Abstract

Recently, a light sheet-based technique called objective-coupled planar illumination (OCPI) microscopy [ Holekamp et al., Neuron 57, 661 (2008) ] was shown to permit low-phototoxicity, high-speed, three-dimensional fluorescence imaging of extended tissue samples. Here, we introduce two major improvements in OCPI microscopy. First, we miniaturize the objective coupler by using a uniaxial gradient-index lens to produce the light sheet. Second, we demonstrate theoretically and experimentally that refractive index mismatch at the fluid/tissue interface causes a significant defocus aberration. By introducing the ability to tune the angle of the light sheet, we show that defocus correction in a miniaturized OCPI microscope leads to a significant improvement in image sharpness deeper into tissue.

© 2008 Optical Society of America

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References

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  1. T. F. Holekamp, D. Turaga, and T. E. Holy, Neuron 57, 661 (2008).
    [CrossRef] [PubMed]
  2. H. Siedentopf and R. Zsigmondy, Ann. Phys. 10, 1 (1903).
  3. A. H. Voie, D. H. Burns, and F. A. Spelman, J. Microsc. 170, 229 (1993).
    [CrossRef] [PubMed]
  4. E. Fuchs, J. S. Jaffe, R. A. Long, and F. Adam, Opt. Express 10, 145 (2002).
    [PubMed]
  5. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodth, and E. H. K. Stelzer, Science 31, 1007 (2004).
    [CrossRef]
  6. H.-U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007).
    [CrossRef] [PubMed]
  7. J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
    [CrossRef] [PubMed]
  8. J. J. Dirckx, L. C. Kuypers, and W. F. Decraemer, J. Biomed. Opt. 10, 44014 (2005).
    [CrossRef] [PubMed]
  9. G. Lopez-Bendito, K. Sturgess, F. Erdélyi, G. Szabó, Z. Molnar, O. Paulsen, Cereb. Cortex 14, 1122 (2004).
    [CrossRef] [PubMed]

2008

T. F. Holekamp, D. Turaga, and T. E. Holy, Neuron 57, 661 (2008).
[CrossRef] [PubMed]

2007

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

2005

J. J. Dirckx, L. C. Kuypers, and W. F. Decraemer, J. Biomed. Opt. 10, 44014 (2005).
[CrossRef] [PubMed]

2004

G. Lopez-Bendito, K. Sturgess, F. Erdélyi, G. Szabó, Z. Molnar, O. Paulsen, Cereb. Cortex 14, 1122 (2004).
[CrossRef] [PubMed]

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodth, and E. H. K. Stelzer, Science 31, 1007 (2004).
[CrossRef]

2002

1993

A. H. Voie, D. H. Burns, and F. A. Spelman, J. Microsc. 170, 229 (1993).
[CrossRef] [PubMed]

1903

H. Siedentopf and R. Zsigmondy, Ann. Phys. 10, 1 (1903).

Adam, F.

Aksay, E.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[CrossRef] [PubMed]

Becker, K.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Burns, D. H.

A. H. Voie, D. H. Burns, and F. A. Spelman, J. Microsc. 170, 229 (1993).
[CrossRef] [PubMed]

Decraemer, W. F.

J. J. Dirckx, L. C. Kuypers, and W. F. Decraemer, J. Biomed. Opt. 10, 44014 (2005).
[CrossRef] [PubMed]

Deininger, K.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodth, and E. H. K. Stelzer, Science 31, 1007 (2004).
[CrossRef]

Deussing, J. M.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Dirckx, J. J.

J. J. Dirckx, L. C. Kuypers, and W. F. Decraemer, J. Biomed. Opt. 10, 44014 (2005).
[CrossRef] [PubMed]

Dodt, H.-U.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Eder, M.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Erdélyi, F.

G. Lopez-Bendito, K. Sturgess, F. Erdélyi, G. Szabó, Z. Molnar, O. Paulsen, Cereb. Cortex 14, 1122 (2004).
[CrossRef] [PubMed]

Fuchs, E.

Holekamp, T. F.

T. F. Holekamp, D. Turaga, and T. E. Holy, Neuron 57, 661 (2008).
[CrossRef] [PubMed]

Holy, T. E.

T. F. Holekamp, D. Turaga, and T. E. Holy, Neuron 57, 661 (2008).
[CrossRef] [PubMed]

Huisken, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodth, and E. H. K. Stelzer, Science 31, 1007 (2004).
[CrossRef]

Jaffe, J. S.

Jahrling, N.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Jung, J. C.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[CrossRef] [PubMed]

Kuypers, L. C.

J. J. Dirckx, L. C. Kuypers, and W. F. Decraemer, J. Biomed. Opt. 10, 44014 (2005).
[CrossRef] [PubMed]

Leischner, U.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Long, R. A.

Lopez-Bendito, G.

G. Lopez-Bendito, K. Sturgess, F. Erdélyi, G. Szabó, Z. Molnar, O. Paulsen, Cereb. Cortex 14, 1122 (2004).
[CrossRef] [PubMed]

Mauch, C. P.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Mehta, A. D.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[CrossRef] [PubMed]

Molnar, Z.

