Abstract

We report the development of a high-throughput whole slide imaging (WSI) system by adapting a cost-effective optomechanical add-on kit to existing microscopes. Inspired by the phase detection concept in professional photography, we attached two pinhole-modulated cameras at the eyepiece ports for instant focal plane detection. By adjusting the positions of the pinholes, we can effectively change the view angle for the sample, and as such, we can use the translation shift of the two pinhole-modulated images to identify the optimal focal position. By using a small pinhole size, the focal-plane-detection range is on the order of millimeter, orders of magnitude longer than the objective’s depth of field. We also show that, by analyzing the phase correlation of the pinhole-modulated images, we can determine whether the sample contains one thin section, folded sections, or multiple layers separated by certain distances – an important piece of information prior to a detailed z scan. In order to achieve system automation, we deployed a low-cost programmable robotic arm to perform sample loading and $14 stepper motors to drive the microscope stage to perform x-y scanning. Using a 20X objective lens, we can acquire a 2 gigapixel image with 14 mm by 8 mm field of view in 90 seconds. The reported platform may find applications in biomedical research, telemedicine, and digital pathology. It may also provide new insights for the development of high-content screening instruments.

© 2015 Optical Society of America

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References

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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref]
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2015 (1)

2011 (2)

M. C. Montalto, R. R. McKay, and R. J. Filkins, “Autofocus methods of whole slide imaging systems and the introduction of a second-generation independent dual sensor scanning method,” J. Pathol. Inform. 2(1), 44 (2011).
[Crossref] [PubMed]

G. Zheng, C. Kolner, and C. Yang, “Microscopy refocusing and dark-field imaging by using a simple LED array,” Opt. Lett. 36(20), 3987–3989 (2011).
[Crossref] [PubMed]

2010 (1)

2008 (1)

2006 (1)

Y. Liron, Y. Paran, N. G. Zatorsky, B. Geiger, and Z. Kam, “Laser autofocusing system for high-resolution cell biological imaging,” J. Microsc. 221(2), 145–151 (2006).
[Crossref] [PubMed]

2002 (1)

H. Foroosh, J. B. Zerubia, and M. Berthod, “Extension of phase correlation to subpixel registration,” IEEE Trans. Image Process. 11(3), 188–200 (2002).
[Crossref] [PubMed]

1995 (1)

L. McKeogh, J. Sharpe, and K. Johnson, “A low-cost automatic translation and autofocusing system for a microscope,” Meas. Sci. Technol. 6(5), 583–587 (1995).
[Crossref]

1991 (1)

L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston., “Comparison of autofocus methods for automated microscopy,” Cytometry 12(3), 195–206 (1991).
[Crossref] [PubMed]

Awwal, A. A. S.

Berthod, M.

H. Foroosh, J. B. Zerubia, and M. Berthod, “Extension of phase correlation to subpixel registration,” IEEE Trans. Image Process. 11(3), 188–200 (2002).
[Crossref] [PubMed]

Bian, Z.

Cook, K.

L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston., “Comparison of autofocus methods for automated microscopy,” Cytometry 12(3), 195–206 (1991).
[Crossref] [PubMed]

Corwin, A. D.

Culp, K.

L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston., “Comparison of autofocus methods for automated microscopy,” Cytometry 12(3), 195–206 (1991).
[Crossref] [PubMed]

Dixon, E. L.

Dong, S.

Filkins, R. J.

M. C. Montalto, R. R. McKay, and R. J. Filkins, “Autofocus methods of whole slide imaging systems and the introduction of a second-generation independent dual sensor scanning method,” J. Pathol. Inform. 2(1), 44 (2011).
[Crossref] [PubMed]

S. Yazdanfar, K. B. Kenny, K. Tasimi, A. D. Corwin, E. L. Dixon, and R. J. Filkins, “Simple and robust image-based autofocusing for digital microscopy,” Opt. Express 16(12), 8670–8677 (2008).
[Crossref] [PubMed]

Firestone, L.

L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston., “Comparison of autofocus methods for automated microscopy,” Cytometry 12(3), 195–206 (1991).
[Crossref] [PubMed]

Foroosh, H.

H. Foroosh, J. B. Zerubia, and M. Berthod, “Extension of phase correlation to subpixel registration,” IEEE Trans. Image Process. 11(3), 188–200 (2002).
[Crossref] [PubMed]

Geiger, B.

Y. Liron, Y. Paran, N. G. Zatorsky, B. Geiger, and Z. Kam, “Laser autofocusing system for high-resolution cell biological imaging,” J. Microsc. 221(2), 145–151 (2006).
[Crossref] [PubMed]

Guo, K.

Johnson, K.

L. McKeogh, J. Sharpe, and K. Johnson, “A low-cost automatic translation and autofocusing system for a microscope,” Meas. Sci. Technol. 6(5), 583–587 (1995).
[Crossref]

Kam, Z.

Y. Liron, Y. Paran, N. G. Zatorsky, B. Geiger, and Z. Kam, “Laser autofocusing system for high-resolution cell biological imaging,” J. Microsc. 221(2), 145–151 (2006).
[Crossref] [PubMed]

Kenny, K. B.

Kolner, C.

Liron, Y.

Y. Liron, Y. Paran, N. G. Zatorsky, B. Geiger, and Z. Kam, “Laser autofocusing system for high-resolution cell biological imaging,” J. Microsc. 221(2), 145–151 (2006).
[Crossref] [PubMed]

McKay, R. R.

M. C. Montalto, R. R. McKay, and R. J. Filkins, “Autofocus methods of whole slide imaging systems and the introduction of a second-generation independent dual sensor scanning method,” J. Pathol. Inform. 2(1), 44 (2011).
[Crossref] [PubMed]

McKeogh, L.

