Abstract

Current implementations of structured illumination microscopy for depth-resolved (three-dimensional) imaging have limitations that restrict its use; specifically, they are not applicable to non-stationary objects imaged with relatively poor condenser optics and in non-fluorescent mode. This includes in-vivo retinal imaging. A novel implementation of structured illumination microscopy is presented that overcomes these issues. A three-wavelength illumination technique is used to obtain the three sub-images required for structured illumination simultaneously rather than sequentially, enabling use on non-stationary objects. An illumination method is presented that produces an incoherent pattern through interference, bypassing the limitations imposed by the aberrations of the condenser lens and thus enabling axial sectioning in non-fluorescent imaging. The application to retinal imaging can lead to a device with similar sectioning capabilities to confocal microscopy without the optical complexity (and cost) required for scanning systems.

© 2011 OSA

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References

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  1. M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997).
    [CrossRef] [PubMed]
  2. D. Karadaglić, “Image formation in conventional brightfield reflection microscopes with optical sectioning property via structured illumination,” Micron 39(3), 302–310 (2008).
    [CrossRef] [PubMed]
  3. D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron 39(7), 808–818 (2008).
    [CrossRef] [PubMed]
  4. M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
    [CrossRef] [PubMed]
  5. M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
    [CrossRef] [PubMed]
  6. T. Wilson, and C. Sheppard, eds., Theory and Practice of Scanning Optical Microscopy (Academic Press, 1984).
  7. S. A. Shroff, J. R. Fienup, and D. R. Williams, “OTF compensation in structured illumination superresolution images,” Proc. SPIE 7094, 1–11 (2008).
  8. S. A. Shroff, D. Williams, and J. R. Fienup, “Structured illumination for imaging of stationary and non-stationary, fluorescent and non-fluorescent objects,” Patent WO/2008/124832, PCT/US2008/059922 (2008).
  9. S. A. Shroff, J. R. Fienup, and D. R. Williams, “Phase-shift estimation in sinusoidally illuminated images for lateral superresolution,” J. Opt. Soc. Am. A 26(2), 413–424 (2009).
    [CrossRef] [PubMed]
  10. L. G. Krzewina and M. K. Kim, “Single-exposure optical sectioning by color structured illumination microscopy,” Opt. Lett. 31(4), 477–479 (2006).
    [CrossRef] [PubMed]
  11. D. A. Atchinson, and G. Smith, Optics of the Human Eye (Butterworth Heinemann, 2000).
  12. E. Hecht, Optics (Addison-Wesley, 2002), 4th ed.
  13. M. Born, and E. Wolf, Principles of Optics (Cambridge University Press, 1999), seventh ed.
  14. J. W. Goodman, Introduction to Fourier Optics (McGraw-Hill, 1996), 2nd ed.
  15. R. N. Bracewell, The Fourier Transform and its Applications (McGraw-Hill, 2000), 3rd ed.

2009

2008

D. Karadaglić, “Image formation in conventional brightfield reflection microscopes with optical sectioning property via structured illumination,” Micron 39(3), 302–310 (2008).
[CrossRef] [PubMed]

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron 39(7), 808–818 (2008).
[CrossRef] [PubMed]

S. A. Shroff, J. R. Fienup, and D. R. Williams, “OTF compensation in structured illumination superresolution images,” Proc. SPIE 7094, 1–11 (2008).

2006

2005

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

2000

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[CrossRef] [PubMed]

1997

Fienup, J. R.

S. A. Shroff, J. R. Fienup, and D. R. Williams, “Phase-shift estimation in sinusoidally illuminated images for lateral superresolution,” J. Opt. Soc. Am. A 26(2), 413–424 (2009).
[CrossRef] [PubMed]

S. A. Shroff, J. R. Fienup, and D. R. Williams, “OTF compensation in structured illumination superresolution images,” Proc. SPIE 7094, 1–11 (2008).

Gustafsson, M. G. L.

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[CrossRef] [PubMed]

Juskaitis, R.

Karadaglic, D.

D. Karadaglić, “Image formation in conventional brightfield reflection microscopes with optical sectioning property via structured illumination,” Micron 39(3), 302–310 (2008).
[CrossRef] [PubMed]

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron 39(7), 808–818 (2008).
[CrossRef] [PubMed]

Kim, M. K.

Krzewina, L. G.

Neil, M. A. A.

Shroff, S. A.

S. A. Shroff, J. R. Fienup, and D. R. Williams, “Phase-shift estimation in sinusoidally illuminated images for lateral superresolution,” J. Opt. Soc. Am. A 26(2), 413–424 (2009).
[CrossRef] [PubMed]

S. A. Shroff, J. R. Fienup, and D. R. Williams, “OTF compensation in structured illumination superresolution images,” Proc. SPIE 7094, 1–11 (2008).

Williams, D. R.

S. A. Shroff, J. R. Fienup, and D. R. Williams, “Phase-shift estimation in sinusoidally illuminated images for lateral superresolution,” J. Opt. Soc. Am. A 26(2), 413–424 (2009).
[CrossRef] [PubMed]

S. A. Shroff, J. R. Fienup, and D. R. Williams, “OTF compensation in structured illumination superresolution images,” Proc. SPIE 7094, 1–11 (2008).

Wilson, T.

