] [PubMed]
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2006 (1)

K. Svoboda and R. Yasuda, "Principles of two-photon excitation microscopy and its applications to neuroscience," Neuron 50, 823-829 (2006).
[CrossRef] [PubMed]

2005 (4)

2003 (4)

2002 (2)

J. Perreault, T. G. Bifano, B. Martin Levine,M. N. Horenstein, "Adaptive optic correction using microelectromechanical deformable mirrors," Opt. Eng. 41, 561-566 (2002)
[CrossRef]

E. Beaurepaire and J. Mertz, "Epi-fluorescence collection in two-photon microscopy," Appl. Opt. 41, 5376-5382 (2002).
[CrossRef] [PubMed]

2001 (2)

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth," J. Neurosci. Meth. 111, 29-37 (2001).
[CrossRef]

E. Beaurepaire, M. Oheim, J. Mertz "Ultra-deep two-photon fluorescence excitation in turbid media," Opt. Commun. 188, 25-29 (2001).
[CrossRef]

2000 (1)

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, S. Kawata, "Adaptive aberration correction in a two-photon microscope", J. Microsc. 200, 105-108 (2000).
[CrossRef] [PubMed]

1999 (1)

1995 (1)

1990 (1)

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

1967 (1)

1964 (1)

Alfano, R. R.

Beaurepaire, E.

E. Beaurepaire and J. Mertz, "Epi-fluorescence collection in two-photon microscopy," Appl. Opt. 41, 5376-5382 (2002).
[CrossRef] [PubMed]

E. Beaurepaire, M. Oheim, J. Mertz "Ultra-deep two-photon fluorescence excitation in turbid media," Opt. Commun. 188, 25-29 (2001).
[CrossRef]

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth," J. Neurosci. Meth. 111, 29-37 (2001).
[CrossRef]

Bifano, T. G.

J. Perreault, T. G. Bifano, B. Martin Levine,M. N. Horenstein, "Adaptive optic correction using microelectromechanical deformable mirrors," Opt. Eng. 41, 561-566 (2002)
[CrossRef]

Booth, M. J.

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, S. Kawata, "Adaptive aberration correction in a two-photon microscope", J. Microsc. 200, 105-108 (2000).
[CrossRef] [PubMed]

Burns, D.

Chaigneau, E.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth," J. Neurosci. Meth. 111, 29-37 (2001).
[CrossRef]

Charpak, S.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth," J. Neurosci. Meth. 111, 29-37 (2001).
[CrossRef]

Denk, W.

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Meth. 2, 932-940 (2005).
[CrossRef]

P. Theer,M. T. Hasan,W. Denk, "Two-photon imaging to a depth of 1000?min living brains by use of a Ti:Al2O3 regenerative amplifier," Opt. Lett. 28, 1022-1024 (2003).
[CrossRef] [PubMed]

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

Durst, M.

Frieden, B. R.

Frumker, E.

Girkin, J.

Hanley, Q. S.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, T. M. Jovin, "Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes," Micron 34, 293-300 (2003).
[CrossRef] [PubMed]

Hasan, M. T.

Heintzmann, R.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, T. M. Jovin, "Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes," Micron 34, 293-300 (2003).
[CrossRef] [PubMed]

Helmchen, F.

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Meth. 2, 932-940 (2005).
[CrossRef]

Horenstein, M. N.

J. Perreault, T. G. Bifano, B. Martin Levine,M. N. Horenstein, "Adaptive optic correction using microelectromechanical deformable mirrors," Opt. Eng. 41, 561-566 (2002)
[CrossRef]

Jovin, T. M.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, T. M. Jovin, "Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes," Micron 34, 293-300 (2003).
[CrossRef] [PubMed]

Juskaitis, R.

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, S. Kawata, "Adaptive aberration correction in a two-photon microscope", J. Microsc. 200, 105-108 (2000).
[CrossRef] [PubMed]

Kawata, S.

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, S. Kawata, "Adaptive aberration correction in a two-photon microscope", J. Microsc. 200, 105-108 (2000).
[CrossRef] [PubMed]

Liu, F.

Mandelik, D.

Marsh, P.

Martin Levine, B.

J. Perreault, T. G. Bifano, B. Martin Levine,M. N. Horenstein, "Adaptive optic correction using microelectromechanical deformable mirrors," Opt. Eng. 41, 561-566 (2002)
[CrossRef]

McCutchen, C. W.

Mertz, J.