L. McKeogh, J. Sharpe, and K. Johnson, “A low-cost automatic translation and autofocusing system for a microscope,” Meas. Sci. Technol. 6(5), 583–587 (1995).
[Crossref]

Montalto, M. C.

M. C. Montalto, R. R. McKay, and R. J. Filkins, “Autofocus methods of whole slide imaging systems and the introduction of a second-generation independent dual sensor scanning method,” J. Pathol. Inform. 2(1), 44 (2011).
[Crossref] [PubMed]

Nanda, P.

Paran, Y.

Y. Liron, Y. Paran, N. G. Zatorsky, B. Geiger, and Z. Kam, “Laser autofocusing system for high-resolution cell biological imaging,” J. Microsc. 221(2), 145–151 (2006).
[Crossref] [PubMed]

Preston, K.

L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston., “Comparison of autofocus methods for automated microscopy,” Cytometry 12(3), 195–206 (1991).
[Crossref] [PubMed]

Sharpe, J.

L. McKeogh, J. Sharpe, and K. Johnson, “A low-cost automatic translation and autofocusing system for a microscope,” Meas. Sci. Technol. 6(5), 583–587 (1995).
[Crossref]

Talsania, N.

L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston., “Comparison of autofocus methods for automated microscopy,” Cytometry 12(3), 195–206 (1991).
[Crossref] [PubMed]

Tasimi, K.

Wang, Y. M.

Yang, C.

Yazdanfar, S.

Zatorsky, N. G.

Y. Liron, Y. Paran, N. G. Zatorsky, B. Geiger, and Z. Kam, “Laser autofocusing system for high-resolution cell biological imaging,” J. Microsc. 221(2), 145–151 (2006).
[Crossref] [PubMed]

Zerubia, J. B.

H. Foroosh, J. B. Zerubia, and M. Berthod, “Extension of phase correlation to subpixel registration,” IEEE Trans. Image Process. 11(3), 188–200 (2002).
[Crossref] [PubMed]

Zheng, G.

Appl. Opt. (1)

Biomed. Opt. Express (1)

Cytometry (1)

L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston., “Comparison of autofocus methods for automated microscopy,” Cytometry 12(3), 195–206 (1991).
[Crossref] [PubMed]

IEEE Trans. Image Process. (1)

H. Foroosh, J. B. Zerubia, and M. Berthod, “Extension of phase correlation to subpixel registration,” IEEE Trans. Image Process. 11(3), 188–200 (2002).
[Crossref] [PubMed]

J. Microsc. (1)

Y. Liron, Y. Paran, N. G. Zatorsky, B. Geiger, and Z. Kam, “Laser autofocusing system for high-resolution cell biological imaging,” J. Microsc. 221(2), 145–151 (2006).
[Crossref] [PubMed]

J. Pathol. Inform. (1)

M. C. Montalto, R. R. McKay, and R. J. Filkins, “Autofocus methods of whole slide imaging systems and the introduction of a second-generation independent dual sensor scanning method,” J. Pathol. Inform. 2(1), 44 (2011).
[Crossref] [PubMed]

Meas. Sci. Technol. (1)

L. McKeogh, J. Sharpe, and K. Johnson, “A low-cost automatic translation and autofocusing system for a microscope,” Meas. Sci. Technol. 6(5), 583–587 (1995).
[Crossref]

Opt. Express (1)

Opt. Lett. (1)

Other (2)

A. Kinba, M. Hamada, H. Ueda, K. Sugitani, and H. Ootsuka, “Auto focus detecting device comprising both phase-difference detecting and contrast detecting methods,” (Google Patents, 1997).

C.-S. Liu, P.-H. Hu, Y.-H. Wang, S.-S. Ke, Y.-C. Lin, Y.-H. Chang, and J.-B. Horng, “Novel fast laser-based auto-focusing microscope,” in Sensors, 2010 IEEE, (IEEE, 2010), 481–485.

Supplementary Material (2)

NameDescription
» Visualization 1: MP4 (13377 KB)      Media 1
» Visualization 2: MP4 (7356 KB)      Media 2

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Figures (5)

Fig. 1
Fig. 1

(a) Pinhole-modulated cameras for instant focal plane detection. (b) By inserting an off-axis pinhole at the Fourier plane, we can effectively change the view angle of the sample. (c1) A 3D-printed plastic case was used to assemble the pinhole-modulated camera. (c2) The off-axis pinhole was punched by a needle on a printing paper. (d) We attached the assembly to the eyepiece ports of a microscope platform.

Fig. 2
Fig. 2

The captured images through the pinhole-modulated cameras (a)-(b), and the main camera (c). (d) The measured relationship between the translational shift of the two pinhole-modulated images and the defocus distance.

Fig. 3
Fig. 3

Using the phase correlation curve for exploring sample structures at the z direction. Samples with one thin section (a), folded section (b), and two different layers separated by certain distance (c).

Fig. 4
Fig. 4

Sample loading and mechanical scanning schemes in the reported platform. (a) 3D-printed plastic gear for controlling focus knob. (b) Sample scanning using a mechanical kit and sample loading using a programmable robotic arm. XM: x-axis motor; YM: y-axis motor; XYG: x-y scanning gear group; ZM: z-axis motor; ZG: z-axis scanning gear. Also refer to Visualization 1 and Visualization 2.

Fig. 5
Fig. 5

Gigapixel images captured by using the reported platform. (a) A captured image of a pathology slide using a 9 megapixel CCD. The field of view is 14 mm by 8 mm and the acquisition time is 90 seconds. (b) A captured image of a blood smear using a 1.5 megapixel color CMOS sensor. The field of view is 15 mm by 15 mm and the acquisition time is 16 minutes. These images can be viewed at: http://gigapan.com/profiles/SmartImagingLab.

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