J. Microsc.

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A

Micron

D. Karadaglić, “Image formation in conventional brightfield reflection microscopes with optical sectioning property via structured illumination,” Micron 39(3), 302–310 (2008).
[CrossRef] [PubMed]

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron 39(7), 808–818 (2008).
[CrossRef] [PubMed]

Opt. Lett.

Proc. Natl. Acad. Sci. U.S.A.

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

Proc. SPIE

S. A. Shroff, J. R. Fienup, and D. R. Williams, “OTF compensation in structured illumination superresolution images,” Proc. SPIE 7094, 1–11 (2008).

Other

S. A. Shroff, D. Williams, and J. R. Fienup, “Structured illumination for imaging of stationary and non-stationary, fluorescent and non-fluorescent objects,” Patent WO/2008/124832, PCT/US2008/059922 (2008).

D. A. Atchinson, and G. Smith, Optics of the Human Eye (Butterworth Heinemann, 2000).

E. Hecht, Optics (Addison-Wesley, 2002), 4th ed.

M. Born, and E. Wolf, Principles of Optics (Cambridge University Press, 1999), seventh ed.

J. W. Goodman, Introduction to Fourier Optics (McGraw-Hill, 1996), 2nd ed.

R. N. Bracewell, The Fourier Transform and its Applications (McGraw-Hill, 2000), 3rd ed.

T. Wilson, and C. Sheppard, eds., Theory and Practice of Scanning Optical Microscopy (Academic Press, 1984).

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Figures (3)

Fig. 1.
Fig. 1.

Schematic representation of the Michelson interferometry set up used for the Fizeau fringe projection technique. The schematic superimposes all branches onto the same axis for clarity. The ray shown coming from S will reflect off both mirrors such that the optical path difference of the reflected rays is only dependant on the thickness of the optical air wedge formed by the mirror, for small tilt angles α. In relation to Fig. 2, Σ represents the plane of the rotating diffuser, M 2 is the common reference mirror and M 1 the wavelength specific mirror. P and R are the pupil plane and retinal plane respectively, representing the eye. The plane containing the mirrors, R′, is conjugate to R.

Fig. 2.
Fig. 2.

Schematic representation of the proposed Structured Illumination Ophthalmoscope using the Fizeau fringe projection technique and three wavelengths for illumination. Red, green and blue incoherent light sources are used. These are matched to the detection peaks for the detectors in the colour CCD camera and, with appropriate filtering, cross-talk is minimised. A rotating diffuser is used to enhance spatial incoherence. A beamsplitter splits the light into the two branches of a Michelson interferometer, one containing a mirror common to all wavelengths, the other containing a dispersing prism and three mirrors, one for each of the illuminating wavelengths. The fringes produced are projected onto the retina by the optics of the eye. Incoming and outgoing light from the eye is split using a polarizing beamplitter and a quarter-wave plate in front of the eye to maximise the returning light based on the eye’s birefringence. A half-wave plate in the common branch of the interferometer controls fringe modulation.

Fig. 3.
Fig. 3.

Simplified geometry for the imaging system showing the object and image planes with their normalised coordinate pairs (to,wo ) and (ti,wi ) respectively, and the pupil plane with coordinates (ξ,η) with are proportional to the spatial frequency coordinates (m,n) [6, 14]. Focussing optics is not shown.

Equations (15)

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I illumination ( t o , w o ) = 1 + μ cos ( ν t o + ϕ ) ,
I object ( t o , w o ) = [ 1 + μ cos ( ν t o + ϕ ) ] ρ ( t o , w o ) ,
I ( t i , w i ) = [ 1 + μ cos ( ν t o + ϕ ) ] ρ ( t o , w o ) h ( t i + t o , w i + w o ) 2 d t o d w o ,
I 0 ( t i , w i ) = ρ ( t o , w o ) h ( t i + t o , w i + w o ) 2 d t o d w o ,
I ν ( t i , w i ) = e i ν t o ρ ( t o , w o ) h ( t i + t o , w i + w o ) 2 d t o d w o ,
I ν ( t i , w i ) = e i ν t o ρ ( t o , w o ) h ( t i + t o , w i + w o ) 2 d t o d w o ,
I ( t i , w i ) = I 0 ( t i , w i ) + μ 2 e i ϕ I ν ( t i , w i ) + μ 2 e i ϕ I ν ( t i , w i ) .
I ± ν = I 1 + I 2 e i 2 π 3 + I 3 e ± i 2 π 3 ,
I ± ν = ( ( I 1 I 2 ) 2 + ( I 1 I 3 ) 2 + ( I 2 I 3 ) 2 2 ) 1 2 .
I 0 = 1 3 ( I 1 + I 2 + I 3 ) .
I ν ( t i , w i ) = e i ν t o ( m , n ) e i ( m t o + n w o ) h ( t i + t o , w i + w o ) 2 d t o d w o d m d n .
e i ( m t i + n w i ) P ( m , n ) P * ( m , n ) = 1 { h ( t 0 + t i , w o + w i ) 2 }
= h ( t o + t i , w o + w i ) 2 e i ( m t o + n w o ) d t o n w o .
I ν ( t i , w i ) = e i v t i ( m , n ) C ( m + ν , n ) e i ( m t i + n w i ) d m d n .
I ( t i , w i ; u ) = [ 1 + μ cos ( ν t o + ϕ ) ] ρ ( t o , w o ; u ) h ( t i + t o , w i + w o ; u ) 2 d t o d w o ,

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