Abstract

Autoconfocal microscopy (ACM) is a simple implementation of a transmitted-light confocal microscopy where a nonlinear detector plays the role of a virtual self-aligned pinhole. We report here a significant improvement of ACM based on the use of graded-field (GF) imaging. The technique of GF imaging involves introducing partial beam blocks in the illumination and detection apertures of an imaging system. These partial beam blocks confer phase-gradient sensitivity to the imaging system and allow control over its background level. We present the theory of the GF contrast in the context of ACM, comparing it to GF contrast in a non-scanning widefield microscope, and discuss various performance characteristics of GF-ACM in terms of resolution, sectioning strength, and an “under-detection” light collection geometry. An advantage of ACM is that it can be readily combined with two-photon excited fluorescence (TPEF) microscopy. We present images of rat brain hippocampus using simultaneous GF-ACM and TPEF microscopy. These images are inherently co-registered.

© 2007 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. A.E. Dixon and C. Cogswell, "Confocal microscopy with transmitted light," in Handbook of Biological Confocal Microscopy, J.B. Pawley, ed. (Plenum Press, 1995), pp. 479-490.
  2. C. Yang and J. Mertz, "Transmission confocal laser scanning microscopy with a virtual pinhole based on nonlinear detection," Opt. Lett. 28,224-227 (2003).
    [CrossRef] [PubMed]
  3. T. Pons and J. Mertz, "Autoconfocal microscopy with nonlinear transmitted light detection," J. Opt. Soc. Am. B 21,1486-1493 (2004).
    [CrossRef]
  4. R. Yi, K.K. Chu, and J. Mertz, "Graded-field microscopy with white light," Opt. Express 14,5191-5200 (2006).
    [CrossRef] [PubMed]
  5. G. Nomarski, "Microinterferometre differentiel a ondes polarisees [in French]," J. Phys. Radium 16,S9 (1955).
  6. R.D. Allen, G.B. David, G. Nomarski, "The Zeiss-Nomarski differential interference equipment for transmittedlight microscopy," Z. Wiss. Mikrosk. 69,193-221 (1969).
    [PubMed]
  7. W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
    [CrossRef] [PubMed]
  8. Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
    [CrossRef] [PubMed]
  9. T. Wilson, "The role of the pinhole in confocal imaging system," in Handbook of Biological Confocal Microscopy, J.B. Pawley, ed. (Plenum Press, 1995), pp. 167-182.
  10. N. Streibl, "Depth transfer by an imaging system," Opt. Acta 31,1233-1241 (1984).
    [CrossRef]
  11. M. Born and E. Wolf, Principles of optics (Cambridge University Press, Cambridge, UK, 1999).

2006 (1)

2004 (1)

2003 (2)

C. Yang and J. Mertz, "Transmission confocal laser scanning microscopy with a virtual pinhole based on nonlinear detection," Opt. Lett. 28,224-227 (2003).
[CrossRef] [PubMed]

W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
[CrossRef] [PubMed]

1999 (1)

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef] [PubMed]

1984 (1)

N. Streibl, "Depth transfer by an imaging system," Opt. Acta 31,1233-1241 (1984).
[CrossRef]

1969 (1)

R.D. Allen, G.B. David, G. Nomarski, "The Zeiss-Nomarski differential interference equipment for transmittedlight microscopy," Z. Wiss. Mikrosk. 69,193-221 (1969).
[PubMed]

1955 (1)

G. Nomarski, "Microinterferometre differentiel a ondes polarisees [in French]," J. Phys. Radium 16,S9 (1955).

Allen, R.D.

R.D. Allen, G.B. David, G. Nomarski, "The Zeiss-Nomarski differential interference equipment for transmittedlight microscopy," Z. Wiss. Mikrosk. 69,193-221 (1969).
[PubMed]

Amos, W.B.

W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
[CrossRef] [PubMed]

Cattermole, D.M.

W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
[CrossRef] [PubMed]

Chu, K.K.

David, G.B.

R.D. Allen, G.B. David, G. Nomarski, "The Zeiss-Nomarski differential interference equipment for transmittedlight microscopy," Z. Wiss. Mikrosk. 69,193-221 (1969).
[PubMed]

Hayashi, Y.

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef] [PubMed]

Laufer, J.

W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
[CrossRef] [PubMed]

Mainen, Z.F.

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef] [PubMed]

Maletic-Savatic, M.

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef] [PubMed]

Malinow, R.

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef] [PubMed]

Mertz, J.

Nomarski, G.

R.D. Allen, G.B. David, G. Nomarski, "The Zeiss-Nomarski differential interference equipment for transmittedlight microscopy," Z. Wiss. Mikrosk. 69,193-221 (1969).
[PubMed]

G. Nomarski, "Microinterferometre differentiel a ondes polarisees [in French]," J. Phys. Radium 16,S9 (1955).

Pons, T.

Reichelt, S.

W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
[CrossRef] [PubMed]

Shi, S.H.

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef] [PubMed]

Streibl, N.

N. Streibl, "Depth transfer by an imaging system," Opt. Acta 31,1233-1241 (1984).
[CrossRef]

Svoboda, K.