G. Lopez-Bendito, K. Sturgess, F. Erdélyi, G. Szabó, Z. Molnar, O. Paulsen, Cereb. Cortex 14, 1122 (2004).
[CrossRef] [PubMed]

Paulsen, O.

G. Lopez-Bendito, K. Sturgess, F. Erdélyi, G. Szabó, Z. Molnar, O. Paulsen, Cereb. Cortex 14, 1122 (2004).
[CrossRef] [PubMed]

Schierloh, A.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Schnitzer, M. J.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[CrossRef] [PubMed]

Siedentopf, H.

H. Siedentopf and R. Zsigmondy, Ann. Phys. 10, 1 (1903).

Spelman, F. A.

A. H. Voie, D. H. Burns, and F. A. Spelman, J. Microsc. 170, 229 (1993).
[CrossRef] [PubMed]

Stelzer, E. H. K.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodth, and E. H. K. Stelzer, Science 31, 1007 (2004).
[CrossRef]

Stepnoski, R.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[CrossRef] [PubMed]

Sturgess, K.

G. Lopez-Bendito, K. Sturgess, F. Erdélyi, G. Szabó, Z. Molnar, O. Paulsen, Cereb. Cortex 14, 1122 (2004).
[CrossRef] [PubMed]

Swoger, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodth, and E. H. K. Stelzer, Science 31, 1007 (2004).
[CrossRef]

Szabó, G.

G. Lopez-Bendito, K. Sturgess, F. Erdélyi, G. Szabó, Z. Molnar, O. Paulsen, Cereb. Cortex 14, 1122 (2004).
[CrossRef] [PubMed]

Turaga, D.

T. F. Holekamp, D. Turaga, and T. E. Holy, Neuron 57, 661 (2008).
[CrossRef] [PubMed]

Voie, A. H.

A. H. Voie, D. H. Burns, and F. A. Spelman, J. Microsc. 170, 229 (1993).
[CrossRef] [PubMed]

Wittbrodth, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodth, and E. H. K. Stelzer, Science 31, 1007 (2004).
[CrossRef]

Zieglgansberger, W.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Zsigmondy, R.

H. Siedentopf and R. Zsigmondy, Ann. Phys. 10, 1 (1903).

Ann. Phys.

H. Siedentopf and R. Zsigmondy, Ann. Phys. 10, 1 (1903).

Cereb. Cortex

G. Lopez-Bendito, K. Sturgess, F. Erdélyi, G. Szabó, Z. Molnar, O. Paulsen, Cereb. Cortex 14, 1122 (2004).
[CrossRef] [PubMed]

J. Biomed. Opt.

J. J. Dirckx, L. C. Kuypers, and W. F. Decraemer, J. Biomed. Opt. 10, 44014 (2005).
[CrossRef] [PubMed]

J. Microsc.

A. H. Voie, D. H. Burns, and F. A. Spelman, J. Microsc. 170, 229 (1993).
[CrossRef] [PubMed]

J. Neurophysiol.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004).
[CrossRef] [PubMed]

Nat. Methods

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Neuron

T. F. Holekamp, D. Turaga, and T. E. Holy, Neuron 57, 661 (2008).
[CrossRef] [PubMed]

Opt. Express

Science

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodth, and E. H. K. Stelzer, Science 31, 1007 (2004).
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

Schematic and calibration of OCPI microscope. (a) Schematic of miniaturized objective coupler. Uniaxial GRIN lens is positioned such that a sheet of light is formed at the focal plane of the objective. (b) Angular defocus adjustment is obtained by changing the angle of incident light. (c) Example axial image of a 0.2 μ m fluorescent bead in agar.

Fig. 2
Fig. 2

Sources and magnitude of defocus in tilted configurations of planar illumination microscopy. (a) Illumination strikes the sample/fluid interface at an angle; the optic axis is also tilted, at an angle α. The fluid has an index of refraction n f , the sample n s . (b) Illumination plane bends at the fluid/sample interface through an angle θ i . (c) Emitted rays are bent at the interface, resulting in a virtual object location that resides on a different plane. (d) Magnitude of both effects, and their combination ( Δ β ) , for n f = 1.33 and n s = 1.40 .

Fig. 3
Fig. 3

Defocus measurement. (a) Beads in water ( n = 1.33 ) were imaged under OCPI. At this illumination angle, the image of beads is diffraction limited. Insets are three different beads at three corresponding depths. The dotted line represents water/PDMS interface. (b) Beads in PDMS ( n = 1.40 ) at same angle as (a). As expected, as depth increases, the defocus increases. (c) Beads in water. At an angle tuned for beads in PDMS, the beads in water are increasingly defocused as the depth increases. (d) Beads in PDMS at the same angle as (c). Although there are some higher-order aberrations, the beads are in focus at all depths. Scale bar in (a), 25 μ m . Scale bar in (a1), 5 μ m .

Fig. 4
Fig. 4

Resolution enhancement with miniaturized objective coupler with defocus correction. Images of a slice of olfactory bulb were taken under the same conditions with (a) old and (b) new OCPI microscopes. Scale bar, 50 μ m .

Equations (1)

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tan θ c Δ n n f ( 1 + 2 tan 2 α ) tan α .

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