Abstract

We describe an optical sectioning microscopy system with no moving parts based on a micro-structured stripe-array light emitting diode (LED). By projecting arbitrary line or grid patterns onto the object, we are able to implement a variety of optical sectioning microscopy techniques such as grid-projection structured illumination and line scanning confocal microscopy, switching from one imaging technique to another without modifying the microscope setup. The micro-structured LED and driver are detailed and depth discrimination capabilities are measured and calculated. ©2007 Optical Society of America

© 2007 Optical Society of America

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References

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  1. T. Wilson and Barry R. Masters, Confocal Microscopy, (Academic Press, San Diego, 1990).
  2. W. B. Amos and J. G. White, "Direct view confocal imaging systems using a slit aperture," in Handbook of Biological Confocal Microscopy, J. B. Pawley (Plenum, New York, 1995), pp. 403-415.
  3. C. J. R. Sheppard and X. Q. Mao, "Confocal microscopes with slit apertures," J. Mod. Opt. 25, 1169-1185 (1988).
    [CrossRef]
  4. 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, 1905-1907 (1997).
    [CrossRef]
  5. OptiGrid, QiOptiq Imaging Solutions, http://www.qioptiqimaging.com>
  6. ApoTome, Carl Zeiss MicroImaging GmbH, http://www.zeiss.com>
  7. P. Herman, B. P. Maliwal, H.-J. Lin, and J. R. Lakowicz, "Frequency-domain fluorescence microscopy with the LED as a light source," J. Microsc. 203, 176-181 (2001).
    [CrossRef] [PubMed]
  8. C. Moser, T. Mayr, and I. Klimant, "Filter cubes with built-in ultrabright light-emitting diodes as exchangeable excitation light sources in fluorescence microscopy," J. Microsc. 222, 135-140 (2006).
    [CrossRef] [PubMed]
  9. O. Bormuth, J. Howard, and E. Schaffer, "LED illumination for video-enhanced DIC imaging of single microtubules," J. Microsc. 226, 1-5 (2007).
    [CrossRef] [PubMed]
  10. S. X. Jin, J. Li, J. Y. Lin, and H. X. Jiang, "InGaN/GaN quantum well interconnected microdisk light emitting diodes," Appl. Phys. Lett. 77, 3236-3238 (2000).
  11. H. W. Choi, C. W. Jeon, M. D. Dawson, P. R. Edwards, and R. W. Martin, "Fabrication and performance of parallel-addressed InGaN micro-LED arrays," IEEE Photon. Technol. Lett. 15, 510-512 (2003).
    [CrossRef]
  12. C. W. Jeon, H. W. Choi, E. Gu, and M. D. Dawson, "High-density matrix-addressable AlInGaN-based 368-nm microarray light-emitting diodes," IEEE Photon. Technol. Lett. 16, 2421-2423 (2004).
    [CrossRef]
  13. H. X. Zhang, E. Gu, C. W Jeon, Z. Gong, M. D. Dawson, M. A. A. Neil, and P. W. M. French, "Microstripe-array InGaN light-emitting diodes with individually addressable elements," IEEE Photon. Technol. Lett. 18, 1681-1683, (2006).
    [CrossRef]
  14. M. Born, E. Wolf, Principles of Optics, (Pergamon Press, Oxford, 1975).
  15. M. A. A. Neil, R. Juskaitis, and T. Wilson, "Real time 3D fluorescence microscopy by two beam interference illumination," Opt. Commun. 153, 1-4 (1998).
    [CrossRef]
  16. L. H. Schaeffer, D. Schuster, and J. Schaffer, "Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach," J. Microsc. 216, 165-174 (2004).
    [CrossRef]
  17. P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Electronic multiconfocal points microscopy," Three dimensional microscopy: Image acquisition and processing II, Proc. SPIE 2412, 56-62 (1995).
  18. P. A. Stokseth, "Properties of a defocused optical system," J. Opt. Soc. Am. 59, 1314-1321 (1969).
    [CrossRef]

2007

O. Bormuth, J. Howard, and E. Schaffer, "LED illumination for video-enhanced DIC imaging of single microtubules," J. Microsc. 226, 1-5 (2007).
[CrossRef] [PubMed]

