Abstract

Structured illumination microscopy provides a simple and cheap mean to obtain optical sections of a sample. It can be implemented easily on a regular fluorescent microscope and is a scanning free alternative to confocal microscopy. We have analyzed theoretically the performances of the technique in terms of sectioning strength, resolution enhancement along the optical axis, and signal to background as a function of the objective used and the grid’s characteristics (pitch and contrast). We show that under optimized conditions, the axial resolution can be improved by a factor of 1.5 in comparison with an epifluorescence microscope, and that optical cuts with a thickness of less than 400nm can be obtained with a 1.4 numerical aperture objective. We modified the original grid in-step modulation pattern and used a sinusoidal modulation for the grid displacement. Optical sections are computed by combining four images acquired during one modulation period. This algorithm is very stable even for modulations at high frequencies. The speed of the acquisition is thus only limited by the performance of the detector and the signal/background ratio of the sample. Finally, we compared our technique to commercial setups: a confocal microscope, a Spinning Disk Microscope and a Zeiss Apotome.

© 2007 Optical Society of America

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References

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    [CrossRef] [PubMed]
  7. M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Daye, D. Parksons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, and T. Wilson, "Time-domain whole-field fluorescence lifetime imaging with optical sectioning," J. Microsc. 203, 246-257 (2001).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  13. M. G. L. Gustafsson, "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution," Proc. Natl. Acad. Sci. USA 102, 13,081-13,086 (2005).
    [CrossRef]
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    [CrossRef]

2005 (2)

J. A. Conchello and J. W. Lichtmann, "Optical sectioning microscopy," Nat. Methods 2, 920-931 (2005).
[CrossRef] [PubMed]

M. G. L. Gustafsson, "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution," Proc. Natl. Acad. Sci. USA 102, 13,081-13,086 (2005).
[CrossRef]

2001 (2)

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Daye, D. Parksons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, and T. Wilson, "Time-domain whole-field fluorescence lifetime imaging with optical sectioning," J. Microsc. 203, 246-257 (2001).
[CrossRef] [PubMed]

A. Dubois, "Phase-map measurements by interferometry with sinusoidal phase modulation and four integrating buckets," J. Opt. Soc. Am. A 18, 1972-1979 (2001).
[CrossRef]

2000 (2)

M. A. A. Neil, A. Squire, R. Juskaitis, P. I. H. Bastiaens, and T. Wilson, "Wide-field optically sectioning fluorescence microscopy with laser illumination," J. Microsc. 197, 1-4 (2000).
[CrossRef] [PubMed]

M. G. L. Gustafsson, "Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy," J. Microsc. 198, 82-87 (2000).
[CrossRef] [PubMed]

1997 (1)

1994 (1)

1993 (1)

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, "Aberrations in confocal fluorescence microscopy induced by mismatches in refractive-index," J. Microsc. 169, 391-405 (1993).
[CrossRef]

1988 (1)

C. J. R. Sheppard and X. Q. Mao, "Confocal microscopes with slit apertures," J. Mod. Opt. 35, 1169-1185 (1988).
[CrossRef]

1987 (1)

1969 (1)

1968 (1)

1963 (1)

W. Lukosz and M. Marchand "Optischen Abbildung unter berschreitung der Beugungsbedingten Auflsungsgrenze," Opt. Acta 10, 241-255 (1963).
[CrossRef]

J. Microsc. (4)

M. A. A. Neil, A. Squire, R. Juskaitis, P. I. H. Bastiaens, and T. Wilson, "Wide-field optically sectioning fluorescence microscopy with laser illumination," J. Microsc. 197, 1-4 (2000).
[CrossRef] [PubMed]

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Daye, D. Parksons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, and T. Wilson, "Time-domain whole-field fluorescence lifetime imaging with optical sectioning," J. Microsc. 203, 246-257 (2001).
[CrossRef] [PubMed]

M. G. L. Gustafsson, "Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy," J. Microsc. 198, 82-87 (2000).
[CrossRef] [PubMed]

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, "Aberrations in confocal fluorescence microscopy induced by mismatches in refractive-index," J. Microsc. 169, 391-405 (1993).
[CrossRef]

J. Mod. Opt. (1)

C. J. R. Sheppard and X. Q. Mao, "Confocal microscopes with slit apertures," J. Mod. Opt. 35, 1169-1185 (1988).
[CrossRef]

J. Opt. Soc. Am. (2)

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

Nat. Methods (1)

J. A. Conchello and J. W. Lichtmann, "Optical sectioning microscopy," Nat. Methods 2, 920-931 (2005).
[CrossRef] [PubMed]

Opt. Acta (1)

W. Lukosz and M. Marchand "Optischen Abbildung unter berschreitung der Beugungsbedingten Auflsungsgrenze," Opt. Acta 10, 241-255 (1963).
[CrossRef]

Opt. Lett. (1)

Proc. Natl. Acad. Sci. USA (1)

M. G. L. Gustafsson, "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution," Proc. Natl. Acad. Sci. USA 102, 13,081-13,086 (2005).
[CrossRef]

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Figures (5)

Fig. 1.
Fig. 1.