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef] [PubMed]

Yang, C.

Yi, R.

J. Microsc. (1)

W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B (1)

J. Phys. Radium (1)

G. Nomarski, "Microinterferometre differentiel a ondes polarisees [in French]," J. Phys. Radium 16,S9 (1955).

Methods (1)

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef] [PubMed]

Opt. Acta (1)

N. Streibl, "Depth transfer by an imaging system," Opt. Acta 31,1233-1241 (1984).
[CrossRef]

Opt. Express (1)

Opt. Lett. (1)

Z. Wiss. Mikrosk. (1)

R.D. Allen, G.B. David, G. Nomarski, "The Zeiss-Nomarski differential interference equipment for transmittedlight microscopy," Z. Wiss. Mikrosk. 69,193-221 (1969).
[PubMed]

Other (3)

T. Wilson, "The role of the pinhole in confocal imaging system," in Handbook of Biological Confocal Microscopy, J.B. Pawley, ed. (Plenum Press, 1995), pp. 167-182.

A.E. Dixon and C. Cogswell, "Confocal microscopy with transmitted light," in Handbook of Biological Confocal Microscopy, J.B. Pawley, ed. (Plenum Press, 1995), pp. 479-490.

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

Supplementary Material (3)

» Media 1: AVI (2340 KB)     
» Media 2: AVI (818 KB)     
» Media 3: AVI (2338 KB)     

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (7)

Fig. 1.
Fig. 1.

Autoconfocal setup (left) and with graded-field enhancement (right). Plane 0: Illumination aperture. Plane 1: Sample plane. Plane 2: Detection aperture. Plane 3: Detector plane. The beam is angularly scanned across the illumination aperture (plane 0) but without spatial translation in that plane. Straight-edged partial beam blocks are introduced into planes 0 and 2 in a complementary fashion such that together they intercept nearly all of the light.

Fig. 2.
Fig. 2.

Functions Ke (left) and Ko (right) from Eqs. (24a) and (24b). αd = 1 and αc = 0.5 in units of ka/fa , and the z-axis has been normalized to a maximum of 1. x 1c and x 1d are in units of fa/ka. xs has been set to 0. Ke is even in both x 1c and x 1d , while Ko is even in x 1c but odd in x 1d . It is this odd parity that makes Ko a derivative finder and leads to phase-gradient contrast in GF imaging.

Fig. 3.
Fig. 3.

Illustration of underfilled detection. When NA is lower on detection side, some light is lost (left). However objects in sample can act like a lens and bring all of the light into the detectable region (right).

Fig. 4.
Fig. 4.

Top: Intensity measured at center of 3μm polystyrene bead (see bottom image) using SHG detector (solid blue) and linear photodiode (dashed red) as a function of focal depth z. Both are normalized to an asymptotic value of 1, indicated by the dotted line, that is approached when the bead is far removed from the focal plane. Bottom: Images of the bead at depths of -6μm (left), 0μm (center), and 6μm (right).

Fig. 5.
Fig. 5.

The angular response profile of a 200 μm-thick LBO crystal . The crystal is less responsive to incident light at off-normal angles, effectively limiting the detection NA.

Fig. 6.
Fig. 6.

Single pyramidal neuron in rat brain hippocampus slice (400 μm thickness) imaged with ordinary ACM (left) and GF ACM (right). Depth in sample is approximately 40 μm. Phase contrast is very poor without GF, but is revealed when GF is added. Scale bar is 20 μm.

Fig. 7.
Fig. 7.

Simultaneous GF-ACM and TPEF images of a rat hippocampus slice (400 μm thickness) stained with sulforhodamine 101, which preferentially labels astrocytes. GF-ACM (top) [Media 1] and TPEF (middle) [Media 2] images are overlayed (bottom) [Media 3]. Scale bar is 30 μm. GF-ACM images appear similar in character to DIC. Objects shown in relief are mainly pyramidal neurons. Overlaid images show locations of astrocytes relative to neurons. Full movie (2.3 MB) of the 3D stack of images is presented in supplementary material, scanning in z from 0 μm to 105 μm.

Equations (32)

Equations on this page are rendered with MathJax. Learn more.