2006

C. Moser, T. Mayr, and I. Klimant, "Filter cubes with built-in ultrabright light-emitting diodes as exchangeable excitation light sources in fluorescence microscopy," J. Microsc. 222, 135-140 (2006).
[CrossRef] [PubMed]

H. X. Zhang, E. Gu, C. W Jeon, Z. Gong, M. D. Dawson, M. A. A. Neil, and P. W. M. French, "Microstripe-array InGaN light-emitting diodes with individually addressable elements," IEEE Photon. Technol. Lett. 18, 1681-1683, (2006).
[CrossRef]

2004

L. H. Schaeffer, D. Schuster, and J. Schaffer, "Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach," J. Microsc. 216, 165-174 (2004).
[CrossRef]

C. W. Jeon, H. W. Choi, E. Gu, and M. D. Dawson, "High-density matrix-addressable AlInGaN-based 368-nm microarray light-emitting diodes," IEEE Photon. Technol. Lett. 16, 2421-2423 (2004).
[CrossRef]

2003

H. W. Choi, C. W. Jeon, M. D. Dawson, P. R. Edwards, and R. W. Martin, "Fabrication and performance of parallel-addressed InGaN micro-LED arrays," IEEE Photon. Technol. Lett. 15, 510-512 (2003).
[CrossRef]

2001

P. Herman, B. P. Maliwal, H.-J. Lin, and J. R. Lakowicz, "Frequency-domain fluorescence microscopy with the LED as a light source," J. Microsc. 203, 176-181 (2001).
[CrossRef] [PubMed]

2000

S. X. Jin, J. Li, J. Y. Lin, and H. X. Jiang, "InGaN/GaN quantum well interconnected microdisk light emitting diodes," Appl. Phys. Lett. 77, 3236-3238 (2000).

1998

M. A. A. Neil, R. Juskaitis, and T. Wilson, "Real time 3D fluorescence microscopy by two beam interference illumination," Opt. Commun. 153, 1-4 (1998).
[CrossRef]

1997

1995

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Electronic multiconfocal points microscopy," Three dimensional microscopy: Image acquisition and processing II, Proc. SPIE 2412, 56-62 (1995).

1988

C. J. R. Sheppard and X. Q. Mao, "Confocal microscopes with slit apertures," J. Mod. Opt. 25, 1169-1185 (1988).
[CrossRef]

1969

Appl. Phys. Lett.

S. X. Jin, J. Li, J. Y. Lin, and H. X. Jiang, "InGaN/GaN quantum well interconnected microdisk light emitting diodes," Appl. Phys. Lett. 77, 3236-3238 (2000).

IEEE Photon. Technol. Lett.

H. W. Choi, C. W. Jeon, M. D. Dawson, P. R. Edwards, and R. W. Martin, "Fabrication and performance of parallel-addressed InGaN micro-LED arrays," IEEE Photon. Technol. Lett. 15, 510-512 (2003).
[CrossRef]

H. X. Zhang, E. Gu, C. W Jeon, Z. Gong, M. D. Dawson, M. A. A. Neil, and P. W. M. French, "Microstripe-array InGaN light-emitting diodes with individually addressable elements," IEEE Photon. Technol. Lett. 18, 1681-1683, (2006).
[CrossRef]

J. Microsc.

L. H. Schaeffer, D. Schuster, and J. Schaffer, "Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach," J. Microsc. 216, 165-174 (2004).
[CrossRef]

P. Herman, B. P. Maliwal, H.-J. Lin, and J. R. Lakowicz, "Frequency-domain fluorescence microscopy with the LED as a light source," J. Microsc. 203, 176-181 (2001).
[CrossRef] [PubMed]

C. Moser, T. Mayr, and I. Klimant, "Filter cubes with built-in ultrabright light-emitting diodes as exchangeable excitation light sources in fluorescence microscopy," J. Microsc. 222, 135-140 (2006).
[CrossRef] [PubMed]

O. Bormuth, J. Howard, and E. Schaffer, "LED illumination for video-enhanced DIC imaging of single microtubules," J. Microsc. 226, 1-5 (2007).
[CrossRef] [PubMed]

J. Mod. Opt.

C. J. R. Sheppard and X. Q. Mao, "Confocal microscopes with slit apertures," J. Mod. Opt. 25, 1169-1185 (1988).
[CrossRef]

J. Opt. Soc. Am.

Opt. Commun.

