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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 20, Iss. 21 — Oct. 8, 2012
  • pp: 23480–23488
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Analysis on enhanced depth of field for integral imaging microscope

Young-Tae Lim, Jae-Hyeung Park, Ki-Chul Kwon, and Nam Kim  »View Author Affiliations


Optics Express, Vol. 20, Issue 21, pp. 23480-23488 (2012)
http://dx.doi.org/10.1364/OE.20.023480


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Abstract

Depth of field of the integral imaging microscope is studied. In the integral imaging microscope, 3-D information is encoded as a form of elemental images Distance between intermediate plane and object point decides the number of elemental image and depth of field of integral imaging microscope. From the analysis, it is found that depth of field of the reconstructed depth plane image by computational integral imaging reconstruction is longer than depth of field of optical microscope. From analyzed relationship, experiment using integral imaging microscopy and conventional microscopy is also performed to confirm enhanced depth of field of integral imaging microscopy.

© 2012 OSA

1. Introduction

Three dimensional (3-D) information capture techniques including pickup, display, and image processing have been widely studied in the fields of science, industry, the military, broadcasting, and bio-medicine. Especially, 3-D information capture techniques of microscopic objects have become a main concern of microscopy. The objective lens of the optical microscope has a shallow depth of field (DOF) so that a thin section of the object can be imaged [1

1. D. A. Agard, “Optical sectioning microscopy: cellular architecture in three dimensions,” Annu. Rev. Biophys. Bioeng. 13(1), 191–219 (1984). [CrossRef] [PubMed]

5

5. C. J. R. Sheppard and X. Q. Mao, “Confocal microscopes with slit apertures,” J. Mod. Opt. 35(7), 1169–1185 (1988). [CrossRef]

]. Hence, a stack of focal images are required to reconstruct the 3-D information of an object [6

6. S. Inoue and R. Oldenbourg, Handbook of Optics (McGrawHill, 1995), Chap. 17.

,7

7. Q. Wu, F. A. Merchant, and K. R. Castleman, Microscope Image Processing (Academic press, 2008), Chap. 2.

]. A representative 3-D information capture technique of microscopy is confocal microscopy. Confocal microscopy is a technique whereby a researcher captures different depth images of an object by axial scanning. Then these image sets are synthesized in depth plane order. A synthesized depth plane image set has extended DOF. Another common technique of 3-D microscopy is digital holographic microscopy (DHM) [8

8. J. Sheng, E. Malkiel, and J. Katz, “Digital holographic microscope for measuring three-dimensional particle distributions and motions,” Appl. Opt. 45(16), 3893–3901 (2006). [CrossRef] [PubMed]

]. DHM offers full volumetric information of the object by numerical processing. However, it has been limited by difficulties in practical use, such as those encountered when capturing full color images and requiring coherent illumination. Integral imaging microscopy is the 3-D microscopy that provides different depth plane information; with it you view images and full-color information of the object by numerical processing. An integral imaging microscope (IIM) acquires 3-D information in an elemental form using a micro lens array. Due to the micro lens array, the DOF of the IIM can be extended to capture a thick object. Acquired 3-D information, i.e. elemental images, of the object is encoded as a form of the disparity between the elemental images, and simple pixel mapping methods are used to generate views and depth plane images. Thus, generated depth plane images are free from off-axis distortion caused by mechanical movements [9

9. Y.-T. Lim, J.-H. Park, N. Kim, and K.-C. Kwon, “Dense light field microscopy,” Proc. SPIE 7237, 72371Q, 72371Q-12 (2009). [CrossRef]

,10

10. Y.-T. Lim, J.-H. Park, K.-C. Kwon, and N. Kim, “Resolution-enhanced integral imaging microscopy that uses lens array shifting,” Opt. Express 17(21), 19253–19263 (2009). [CrossRef] [PubMed]

].

2. Lateral resolution in integral imaging microscope

The IIM is composed of the objective lens, tube lens, micro lens array, and charge coupled device (CCD). The structure of the IIM is shown in Fig. 1
Fig. 1 Structure of the integral imaging microscope.
.

In Fig. 2 fla is the focal length of the micro lens array. To capture more views of the object, third case has to be satisfied.

3. DOF in IIM

DOF by lens array around the intermediate image plane can be express as
Dla=λNAla2+dgNAlae.
(6)
It is magnified as 1/M2 by the objective and tube lenses. DOF around the object plane is given by
D=1M2Dla=λM2NAla2+dM2gNAlae.
(7)
From Eq. (4) and Eq. (7), both lateral and axial resolutions are determined by NAla. Note that extended DOF of the IIM compared to conventional optical microscope means that area of focused depth plane image is extended by micro lens array. The resolution of reconstructed images in the extended DOF has low quality than conventional optical microscope. The IIM still has resolution tradeoff by micro lens array [10

10. Y.-T. Lim, J.-H. Park, K.-C. Kwon, and N. Kim, “Resolution-enhanced integral imaging microscopy that uses lens array shifting,” Opt. Express 17(21), 19253–19263 (2009). [CrossRef] [PubMed]

].