SHG [ I 3 ( x 3 ) ] 2 d x 3 = B ( x 3 ) + S x 3 z 4 d x 3
SHG [ I 3 ( 0 ) ] 2 = B ( 0 ) + S 0 z 4
SHG B ( 0 ) 4 + B ( 0 ) 3 S 0 z + B ( 0 ) 2 S 0 z 2 + B ( 0 ) S 0 z 3 + S 0 z 4
E 0 ( ξ 0 ; x s ) = E 0 e i x s ξ 0
E 1 ( x 1 ; x s ) = α 1 α 2 E 0 ( ξ 0 ; x s ) e i x 1 ξ 0 d ξ 0 = E 0 α 1 α 2 e i ( x 1 x s ) ξ 0 d ξ 0
E 1 ( x 1 ; x s ) = ( α 2 α 1 ) e i ( α 1 + α 2 ) x 1 s 2 sinc ( 1 2 ( α 2 α 1 ) x 1 s )
E 1 ( x 1 ; x s ) = α d e i α c x 1 s sinc ( 1 2 α d x 1 s )
J ( x , x ; x s ) = E ( x ; x s ) E * ( x ; x s ) ¯
J 1 ( x 1 , x 1 ; x s ) = α d 2 e i α c x 1 d sin c ( 1 2 α d x 1 s ) sinc ( 1 2 α d x 1 s )
x 1 c = 1 2 ( x 1 + x 1 )
x 1 d = x 1 x 1
J 1 in ( x 1 c , x 1 d ; x s ) = α d 2 e i α c x 1 d sinc ( 1 2 α d ( x 1 c x s + 1 2 x 1 d ) ) sinc ( 1 2 α d ( x 1 c x s 1 2 x 1 d ) )
I 3 ( x 3 , x s ) = d x 1 c d x 1 d G 13 ( x 3 + x 1 c , x 1 d ) J 1 out ( x 1 c , x 1 d )
G 13 ( x 1 c , x 1 d ; x 3 , x s ) = β d 2 e i β c x 1 d sinc ( 1 2 β d ( x 3 + x 1 c 1 2 x 1 d ) ) sinc ( 1 2 β d ( x 3 + x 1 c + 1 2 x 1 d ) )
E 1 out ( x 1 ; x s ) = t ( x 1 ) E 1 in ( x 1 ; x s )
J 1 out ( x 1 c , x 1 d ; x s ) = T ( x 1 c , x 1 d ) J 1 in ( x 1 c , x 1 d ; x s )
T ( x 1 c , x 1 d ) = t ( x 1 c 1 2 x 1 d ) t * ( x 1 c + 1 2 x 1 d )
I 3 ( x 3 , x s ) = d x 1 c d x 1 d G 13 ( x 1 c , x 1 d ; x 3 , x s ) T ( x 1 c , x 1 d ) J 1 in ( x 1 c , x 1 d ; x s )
I 3 ( x 3 , x s ) = d x 1 c d x 1 d T r ( x 1 c , x 1 d ) K e ( x 1 c , x 1 d ; x 3 , x s ) + T i ( x 1 c , x 1 d ) K o ( x 1 c , x 1 d ; x 3 , x s )
K e ( x 1 c , x 1 d ; x 3 , x s ) = α d 2 β d 2 cos ( ( α c + β c ) x 1 d ) sinc ( α d 2 ( x 1 c x s + 1 2 x 1 d ) ) sinc ( α d 2 ( x 1 c x s 1 2 x 1 d ) )
× sinc ( β d 2 ( x 1 c + x 3 + 1 2 x 1 d ) ) sinc ( β d 2 ( x 1 c + x 3 1 2 x 1 d ) )
K o ( x 1 c , x 1 d ; x 3 , x s ) = α d 2 β d 2 sin ( ( α c + β c ) x 1 d ) sinc ( α d 2 ( x 1 c x s + 1 2 x 1 d ) ) sinc ( α d 2 ( x 1 c x s 1 2 x 1 d ) )
× sinc ( β d 2 ( x 1 c + x 3 + 1 2 x 1 d ) ) sinc ( β d 2 ( x 1 c + x 3 1 2 x 1 d ) )
P 3 ( x s ) = I 3 x 3 x s d x 3
sinc ( 1 2 β d ( x 13 c + 1 2 x 1 d ) ) sinc ( 1 2 β d ( x 13 c 1 2 x 1 d ) ) d x 1 c = 2 π β d sinc ( 1 2 β d x 1 d )
P 3 ( x s ) = d x 1 c d x 1 d T r ( x 1 c , x 1 d ) K ˜ e ( x 1 c , x 1 d ; x 3 , x s ) + T i ( x 1 c , x 1 d ) K ˜ o ( x 1 c , x 1 d ; x 3 , x s )
K ˜ e ( x 1 c , x 1 d ; x 3 , x s ) = β d α d 2 cos ( ( α c + β c ) x 1 d ) sinc ( 1 2 β d x 1 d )
× sinc ( 1 2 α d ( x 1 c x s + 1 2 x 1 d ) ) sinc ( 1 2 α d ( x 1 c x s 1 2 x 1 d ) )
K ˜ o ( x 1 c , x 1 d ; x 3 , x s ) = β d α d 2 sin ( ( α c + β c ) x 1 d ) sin c ( 1 2 β d x 1 d )
× sin c ( 1 2 α d ( x 1 c x s + 1 2 x 1 d ) ) sin c ( 1 2 α d ( x 1 c x s 1 2 x 1 d ) )
K e ( x 1 c , x 1 d ) = α d 4 cos ( 2 α c x 1 d ) sin c 2 ( α d 2 ( x 1 c x s + 1 2 x 1 d ) ) sin c 2 ( α d 2 ( x 1 c x s 1 2 x 1 d ) )
K o ( x 1 c , x 1 d ) = α d 4 sin ( 2 α c x 1 d ) sin c 2 ( α d 2 ( x 1 c x s + 1 2 x 1 d ) ) sin c 2 ( α d 2 ( x 1 c x s 1 2 x 1 d ) )

Metrics