E. Beaurepaire and J. Mertz, "Epi-fluorescence collection in two-photon microscopy," Appl. Opt. 41, 5376-5382 (2002).
[CrossRef] [PubMed]

E. Beaurepaire, M. Oheim, J. Mertz "Ultra-deep two-photon fluorescence excitation in turbid media," Opt. Commun. 188, 25-29 (2001).
[CrossRef]

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth," J. Neurosci. Meth. 111, 29-37 (2001).
[CrossRef]

J. Mertz, C. Xu, W. W. Webb, "Single-molecule detection by two-photon-excited fluorescence," Opt. Lett. 20, 2532-2534 (1995).
[CrossRef] [PubMed]

Munroe, P.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, T. M. Jovin, "Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes," Micron 34, 293-300 (2003).
[CrossRef] [PubMed]

Nailon, J.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, T. M. Jovin, "Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes," Micron 34, 293-300 (2003).
[CrossRef] [PubMed]

Neil, M. A. A.

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, S. Kawata, "Adaptive aberration correction in a two-photon microscope", J. Microsc. 200, 105-108 (2000).
[CrossRef] [PubMed]

Oheim, M.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth," J. Neurosci. Meth. 111, 29-37 (2001).
[CrossRef]

E. Beaurepaire, M. Oheim, J. Mertz "Ultra-deep two-photon fluorescence excitation in turbid media," Opt. Commun. 188, 25-29 (2001).
[CrossRef]

Oron, D.

Perreault, J.

J. Perreault, T. G. Bifano, B. Martin Levine,M. N. Horenstein, "Adaptive optic correction using microelectromechanical deformable mirrors," Opt. Eng. 41, 561-566 (2002)
[CrossRef]

Sarafis, V.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, T. M. Jovin, "Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes," Micron 34, 293-300 (2003).
[CrossRef] [PubMed]

Silberberg, Y.

Strickler, J. H.

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

Svoboda, K.

K. Svoboda and R. Yasuda, "Principles of two-photon excitation microscopy and its applications to neuroscience," Neuron 50, 823-829 (2006).
[CrossRef] [PubMed]

Tal, E.

Tanaka, T.

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, S. Kawata, "Adaptive aberration correction in a two-photon microscope", J. Microsc. 200, 105-108 (2000).
[CrossRef] [PubMed]

Theer, P.

van Howe, J.

Webb, W. W.

Webb, W.W.

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

Wilson, T.

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, S. Kawata, "Adaptive aberration correction in a two-photon microscope", J. Microsc. 200, 105-108 (2000).
[CrossRef] [PubMed]

Xu, C.

Yasuda, R.

K. Svoboda and R. Yasuda, "Principles of two-photon excitation microscopy and its applications to neuroscience," Neuron 50, 823-829 (2006).
[CrossRef] [PubMed]

Ying, J.

Zhu, G.

Zipfel, W.

Appl. Opt. (2)

J. Microsc. (1)

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, S. Kawata, "Adaptive aberration correction in a two-photon microscope", J. Microsc. 200, 105-108 (2000).
[CrossRef] [PubMed]

J. Neurosci. Meth. (1)

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth," J. Neurosci. Meth. 111, 29-37 (2001).
[CrossRef]

J. Opt. Soc. Am. (2)

Micron (1)

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, T. M. Jovin, "Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes," Micron 34, 293-300 (2003).
[CrossRef] [PubMed]

Nat. Meth. (1)

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Meth. 2, 932-940 (2005).
[CrossRef]

Neuron (1)

K. Svoboda and R. Yasuda, "Principles of two-photon excitation microscopy and its applications to neuroscience," Neuron 50, 823-829 (2006).
[CrossRef] [PubMed]

Opt. Commun. (1)

E. Beaurepaire, M. Oheim, J. Mertz "Ultra-deep two-photon fluorescence excitation in turbid media," Opt. Commun. 188, 25-29 (2001).
[CrossRef]

Opt. Eng. (1)

J. Perreault, T. G. Bifano, B. Martin Levine,M. N. Horenstein, "Adaptive optic correction using microelectromechanical deformable mirrors," Opt. Eng. 41, 561-566 (2002)
[CrossRef]

Opt. Express (3)

Opt. Lett. (4)

Science (1)

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

Other (2)

M. Born and E. Wolf, Principles of Optics (Cambridge University Press, Cambridge, UK, 1999).

J.W. Goodman, Introduction to Fourier Optics, Roberts & Company Publishers, Greenwood Village, CO (2005).

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Figures (4)

Fig. 1.
Fig. 1.

