Abstract

A critical consideration for whole slide imaging (WSI) platform is to perform accurate autofocusing at high speed. Typical WSI systems acquire a z-stack of sample images and determine the best focal position by maximizing a figure of merit. This strategy, however, has suffered from several limitations, including low speed due to multiple image acquisitions, relatively low accuracy of focal plane estimation, short axial range for autofocusing, and difficulties in handling transparent samples. By exploring the autocorrelation property of the tissue sections, we report a novel single-frame autofocusing scheme to address the above challenges. In this approach, we place a two-pinhole-modulated camera at the epi-illumination arm. The captured image contains two copies of the sample separated by a certain distance. By identifying this distance, we can recover the defocus distance of the sample over a long z-range without z-scanning. To handle transparent samples, we set an offset distance to the autofocusing camera for generating out-of-focus contrast in the captured image. The single-frame nature of our scheme allows autofocusing even when the stage is in continuous motion. We demonstrate the use of the our autofocusing scheme for fluorescence WSI and quantify the focusing performance on 1550 different tissue tiles. The average autofocusing error is ~0.11 depth-of-field, 3 folds better than that of conventional methods. We report an autofocusing speed of 0.037 s per tile, which is much faster than that of conventional methods. The autofocusing range is ~80 µm, 8 folds longer than that of conventional methods. The reported scheme is able to solve the autofocusing challenges in WSI systems and may find applications in high-throughput brightfield/fluorescence WSI.

© 2016 Optical Society of America

Full Article  |  PDF Article
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References

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  1. L. Pantanowitz, J. H. Sinard, W. H. Henricks, L. A. Fatheree, A. B. Carter, L. Contis, B. A. Beckwith, A. J. Evans, A. Lal, A. V. Parwani, and College of American Pathologists Pathology and Laboratory Quality Center, “Validating whole slide imaging for diagnostic purposes in pathology: guideline from the College of American Pathologists Pathology and Laboratory Quality Center,” Arch. Pathol. Lab. Med. 137(12), 1710–1722 (2013).
    [Crossref] [PubMed]
  2. J. R. Gilbertson, J. Ho, L. Anthony, D. M. Jukic, Y. Yagi, and A. V. Parwani, “Primary histologic diagnosis using automated whole slide imaging: a validation study,” BMC Clin. Pathol. 6(1), 4 (2006).
    [Crossref] [PubMed]
  3. 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]
  4. 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]
  5. 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]
  6. R. R. McKay, V. A. Baxi, and M. C. Montalto, “The accuracy of dynamic predictive autofocusing for whole slide imaging,” J. Pathol. Inform. 2(1), 38 (2011).
    [Crossref] [PubMed]
  7. K. Guo, J. Liao, Z. Bian, X. Heng, and G. Zheng, “InstantScope: a low-cost whole slide imaging system with instant focal plane detection,” Biomed. Opt. Express 6(9), 3210–3216 (2015).
    [Crossref] [PubMed]
  8. B. D. Lucas and T. Kanade, “An iterative image registration technique with an application to stereo vision,” in IJCAI, 1981), 674–679.
  9. Y. Fan, Y. Gal, and A. P. Bradley, “An algorithm for microscopic specimen delineation and focus candidate selection,” Micron 66, 51–62 (2014).
    [Crossref] [PubMed]

2015 (1)

2014 (1)

Y. Fan, Y. Gal, and A. P. Bradley, “An algorithm for microscopic specimen delineation and focus candidate selection,” Micron 66, 51–62 (2014).
[Crossref] [PubMed]

2013 (1)

L. Pantanowitz, J. H. Sinard, W. H. Henricks, L. A. Fatheree, A. B. Carter, L. Contis, B. A. Beckwith, A. J. Evans, A. Lal, A. V. Parwani, and College of American Pathologists Pathology and Laboratory Quality Center, “Validating whole slide imaging for diagnostic purposes in pathology: guideline from the College of American Pathologists Pathology and Laboratory Quality Center,” Arch. Pathol. Lab. Med. 137(12), 1710–1722 (2013).
[Crossref] [PubMed]

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]

R. R. McKay, V. A. Baxi, and M. C. Montalto, “The accuracy of dynamic predictive autofocusing for whole slide imaging,” J. Pathol. Inform. 2(1), 38 (2011).
[Crossref] [PubMed]

2008 (1)

2006 (1)

J. R. Gilbertson, J. Ho, L. Anthony, D. M. Jukic, Y. Yagi, and A. V. Parwani, “Primary histologic diagnosis using automated whole slide imaging: a validation study,” BMC Clin. Pathol. 6(1), 4 (2006).
[Crossref] [PubMed]

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]

Anthony, L.

J. R. Gilbertson, J. Ho, L. Anthony, D. M. Jukic, Y. Yagi, and A. V. Parwani, “Primary histologic diagnosis using automated whole slide imaging: a validation study,” BMC Clin. Pathol. 6(1), 4 (2006).
[Crossref] [PubMed]

Baxi, V. A.

R. R. McKay, V. A. Baxi, and M. C. Montalto, “The accuracy of dynamic predictive autofocusing for whole slide imaging,” J. Pathol. Inform. 2(1), 38 (2011).
[Crossref] [PubMed]

Beckwith, B. A.

L. Pantanowitz, J. H. Sinard, W. H. Henricks, L. A. Fatheree, A. B. Carter, L. Contis, B. A. Beckwith, A. J. Evans, A. Lal, A. V. Parwani, and College of American Pathologists Pathology and Laboratory Quality Center, “Validating whole slide imaging for diagnostic purposes in pathology: guideline from the College of American Pathologists Pathology and Laboratory Quality Center,” Arch. Pathol. Lab. Med. 137(12), 1710–1722 (2013).
[Crossref] [PubMed]

Bian, Z.

