Abstract

Increasing the depth-of-field (DOF) while maintaining high resolution imaging has been a classical challenge for conventional microscopes. Extended DOF (EDOF) is especially essential for imaging thick specimens. We present a microscope capable of capturing EDOF images in a single shot. A volumetric optical sampling method is applied by rapidly scanning the focus of a vari-focal microscope objective throughout the extended depths of a thick specimen within the duration of a single detector exposure. An EDOF image is reconstructed by deconvolving the captured image with the response function of the system. Design of a vari-focal objective and algorithms for restoring EDOF images are presented. Proof-of-concept experimental results demonstrate significantly extended DOF compared to the conventional microscope counterparts.

© 2010 OSA

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. T. S. Tkaczyk, Field Guide to Microscopy (SPIE Press, 2009).
  2. S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
    [CrossRef] [PubMed]
  3. W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
    [PubMed]
  4. E. R. Dowski and W. T. Cathey, “Extended depth of field through wave-front coding,” Appl. Opt. 34(11), 1859–1866 (1995).
    [CrossRef] [PubMed]
  5. S. C. Tucker, W. T. Cathey, and E. R. Dowski., “Extended depth of field and aberration control for inexpensive digital microscope systems,” Opt. Express 4(11), 467–474 (1999), http://www.opticsinfobase.org/abstract.cfm?URI=oe-4-11-467 .
    [CrossRef] [PubMed]
  6. J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
    [CrossRef] [PubMed]
  7. F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
    [CrossRef] [PubMed]
  8. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
    [CrossRef] [PubMed]
  9. 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]
  10. J. B. Sibarita, “Deconvolution microscopy,” Adv. Biochem. Eng. Biotechnol. 95, 201–243 (2005).
    [PubMed]
  11. F. Aguet, D. Van De Ville, and M. Unser, “Model-based 2.5-d deconvolution for extended depth of field in brightfield microscopy,” IEEE Trans. Image Process. 17(7), 1144–1153 (2008).
    [CrossRef] [PubMed]
  12. J. P. Rolland, P. Meemon, S. Murali, K. P. Thompson, and K. S. Lee, “Gabor-based fusion technique for optical coherence microscopy,” Opt. Express 18(4), 3632–3642 (2010), http://www.opticsinfobase.org/abstract.cfm?URI=oe-18-4-3632 .
    [CrossRef] [PubMed]
  13. http://www.varioptic.com .
  14. J. A. Conchello and M. E. Dresser, “Extended depth-of-focus microscopy via constrained deconvolution,” J. Biomed. Opt. 12(6), 064026 (2007).
    [CrossRef]
  15. S. Murali, K. P. Thompson, and J. P. Rolland, “Three-dimensional adaptive microscopy using embedded liquid lens,” Opt. Lett. 34(2), 145–147 (2009).
    [CrossRef] [PubMed]
  16. S. Liu and H. Hua, “Time-multiplexed dual-focal plane head-mounted display with a liquid lens,” Opt. Lett. 34(11), 1642–1644 (2009).
    [CrossRef] [PubMed]
  17. C. Pan, J. B. Chen, R. Zgang, and S. L. Zhuang, “Extension ratio of depth of field by wavefront coding method,” Opt. Express 16(17), 13364–13371 (2008), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-16-17-13364 .
    [CrossRef] [PubMed]

2010

2009

S. Murali, K. P. Thompson, and J. P. Rolland, “Three-dimensional adaptive microscopy using embedded liquid lens,” Opt. Lett. 34(2), 145–147 (2009).
[CrossRef] [PubMed]

S. Liu and H. Hua, “Time-multiplexed dual-focal plane head-mounted display with a liquid lens,” Opt. Lett. 34(11), 1642–1644 (2009).
[CrossRef] [PubMed]

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

2008

C. Pan, J. B. Chen, R. Zgang, and S. L. Zhuang, “Extension ratio of depth of field by wavefront coding method,” Opt. Express 16(17), 13364–13371 (2008), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-16-17-13364 .
[CrossRef] [PubMed]

F. Aguet, D. Van De Ville, and M. Unser, “Model-based 2.5-d deconvolution for extended depth of field in brightfield microscopy,” IEEE Trans. Image Process. 17(7), 1144–1153 (2008).
[CrossRef] [PubMed]

2007

J. A. Conchello and M. E. Dresser, “Extended depth-of-focus microscopy via constrained deconvolution,” J. Biomed. Opt. 12(6), 064026 (2007).
[CrossRef]

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[PubMed]

2005

J. B. Sibarita, “Deconvolution microscopy,” Adv. Biochem. Eng. Biotechnol. 95, 201–243 (2005).
[PubMed]

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

2004

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

1999

1997

1995

Aguet, F.

F. Aguet, D. Van De Ville, and M. Unser, “Model-based 2.5-d deconvolution for extended depth of field in brightfield microscopy,” IEEE Trans. Image Process. 17(7), 1144–1153 (2008).
[CrossRef] [PubMed]

Basiji, D. A.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[PubMed]

Biteen, J. S.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Cathey, W. T.

Chen, J. B.

Conchello, J. A.

J. A. Conchello and M. E. Dresser, “Extended depth-of-focus microscopy via constrained deconvolution,” J. Biomed. Opt. 12(6), 064026 (2007).
[CrossRef]

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[CrossRef] [PubMed]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Denk, W.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

Dowski, E. R.

Dresser, M. E.