M. A. A. Neil, R. Juskaitis, and T. Wilson, "Real time 3D fluorescence microscopy by two beam interference illumination," Opt. Commun. 153, 1-4 (1998).
[CrossRef]

Opt. Lett.

Photon. Technol. Lett.

C. W. Jeon, H. W. Choi, E. Gu, and M. D. Dawson, "High-density matrix-addressable AlInGaN-based 368-nm microarray light-emitting diodes," IEEE Photon. Technol. Lett. 16, 2421-2423 (2004).
[CrossRef]

Proc. SPIE

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Electronic multiconfocal points microscopy," Three dimensional microscopy: Image acquisition and processing II, Proc. SPIE 2412, 56-62 (1995).

Other

M. Born, E. Wolf, Principles of Optics, (Pergamon Press, Oxford, 1975).

OptiGrid, QiOptiq Imaging Solutions, http://www.qioptiqimaging.com>

ApoTome, Carl Zeiss MicroImaging GmbH, http://www.zeiss.com>

T. Wilson and Barry R. Masters, Confocal Microscopy, (Academic Press, San Diego, 1990).

W. B. Amos and J. G. White, "Direct view confocal imaging systems using a slit aperture," in Handbook of Biological Confocal Microscopy, J. B. Pawley (Plenum, New York, 1995), pp. 403-415.

Supplementary Material (3)

» Media 1: MOV (10 KB)     
» Media 2: MOV (1187 KB)     
» Media 3: MOV (791 KB)     

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Figures (8)

Fig. 1.
Fig. 1.

Picture of the micro-stripe LED

Fig. 2.
Fig. 2.

Driver board

Fig. 3.
Fig. 3.

Microscope setup

Fig. 4.
Fig. 4.

scanning scheme used for grid-projection

Fig. 5.
Fig. 5.

20x images of stained pollen grains acquired with grid-projection structured illumination. (a) Modulated raw image [Media 1], (b) Sectioned image, (c) Conventional image

Fig. 6.
Fig. 6.

20x magnification images of stained pollen grains (a) conventional image, (b) maximum projection confocal image and (c) automatic slit confocal image. Movies of (a) and (b) show how the images evolve as the line is scanned across the sample [Media 2] [Media 3]

Fig. 7.
Fig. 7.

Theoretical and experimental axial responses of the structured illumination system

Fig. 8.
Fig. 8.

Theoretical and experimental sectioning strengths of a slit scanning confocal microscope.

Equations (14)

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I sec = 2 3 ( I 1 I 2 ) 2 + ( I 2 I 3 ) 2 + ( I 3 I 1 ) 2
I conv = I 1 + I 2 + I 3 3
I sec = i = 1 N Mask i · I i
Mask i ( x ) x [ Nx , Ny ] = { 1 if MAXPOS z ( x ) = i 0 else
I sec ( x ) x [ Nx , Ny ] = MAX i = 1 N [ I i ( x ) ]
I WF ( x ) = i = 1 N I i ( x ) N
I sec ( u ) = MTF i ( u , ν ) × MTF d ( u , ν ) MTF ( u , ν ) 2
u = ( 8 π n λ ) z sin 2 ( α 2 ) ν = ( 2 π Λ ) ( n sin ( α ) λ )
MTF ( u , ν ) = { g ( ν ) 2 J 1 [ u ν ( 1 ν 2 ) ] u ν ( 1 ν 2 ) if 0 < ν < 2 0 otherwise
g ( ν ) = 1 0.69 ν + 0.0076 ν 3 + 0.043 ν 3
I ˜ sec ( u ) = 2 J 1 [ u ν ( 1 ν 2 ) ] u ν ( 1 ν 2 ) 2
I ( u ) + OTF ( u , ν x , 0 ) 2 sinc ( ν x d i 2 π ) sinc ( ν x d d 2 π ) d ν x
d i , d = 2 π λ D i , d sin α sinc ( x ) = sin ( x ) x
OTF ( u , ν x , 0 ) = { g ( ν x ) 2 J 1 [ u ν x ( 1 ν x 2 ) ] u ν x ( 1 ν x 2 ) if 2 < ν x < 2 0 otherwise

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