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]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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2006

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

2005

2003

2002

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

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]

2000

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

1995

1990

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

1967

1964

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.

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]

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.

J. Microsc.

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.

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.

Micron

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.

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

Neuron

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.

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

Opt. Eng.

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

Opt. Lett.

Science

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

Other

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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