J. A. Conchello and M. E. Dresser, “Extended depth-of-focus microscopy via constrained deconvolution,” J. Biomed. Opt. 12(6), 064026 (2007).
[CrossRef]

Frost, K.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[PubMed]

Hall, B. E.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[PubMed]

Helmchen, F.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

Hua, H.

Huisken, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Juskaitis, R.

Lee, K. S.

Liang, L.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[PubMed]

Lichtman, J. W.

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[CrossRef] [PubMed]

Liu, N.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Liu, S.

Lord, S. J.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Meemon, P.

Moerner, W. E.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Murali, S.

Neil, M. A. A.

Ortyn, W. E.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[PubMed]

Pan, C.

Pavani, S. R. P.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Perry, D. J.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[PubMed]

Piestun, R.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Rolland, J. P.

Sibarita, J. B.

J. B. Sibarita, “Deconvolution microscopy,” Adv. Biochem. Eng. Biotechnol. 95, 201–243 (2005).
[PubMed]

Stelzer, E. H. K.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Swoger, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Thompson, K. P.

Thompson, M. A.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Tucker, S. C.

Twieg, R. J.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Unser, M.

F. Aguet, D. Van De Ville, and M. Unser, “Model-based 2.5-d deconvolution for extended depth of field in brightfield microscopy,” IEEE Trans. Image Process. 17(7), 1144–1153 (2008).
[CrossRef] [PubMed]

Van De Ville, D.

F. Aguet, D. Van De Ville, and M. Unser, “Model-based 2.5-d deconvolution for extended depth of field in brightfield microscopy,” IEEE Trans. Image Process. 17(7), 1144–1153 (2008).
[CrossRef] [PubMed]

Venkatachalam, V.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[PubMed]

Wilson, T.

Wittbrodt, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Zgang, R.

Zhuang, S. L.

Adv. Biochem. Eng. Biotechnol.

J. B. Sibarita, “Deconvolution microscopy,” Adv. Biochem. Eng. Biotechnol. 95, 201–243 (2005).
[PubMed]

Appl. Opt.

Cytometry A

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[PubMed]

IEEE Trans. Image Process.

F. Aguet, D. Van De Ville, and M. Unser, “Model-based 2.5-d deconvolution for extended depth of field in brightfield microscopy,” IEEE Trans. Image Process. 17(7), 1144–1153 (2008).
[CrossRef] [PubMed]

J. Biomed. Opt.

J. A. Conchello and M. E. Dresser, “Extended depth-of-focus microscopy via constrained deconvolution,” J. Biomed. Opt. 12(6), 064026 (2007).
[CrossRef]

Nat. Methods

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

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

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Science

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Other

T. S. Tkaczyk, Field Guide to Microscopy (SPIE Press, 2009).

http://www.varioptic.com .

Supplementary Material (1)

» Media 1: MOV (4002 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 (8)

Fig. 1
Fig. 1

Schematic illustration of the volumetric optical sampling method for a thick 1-D specimen with an extended depth range from zn to zf .

Fig. 2
Fig. 2

Driving mechanisms of the (a) image sensor and (b) focus of the microscope objective, where 1/T equals to the frame rate of the image sensor and 0.5/T equals to the driving frequency of the liquid lens.

Fig. 3
Fig. 3

Optical layouts of the vari-focal microscope objective integrated with a liquid lens. The focal distance z is set at (a) zn + 0.00 mm, (b) zn + 0.08 mm, and (c) zn + 0.16 mm respectively. The corresponding diffraction MTF at each focal distance is shown in (d), (e), and (f) respectively.

Fig. 4
Fig. 4

Point spread functions of a conventional microscope as the defocus distance, z 0-z, increases at (a) 0.00 mm, (b) 0.04 mm, (c) 0.08 mm, (d) 0.12 mm, and (e) 0.16 mm, respectively; Point spread functions―PSFx’ , y’ (z 0) of the EDOF microscope for z 0 equals to (a) zn + 0.00 mm, (b) zn + 0.04 mm, (c) zn + 0.08 mm, (d) zn + 0.12 mm, and (e) zn + 0.16 mm, respectively.

Fig. 5
Fig. 5

Modulation transfer functions of the vari-focal microscope objective with different object distance of z 0 equals to zn + 0.00 mm (red circle marker), zn + 0.04 mm (green square marker), zn + 0.08 mm (blue inverse triangle marker), zn + 0.12 mm (cyan triangle marker), and zn + 0.16 mm (magenta star marker), respectively.

Fig. 6
Fig. 6

Schematic setup for the proof-of-concept EDOF microscope.

Fig. 7
Fig. 7

Conventional microscopic images of the sample, as the objective focuses at (a) near and (b) far distances. (c) Single-shot image of the same sample captured by applying the volumetric sampling approach. (d) Restored single-shot EDOF image after applying the deconvolution filter. (Media 1)

Fig. 8
Fig. 8

Normalized pixel intensities along the (a) solid and (b) dashed lines in Figs. 7 (a)-(d).

Equations (4)

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

P S F x ' , y ' ( z 0 ) = 1 z f z n z = z n z f d z P S F x ' , y ' ( z 0 , z ) ,
P S F x ' , y ' ( z 0 ) = lim N 1 N + 1 n = 0 N P S F x ' , y ' ( z 0 , z n + n z f z n N ) ,
o ( z 0 ) = i x ' , y ' ( z 0 ) 1 P S F x ' , y ' ( z 0 ) .
o ( z n z 0 z f ) = i x ' , y ' ( z n z 0 z f ) 1 P S F x ' , y ' ( z 0 ) ,

Metrics