Figure 4
Fig. 4 Simulation result: (a) comparison of DOF with a conventional optical microscope and integral imaging microscope; (b) calculated DOF after changing d and g.
shows simulation results of the calculated DOF of a conventional optical microscope and IIM from Eq. (5) and Eq. (7), where NA = 0.2, λ = 532nm, M = 10, fla = 2.4mm, e = 6μm, and φ = 125μm. In Fig. 4(a), DOF of the IIM is longer than DOF of the conventional optical microscope when d = 3.3mm. As mentioned above in section 2, NAla<NA/M, to form an elemental image with multiple view information, d is longer than 3.125mm. In the IIM, the depth information of the object is encoded as a disparity between the elemental images. Hence, the minimum depth deviation which causes a barely detectable change of the disparity between the elemental images can be regarded as a measure of axial resolution. In Fig. 4(b), if d is 3.5mm, then g is 7.6mm. Numerically calculated DOF of the IIM exceed DOF of the conventional optical microscope. In this condition, Rele is 30.5μm and each object point is imaged to four elemental lens. Four view images can be reconstructed via the OVIR method. However, these images are blurred. Rele is larger than pixel size of the CCD e. Hence, effective range of d has to be set.

4. Experiments for generating extended DOF images and comparison with conventional extended depth of field images

The experimental setup was composed of the Olympus BX-51 and a micro lens array module as shown in Fig. 6(a)
Fig. 6 Experimental setup: (a) captured experimental setup; (b) diagram of the micro lens array module.
. The micro lens array module is composed of the micro lens array, focal lens, and CCD. Each part of micro lens array module can be separable as shown in Fig. 6(b). The NA of the objective lens was 0.2, the wavelength was 532nm, the magnification of the objective lens was 10, the focal length of the micro lens array was 2.4mm, the pixel pitch size of the CCD was 6μm, the pitch size of the micro lens was 125μm and d is 10mm.

Figure 7(a)
Fig. 7 (a) 2-D micro scale object image and (b) elemental image.
shows the 2D image of the object used in the experiment. The micro register height is about 140μm. From Eq. (7), calculated D, Do, and Rele are 180μm, 18μm, and 12.6μm, respectively. Figure 7(b) shows the elemental image of Fig. 7(a). The pixel size and number of pixels of the elemental image are 946 × 946 and 11 × 11, respectively. Calculated DOF of the IIM is 18μm, and then 8 images are required to compare the DOF of a conventional optical microscope shown as Fig. 8
Fig. 8 Sequentially-captured 2D microscopic image of Fig. 7(a).
. From Fig. 7(b), depth slice images were generated at a reconstructed distance with z = −3mm to −6mm, and 3mm to 6mm, shown as Fig. 9
Fig. 9 Depth plane image generated from Fig. 7(b) using CIIR.
. In conventional CIIR method, for a given depth plane, specific depth plane can be generated. CIIR method used in this paper is only effective within integer depth plane . If g is same to focal length of the micro lens array, errors occurs for coding. Each local position of pixels is remapped on certain depth plane. In this case, focal length of the micro lens array is 2.4mm, minimum condition to generate depth plane of center region of the object is 3mm vice versa. In our experiment, 3mm to 6mm and −3mm to −6mm on depth plane of CIIR cover the range of 140μm.

In order to verify that the reconstructed images of IIM are equivalent to the 2-D images of conventional microscopy, we first compared PSNR of Figs. 8 and 9. The average PSNR difference between the two images is 11.75dB, and the maximum difference is 2.2dB, shown as Fig. 10
Fig. 10 PSNR of each depth plane image between 2-D and IIM.
. Reconstructed depth plane images using CIIR has low resolution and noise which is caused by a boundary between each lens of the micro lens array. Results show that depth plane images using the CIIR method have similar DOF with sequentially-captured 2-D images of conventional optical microscopes. Second, we analyzed image intensities both of Fig. 8 and Fig. 9. Distribution of maximum pixel intensity of Fig. 11(a)
Fig. 11 Intensities of pixels: (a) z = 3 to 6mm by CIIR, (b) z = 16.6 to 66.4μm by 2-D, (c) z = −3 to −6mm by CIIR, (d) z = −16.6 to −66.4μm by 2-D.
and Fig. 11(b) are 95th, 205th, and 380th, and of Fig. 11(c) and Fig. 11(d) are 89th, 218th, and 376th. The difference of maximum intensity between both images is about 40. It is also affected by boundary distortion of the micro lens array while CIIR is processing. Results show that two image sets have a similar profile.

5. Conclusion

IIM can form elemental images in a unique way. The relationship between lateral resolution and DOF was analyzed to explain the capability to surface reconstructed depth plane images of an object. Corresponding distance from the intermediate plane to the micro lens array and object point around the focal plane of the microscope are main factors in forming an elemental image. These factors determine the disparity, that is, 3-D information of the object. The reconstructed depth plane image has low resolution, but it includes the DOF of the conventional optical microscope. In future work, a resolution-enhancing method such as an intermediate view reconstruction technique will be used for our research. It will be a useful device to acquire 3-D information of the microscope object without mechanical movements.