FWHM of the axial response in optical units as a function of the normalized spatial frequency of the grid. This shows that the narrowest axial response is achieved for a normalized spatial frequency equal to 1

Fig. 2.
Fig. 2.

PSF and OTF for an epifluorescence microscope and for structured illumination: the OTF of structured illumination microscope (bottom right) is enlarged compared to the OTF in the epifluorescence case (top right). This is consistent with the PSF of the structured illumination case that is narrower along the optical axis than that of the epifluorescence case

Fig. 3.
Fig. 3.

evolution of the optical section contrast C as a function of γ for different values of the contrast m

Fig. 4.
Fig. 4.

experimental setup: the use of a microscope objective in the illumination ensures a good contrast of the image of the grid

Fig. 5.
Fig. 5.

Experimental comparison of the wide-field axial PSF and the Structured illumination axial PSF

Tables (1)

Tables Icon

Table 1. Axial response in microns and corresponding υ′g : this table summarize the achievable thicknesses evaluated as the FWHM of the axial response (given in microns) for different grid pitches (given in line pair per millimeter) for different objectives and grids. We also indicate the corresponding normalized spatial frequency of the grid in the plane of the specimen υ′g which was calculated accordingly to our experimental setup. The wavelength λ was set to 0.6µm.

Equations (25)

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I ( x , y ) = I C ( x , y ) + I S ( x , y ) cos ( 2 π υ g x + ϕ )
S = I 1 + I 2 exp ( j 2 π 3 ) + I 3 exp ( j 4 π 3 )
S [ ( I 1 I 2 ) 2 + ( I 1 I 3 ) 2 + ( I 2 I 3 ) 2 ] 1 2
I ( x , y , t ) = I C + I S cos ( 2 π υ g X sin ( t T + ψ ) + φ )
I P ( x , y ) = p T 4 ( p + 1 ) T 4 I ( x , y , t ) d t
Σ S = I 1 + I 2 + I 3 I 4 = ( 4 T π ) Γ S I S sin φ
Σ C = I 1 + I 2 + I 3 + I 4 = ( 4 T π ) Γ C I S cos φ
Γ S = n = 0 + ( 1 ) n J 2 n + 1 ( 2 π υ g X ) 2 n + 1 sin [ ( 2 n + 1 ) φ ]
Γ C = n = 0 + J 4 n + 2 ( 2 π υ g X ) 2 n + 1 sin [ 2 ( 2 n + 1 ) φ ]
S 2 + C 2 = ( 4 T Γ π ) 2 I S 2
S [ ( I 0 I 1 ) 2 + ( I 2 I 3 ) ] 1 2
( ρ x , ρ y ) = k N A ( x , y )
u = 4 k n z sin 2 α 2
( υ x ' , υ y ' ) = ( υ x , υ y ) N A λ
S ( u , υ g ' ) 2 J 1 ( u υ g ' ( 1 υ g ' 2 ) ) ( u υ g ' ( 1 υ g ' 2 ) ) 2
I ( ρ x 1 , ρ y 1 ) = G ( ρ x 0 , ρ y 0 ) f ( ρ x 1 , ρ y 1 ) × h exc ( ρ x 0 ρ x 1 , ρ y 0 ρ y 1 ) 2 d ρ x 0 d ρ y 0
I ( ρ x , ρ y ) = G ( ρ x 0 , ρ y 0 ) f ( ρ x 0 , ρ y 0 )
× h exc ( ρ x 0 ρ x 1 , ρ y 0 ρ y 1 ) 2 d ρ x 0 d ρ y 0
× h em ( ρ x 1 ρ x , ρ y 1 ρ y ) 2 d ρ x 1 d ρ y 1
I ( ρ x , ρ y ) = G ( ρ x 0 , ρ y 0 ) h exc ( ρ x 0 , ρ y 0 ) 2 × h det ( ρ y , ρ y ) 2 d ρ x 0 d ρ y 0
G p = 1 + cos ( υ ˜ ρ x 0 + ( p 1 ) 2 π 3 )
S ( ρ x , ρ y ) h det ( ρ x , ρ y ) 2 × exp ( j υ g ' ρ x 0 ) h exc ( ρ x 0 , ρ y 0 ) 2 d ρ x 0 d ρ y 0
S ( ρ x , ρ y ) h det ( ρ x , ρ y ) 2 × P ( υ x , υ y ) × P * ( υ x υ g ' , υ y ) d υ x d υ y
P SF IS ( ρ x , ρ y ; u ) PSF conv ( ρ x , ρ y ; u ) × 2 J 1 ( u υ g ' ( 1 υ g ' 2 ) ) ( u υ g ' ( 1 υ g ' 2 ) )
I = I C + I S 2 ( 1 + m cos ( υ g ' t + φ ) )

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