Experimental layout. A mode-locked Ti:sapphire laser beam is focused into a sample, and the resulting TPEF is detected in the backward direction with photomultiplier tube (PMT). The layout is the same as a standard TPEF microscope except that a DM has been inserted into the beam path prior to the scan mirrors (4mm clear aperture). A set of unit-magnification afocal lenses image the DM to the scan mirrors (and hence to the objective back aperture). Aberrations are applied by 4-zone or 2-zone voltage patterns at the DM, as shown. The inset is a schematic of the ballistic-light focus profile in the sample without (solid) and with (dashed) DM-induced aberrations. The aberrations provoke a relative increase in the cross-sectional area of this profile (and hence a relative decrease in TPEF) that is more pronounced near the focal plane than far from the focal plane.

Fig. 2.
Fig. 2.

Plots of TPEF from a thin uniform fluorescent slab (6μm thick fluorescein solution sandwiched between two coverslips) as a function of defocus. Measurements were taken without (green triangles) and with 2-zone (red squares) and 4-zone (blue circles) DM-induced aberrations. (a) Plots of F 0(z) (green) and Fϕ (z) (red, blue) and, (b) the corresponding ratios F 0(z)/Fϕ (z). Measurements were acquired with no beam scanning and an Olympus 20 × NA=0.95 objective. Dashed traces in (b) are theoretical evaluations of F 0(z)/Fϕ (z) for an infinitely thin fluorescent plane.

Fig. 3.
Fig. 3.

Demonstration of TPEF background subtraction by differential aberration imaging. A thick tissue sample was mimicked by artificially rendering the objective immersion medium (water-ethanol mixture) both scattering and uniformly fluorescent, resulting in significant TPEF background (the medium scattering length was ∼ 500μm and the Olympus 20 × NA=0.95 objective working distance was 2mm). Images of fluorescently labeled pollen grains (Carolina Biological Supply) were acquired without (a) and with (b) 4-zone DM-induced aberrations (same lookup table). Upon subtraction (c), the background is considerably reduced and the contrast of the pollen grains is enhanced (same lookup table, but autoscaled). Note: for clearer images, averaging was performed over a 10 frame z-stack spanning a 10μm depth; negative values in panel (c) were set to zero post averaging. Qualitative measures of contrast improvement are shown in panel (d) illustrating the ratio of signal+background (averaged over a small zone inside a pollen grain) to background (averaged over a zone in proximity of the pollen grain). The ratio is shown for the uncorrected image (solid green), and after differential aberration correction with 2-zone (dotted red), and 4-zone (dashed blue) aberrations. The depth= 0 reference is arbitrary. The images were obtained with a laser power of ∼70mW (after the objective) at λ= 800nm.

Fig. 4.
Fig. 4.

Illustration of the resolution enhancement occasioned with differential aberration TPEF imaging. A fixed multiply-labeled bovine pulmonary artery endothelial cell (Molecular Probes Fluocell) was imaged without (a) and with (b) 4-zone DM-induced aberrations, and their subtraction is shown in (c). The higher magnification insets were acquired at higher laser power. The resolution of the Bodipy-labeled microtubules in these insets is apparently enhanced upon image subtraction. Images were acquired with an Olympus 40 × NA=1.3 oil immersion objective. The immersion medium here was neither scattering nor fluorescent.

Equations (12)

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I b ( ρ , z ) = W ( z ) PSF ( ρ , z )
CSF ( ρ , z ) = 1 ( 2 π ) 2 e ikz ( P ( k ) e i z k 2 2 k ) e i ρ k d 2 k
PSF ( ρ , z ) = CSF ( ρ , z ) 2 CSF ( ρ , z ) 2 d 2 ρ
P ϕ ( k ) = P 0 ( k ) e i ϕ ( k )
F ( z ) = W 2 ( z ) PSF 2 ( ρ , z ) d 2 ρ
OTF ( k ; z ) = PSF ( ρ , z ) e i ρ k d 2 ρ
F ( z ) = W 2 ( z ) ( 2 π ) 2 OTF ( k ; z ) 2 d 2 k
F ϕ ( 0 ) F 0 ( 0 )
F ϕ ( z ) F 0 ( 0 ) for l arg e z .
Area ( z ) = ( I b ( ρ , z ) d 2 ρ ) 2 I b 2 ( ρ , z ) d 2 ρ = ( 2 π ) 2 OTF ( k ; z ) 2 d 2 k
Area ϕ ( z ) Area 0 ( z ) = F 0 ( z ) F ϕ ( z ) .
Δ F ( z ) = F 0 ( z ) F ϕ ( z )

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