Acknowledgments

This research was supported by the Leaders in INdustry-university Cooperation(LINC) Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (2012-B-0013-010116).

References and links

1.

D. A. Agard, “Optical sectioning microscopy: cellular architecture in three dimensions,” Annu. Rev. Biophys. Bioeng. 13(1), 191–219 (1984). [CrossRef] [PubMed]

2.

J. G. McNally, C. Preza, J. A. Conchello, and L. J. Thomas Jr., “Artifacts in computational optical-sectioning microscopy,” J. Opt. Soc. Am. A 11(3), 1056–1067 (1994). [CrossRef] [PubMed]

3.

S. Liu and H. Hua, “Extended depth-of-field microscopic imaging with a variable focus microscope objective,” Opt. Express 19(1), 353–362 (2011). [CrossRef] [PubMed]

4.

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

5.

C. J. R. Sheppard and X. Q. Mao, “Confocal microscopes with slit apertures,” J. Mod. Opt. 35(7), 1169–1185 (1988). [CrossRef]

6.

S. Inoue and R. Oldenbourg, Handbook of Optics (McGrawHill, 1995), Chap. 17.

7.

Q. Wu, F. A. Merchant, and K. R. Castleman, Microscope Image Processing (Academic press, 2008), Chap. 2.

8.

J. Sheng, E. Malkiel, and J. Katz, “Digital holographic microscope for measuring three-dimensional particle distributions and motions,” Appl. Opt. 45(16), 3893–3901 (2006). [CrossRef] [PubMed]

9.

Y.-T. Lim, J.-H. Park, N. Kim, and K.-C. Kwon, “Dense light field microscopy,” Proc. SPIE 7237, 72371Q, 72371Q-12 (2009). [CrossRef]

10.

Y.-T. Lim, J.-H. Park, K.-C. Kwon, and N. Kim, “Resolution-enhanced integral imaging microscopy that uses lens array shifting,” Opt. Express 17(21), 19253–19263 (2009). [CrossRef] [PubMed]

11.

D.-H. Shin and E.-S. Kim, “Computational integral imaging reconstruction of 3D Object using a depth conversion technique,” J. Opt. Soc. Kor. 12(3), 131–135 (2008). [CrossRef]

OCIS Codes
(100.6890) Image processing : Three-dimensional image processing
(110.0180) Imaging systems : Microscopy
(170.6900) Medical optics and biotechnology : Three-dimensional microscopy

ToC Category:
Microscopy

History
Original Manuscript: July 6, 2012
Revised Manuscript: September 1, 2012
Manuscript Accepted: September 24, 2012
Published: September 27, 2012

Citation
Young-Tae Lim, Jae-Hyeung Park, Ki-Chul Kwon, and Nam Kim, "Analysis on enhanced depth of field for integral imaging microscope," Opt. Express 20, 23480-23488 (2012)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-20-21-23480


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References

  1. D. A. Agard, “Optical sectioning microscopy: cellular architecture in three dimensions,” Annu. Rev. Biophys. Bioeng.13(1), 191–219 (1984). [CrossRef] [PubMed]
  2. J. G. McNally, C. Preza, J. A. Conchello, and L. J. Thomas., “Artifacts in computational optical-sectioning microscopy,” J. Opt. Soc. Am. A11(3), 1056–1067 (1994). [CrossRef] [PubMed]
  3. S. Liu and H. Hua, “Extended depth-of-field microscopic imaging with a variable focus microscope objective,” Opt. Express19(1), 353–362 (2011). [CrossRef] [PubMed]
  4. J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods2(12), 920–931 (2005). [CrossRef] [PubMed]
  5. C. J. R. Sheppard and X. Q. Mao, “Confocal microscopes with slit apertures,” J. Mod. Opt.35(7), 1169–1185 (1988). [CrossRef]
  6. S. Inoue and R. Oldenbourg, Handbook of Optics (McGrawHill, 1995), Chap. 17.
  7. Q. Wu, F. A. Merchant, and K. R. Castleman, Microscope Image Processing (Academic press, 2008), Chap. 2.
  8. J. Sheng, E. Malkiel, and J. Katz, “Digital holographic microscope for measuring three-dimensional particle distributions and motions,” Appl. Opt.45(16), 3893–3901 (2006). [CrossRef] [PubMed]
  9. Y.-T. Lim, J.-H. Park, N. Kim, and K.-C. Kwon, “Dense light field microscopy,” Proc. SPIE7237, 72371Q, 72371Q-12 (2009). [CrossRef]
  10. Y.-T. Lim, J.-H. Park, K.-C. Kwon, and N. Kim, “Resolution-enhanced integral imaging microscopy that uses lens array shifting,” Opt. Express17(21), 19253–19263 (2009). [CrossRef] [PubMed]
  11. D.-H. Shin and E.-S. Kim, “Computational integral imaging reconstruction of 3D Object using a depth conversion technique,” J. Opt. Soc. Kor.12(3), 131–135 (2008). [CrossRef]

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