Abstract

Light-sheet-based microscopy [single-plane illumination microscope (SPIM)] performs very well at low numerical apertures. It complements conventional (FM), confocal (CFM), and two-photon fluorescence microscopy (2hν-FM) currently used in modern life sciences. Lateral and axial SPIM point spread function (PSF) extents are measured by using fluorescent beads to determine the 3D resolution. The results are compared with values derived from an analytical theory and numerical simulations. The discrepancies are found to be less than 5%. The axial extent of a SPIM–PSF (10×0.3W) is approximately 5.7μm. This value is almost a factor of 2 smaller than in CFM, more than 2.5 times smaller than in FM, and more than three times smaller than in 2hν-FM. SPIM outperforms 2hν-FM and FM, while CFM has a better axial resolution at NAs above 0.8.

© 2006 Optical Society of America

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References

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2004 (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef] [PubMed]

2003 (1)

2002 (1)

2001 (1)

1999 (1)

1994 (1)

E. H. K. Stelzer and S. Lindek, Opt. Commun. 111, 536 (1994).
[CrossRef]

1993 (1)

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

1991 (1)

T. D. Visser, F. C. A. Groen, and G. J. Brakenhoff, Optik (Jena) 87, 39 (1991).

1986 (1)

1964 (1)

Azam, F.

Brakenhoff, G. J.

T. D. Visser, F. C. A. Groen, and G. J. Brakenhoff, Optik (Jena) 87, 39 (1991).

Burns, D. H.

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

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef] [PubMed]

Fuchs, E.

Grill, S.

Groen, F. C. A.

T. D. Visser, F. C. A. Groen, and G. J. Brakenhoff, Optik (Jena) 87, 39 (1991).

Huisken, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef] [PubMed]

J. Swoger, J. Huisken, and E. H. K. Stelzer, Opt. Lett. 28, 1654 (2003).
[CrossRef] [PubMed]

Jaffe, J. S.

Lindek, S.

E. H. K. Stelzer and S. Lindek, Opt. Commun. 111, 536 (1994).
[CrossRef]

Long, R. A.

Mansuripur, M.

McCutchen, C. W.

Rohrbach, A.

Spelman, F. A.

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

Stelzer, E. H. K.

Swoger, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef] [PubMed]

J. Swoger, J. Huisken, and E. H. K. Stelzer, Opt. Lett. 28, 1654 (2003).
[CrossRef] [PubMed]

Visser, T. D.

T. D. Visser, F. C. A. Groen, and G. J. Brakenhoff, Optik (Jena) 87, 39 (1991).

Voie, A. H.

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

Wittbrodt, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef] [PubMed]

J. Microsc. (1)

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

J. Opt. Soc. Am. (1)

J. Opt. Soc. Am. A (3)

Opt. Commun. (1)

E. H. K. Stelzer and S. Lindek, Opt. Commun. 111, 536 (1994).
[CrossRef]

Opt. Express (1)

Opt. Lett. (1)

Optik (Jena) (1)

T. D. Visser, F. C. A. Groen, and G. J. Brakenhoff, Optik (Jena) 87, 39 (1991).

Science (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef] [PubMed]

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Figures (2)

Fig. 1
Fig. 1

(a), (b) A conventional wide-field microscope (CCD camera, tube lens, filter, and detection objective lens) is used for fluorescence detection. A collimated laser beam is focused by a cylindrical lens and generates a light sheet. It excites only fluorophores in the focal plane of the detection objective lens, since the illumination axis is rotated by θ = 90 ° relative to the detection axis. The sample can be translated along the three principal axes and rotated around an axis perpendicular to both the illumination and the detection axes. The sample is moved along the z axis to acquire stacks; all other parts are spatially confined. (c) The detection magnification M det , illumination wavelength λ ill , refractive index of the immersion medium n, and the extent of the CCD chip size CCD determine the FOV. The illumination numerical aperture NA ill is adjustable by a rectangular diaphragm and is adapted to the FOV. The width of the light sheet in the center of the FOV w 0 is 1 2 the width at the edge w edge . The detection numerical aperture NA det and wavelength combinations λ det , λ ill are determined by the experiments, lenses, and dyes.

Fig. 2
Fig. 2

(a), (b), and (c) show simulated illumination, detection, and system PSFs for a SPIM with a 10 × 0.3 W detection objective lens. The length of the scale bar is 20 μ m . The parameters are camera pixel pitch 4.65 μ m , λ ill = 0.488 μ m , λ det = 0.565 μ m , n = 1.33 , image size 75 × 76   pixels , NA ill = 0.042 (adapted to a FOV of 476.2 μ m ; only the central part is shown), M det = 10 , NA det = 0.3 . A gamma of 0.4 was applied to images (b) and (c).

Tables (2)

Tables Icon

Table 1 Lateral and Axial PSF Extents and Focal Volumes of Analytical, Simulated, and Measured PSFs in a SPIM a

Tables Icon

Table 2 Lateral and Axial Extents and Focal Volumes of Simulated PSFs in a SPIM Compared with Conventional, Confocal, and Two-Photon Microscopes a

Equations (6)

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h ( x , y , z ) = E 0 ( k ) T ( k ) P ( k ) B ( k ) e i k r d 3 k .
Δ r cylindrical ( λ 0 , n , α ) = λ 0 α 2 n 2 α sin ( 2 α ) ,
Δ z cylindrical ( λ 0 , n , α ) = λ 0 α 2 n 2 α 2 4 sin 2 ( α ) + α sin ( 2 α ) ,
Δ r spherical ( λ 0 , n , α ) = λ 0 n 3 2 cos ( α ) cos ( 2 α ) ,
Δ z spherical ( λ 0 , n , α ) = λ 0 n [ 1 cos ( α ) ] ,
σ 1 + 2 = 1 ( 1 σ 1 ) 2 + ( 1 σ 2 ) 2 .

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