Abstract

Although the single-shot focus scanning technique (SSFS) has been experimentally demonstrated for extended depth of field (EDOF) imaging, few work has been performed to characterize its imaging properties and limitations. In this paper, based on an analytical model of a SSFS system, we examined the properties of the system response and the restored image quality in relation to the axial position of the object, scan range, and signal-to-noise ratio, and demonstrated the properties via a prototype of 10 × 0.25 NA microscope system. We quantified the full range of the achievable EDOF is equivalent to the focus scan range. We further demonstrated that the restored image quality can be improved by extending the focus scan range by a distance equivalent to twice of the standard DOF. For example, in a focus-scanning microscope with a ± 15 μm standard DOF, a 120 μm focus scan range can obtain a ± 60 μm EDOF, but a 150 μm scan range affords noticeably better EDOF images for the same EDOF range. These results provide guidelines for designing and implementing EDOF systems using SSFS technique.

© 2015 Optical Society of America

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References

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2011 (1)

2009 (1)

2008 (3)

2007 (1)

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

2005 (1)

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

2002 (1)

1995 (1)

1972 (1)

G. Häusler, “A method to increase the depth of focus by two step image processing,” Opt. Commun. 6(1), 38–42 (1972).
[Crossref]

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]

Bagheri, S.

Booth, M. J.

Botcherby, E. J.

Cathey, W. T.

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] [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] [PubMed]

Häusler, G.

G. Häusler, “A method to increase the depth of focus by two step image processing,” Opt. Commun. 6(1), 38–42 (1972).
[Crossref]

Hua, H.

Javidi, B.

Juskaitis, R.

Kuthirummal, S.

H. Nagahara, S. Kuthirummal, C. Zhou, and S. K. Nayar, “Flexible depth of field photography,” European Conf. Computer Vision, 60–73 (2008).

Liu, S.

Murali, S.

Nagahara, H.

H. Nagahara, S. Kuthirummal, C. Zhou, and S. K. Nayar, “Flexible depth of field photography,” European Conf. Computer Vision, 60–73 (2008).

Nayar, S. K.

H. Nagahara, S. Kuthirummal, C. Zhou, and S. K. Nayar, “Flexible depth of field photography,” European Conf. Computer Vision, 60–73 (2008).

Rolland, J. P.

Sibarita, J. B.

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

Thompson, K. P.

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]

Wilson, T.

Zhou, C.

H. Nagahara, S. Kuthirummal, C. Zhou, and S. K. Nayar, “Flexible depth of field photography,” European Conf. Computer Vision, 60–73 (2008).

Adv. Biochem. Eng. Biotechnol. (1)

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

Appl. Opt. (2)

IEEE Trans. Image Process. (1)

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. (1)

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

Opt. Commun. (1)

G. Häusler, “A method to increase the depth of focus by two step image processing,” Opt. Commun. 6(1), 38–42 (1972).
[Crossref]

Opt. Express (2)

Opt. Lett. (2)

Other (1)

H. Nagahara, S. Kuthirummal, C. Zhou, and S. K. Nayar, “Flexible depth of field photography,” European Conf. Computer Vision, 60–73 (2008).

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Figures (12)

Fig. 1
Fig. 1 Schematic layout of a single-shot focus scanning (SSFS) microscope objective system
Fig. 2
Fig. 2 Examples of PSFs. (a) and (b) are the PSFs of a standard system at the axial positions of zero (in-focus) and 20 μm (defocus of 20 μm), respectively; (c) and (d) are the PSFaccs of a SSFS system with the scan range of 160 μm at the same axial positions corresponding to (a) and (b). Simulation parameters: λ = 0.5 μm and NA = 0.25.
Fig. 3
Fig. 3 (a) Degradation of the SR at the origin of the axial position versus the increased scan range in a SSFS system; (b) SRs of a standard system and a SSFS system with the scan ranges of 80 μm, 120 μm, and 160 μm. Simulation parameters: λ = 0.5 μm and NA = 0.25.
Fig. 4
Fig. 4 The MTFs of sampled axial positions (0, 1/4, 1/2, 3/4, and 1 of the half-scan range) in a SSFS system with the scan ranges of (a) 80 μm and (b) 160 μm compared with the diffraction-limited MTF of a standard microscope system. Simulation parameters: λ = 0.5 μm; NA = 0.25.
Fig. 5
Fig. 5 NRMSD of the representative kernels in a standard microscope system and a single-shot focus scanning system with the scan ranges of 80 μm, 120 μm, and 160 μm. Simulation parameters: λ = 0.5 μm and NA = 0.25.
Fig. 6
Fig. 6 Cutoff frequency as a function of the scan range while using camera dynamic range of 48 dB for objects located at the midpoint and the edge of the scan range. Simulation parameters: λ = 0.5 μm and NA = 0.25.
Fig. 7
Fig. 7 The overall MTFs of sampled axial positions (0, 1/4, 1/2, 3/4, and 1 of the half-scan range) in a SSFS system with the scan ranges of (a, c, and e) 80 μm and (b, d, and f) 160 μm. Three different levels of NSRs, −48 dB, −30 dB, and −20 dB, were applied in the simulations for the figures (a, b), (c, d), and (e, f), respectively. Simulation parameters: λ = 0.5 μm; NA = 0.25.
Fig. 8
Fig. 8 Experimental results of the axial distributions of the peak value of the PSFs in the prototype system taking from (a) PSF of the standard image captured by fixing focus plane during exposure; (b) PSFacc of the SSFS image captured by scanning focus plane within the scan range of 160 μm during a single exposure.
Fig. 9
Fig. 9 Images of a USAF resolution target. (a) The image of the target placed within the DOF of standard microscope; (b to f) the restored images of the SSFS system with the scan range of 160 μm for the target placed at (b) the midpoint of the scan range, and the axial positions corresponding to (c) 1/4, (d) 1/2, (e) 3/4, and (f) 1 of the half-scan range from the midpoint.
Fig. 10
Fig. 10 (a) Analyzed contrast values of the sampled spatial frequencies for the standard in-focused target image shown in Fig. 9(a) and the restored images of the SSFS system shown in Fig. 9(b) and 9(f); (b) Analyzed CNR values of the sampled spatial frequencies for the standard in-focused target image shown in Fig. 9(a) and the restored images of the SSFS system shown in Fig. 9(b) through 9(f).
Fig. 11
Fig. 11 Summations of CNR values through sampled frequencies in relation to axial positions for the SSFS system with the scan range of 120 μm and 160 μm.
Fig. 12
Fig. 12 Analyzed CNR values of the sampled spatial frequencies for the restored SSFS images of the target placed at the axial position of 60 μm from the midpoint with various scan ranges from 120 μm to 160 μm.

Equations (11)

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PSF(r,z z 0 )= | 2 0 1 J 0 ( 2π λ NArρ)exp{ i 2π λ [ σ ρ 2 +W(ρ) ] }ρdρ | 2 with σ=2n(z z 0 ) sin 2 ( α 2 ) ,
PS F acc (r, z 0 )= 1 S S/2 S/2 PSF(r, z 0 z)dz ,
PS F acc (r, z 0 )= 1 S rect( z S )PSF(r, z 0 z)dz.
PS F rep (r)= 1 S S/2 S/2 PSF(r,z)dz.
o ^ = F 1 { IG },
G= H rep * | H rep | 2 +NSR ,
EDOF=±S/2.
NRMSD( z 0 )= [ 1 N i=1 N | H acc i ( z 0 ) H rep i H rep i | 2 ] 1/2 ,
S'=2(EDO F D +DOF),
MT F z 0 =| H acc ( z 0 )G |.
CNR= I max I min σ ,

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