Bradley, A. P.

Y. Fan, Y. Gal, and A. P. Bradley, “An algorithm for microscopic specimen delineation and focus candidate selection,” Micron 66, 51–62 (2014).
[Crossref] [PubMed]

Carter, A. B.

L. Pantanowitz, J. H. Sinard, W. H. Henricks, L. A. Fatheree, A. B. Carter, L. Contis, B. A. Beckwith, A. J. Evans, A. Lal, A. V. Parwani, and College of American Pathologists Pathology and Laboratory Quality Center, “Validating whole slide imaging for diagnostic purposes in pathology: guideline from the College of American Pathologists Pathology and Laboratory Quality Center,” Arch. Pathol. Lab. Med. 137(12), 1710–1722 (2013).
[Crossref] [PubMed]

Contis, L.

L. Pantanowitz, J. H. Sinard, W. H. Henricks, L. A. Fatheree, A. B. Carter, L. Contis, B. A. Beckwith, A. J. Evans, A. Lal, A. V. Parwani, and College of American Pathologists Pathology and Laboratory Quality Center, “Validating whole slide imaging for diagnostic purposes in pathology: guideline from the College of American Pathologists Pathology and Laboratory Quality Center,” Arch. Pathol. Lab. Med. 137(12), 1710–1722 (2013).
[Crossref] [PubMed]

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.

Evans, A. J.

L. Pantanowitz, J. H. Sinard, W. H. Henricks, L. A. Fatheree, A. B. Carter, L. Contis, B. A. Beckwith, A. J. Evans, A. Lal, A. V. Parwani, and College of American Pathologists Pathology and Laboratory Quality Center, “Validating whole slide imaging for diagnostic purposes in pathology: guideline from the College of American Pathologists Pathology and Laboratory Quality Center,” Arch. Pathol. Lab. Med. 137(12), 1710–1722 (2013).
[Crossref] [PubMed]

Fan, Y.

Y. Fan, Y. Gal, and A. P. Bradley, “An algorithm for microscopic specimen delineation and focus candidate selection,” Micron 66, 51–62 (2014).
[Crossref] [PubMed]

Fatheree, L. A.

L. Pantanowitz, J. H. Sinard, W. H. Henricks, L. A. Fatheree, A. B. Carter, L. Contis, B. A. Beckwith, A. J. Evans, A. Lal, A. V. Parwani, and College of American Pathologists Pathology and Laboratory Quality Center, “Validating whole slide imaging for diagnostic purposes in pathology: guideline from the College of American Pathologists Pathology and Laboratory Quality Center,” Arch. Pathol. Lab. Med. 137(12), 1710–1722 (2013).
[Crossref] [PubMed]

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]

Gal, Y.

Y. Fan, Y. Gal, and A. P. Bradley, “An algorithm for microscopic specimen delineation and focus candidate selection,” Micron 66, 51–62 (2014).
[Crossref] [PubMed]

Gilbertson, J. R.

J. R. Gilbertson, J. Ho, L. Anthony, D. M. Jukic, Y. Yagi, and A. V. Parwani, “Primary histologic diagnosis using automated whole slide imaging: a validation study,” BMC Clin. Pathol. 6(1), 4 (2006).
[Crossref] [PubMed]

Guo, K.

Heng, X.

Henricks, W. H.

L. Pantanowitz, J. H. Sinard, W. H. Henricks, L. A. Fatheree, A. B. Carter, L. Contis, B. A. Beckwith, A. J. Evans, A. Lal, A. V. Parwani, and College of American Pathologists Pathology and Laboratory Quality Center, “Validating whole slide imaging for diagnostic purposes in pathology: guideline from the College of American Pathologists Pathology and Laboratory Quality Center,” Arch. Pathol. Lab. Med. 137(12), 1710–1722 (2013).
[Crossref] [PubMed]

Ho, J.

J. R. Gilbertson, J. Ho, L. Anthony, D. M. Jukic, Y. Yagi, and A. V. Parwani, “Primary histologic diagnosis using automated whole slide imaging: a validation study,” BMC Clin. Pathol. 6(1), 4 (2006).
[Crossref] [PubMed]

Jukic, D. M.

J. R. Gilbertson, J. Ho, L. Anthony, D. M. Jukic, Y. Yagi, and A. V. Parwani, “Primary histologic diagnosis using automated whole slide imaging: a validation study,” BMC Clin. Pathol. 6(1), 4 (2006).
[Crossref] [PubMed]

Kenny, K. B.

Lal, A.

L. Pantanowitz, J. H. Sinard, W. H. Henricks, L. A. Fatheree, A. B. Carter, L. Contis, B. A. Beckwith, A. J. Evans, A. Lal, A. V. Parwani, and College of American Pathologists Pathology and Laboratory Quality Center, “Validating whole slide imaging for diagnostic purposes in pathology: guideline from the College of American Pathologists Pathology and Laboratory Quality Center,” Arch. Pathol. Lab. Med. 137(12), 1710–1722 (2013).
[Crossref] [PubMed]

Liao, J.

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]

R. R. McKay, V. A. Baxi, and M. C. Montalto, “The accuracy of dynamic predictive autofocusing for whole slide imaging,” J. Pathol. Inform. 2(1), 38 (2011).
[Crossref] [PubMed]

Montalto, M. C.

R. R. McKay, V. A. Baxi, and M. C. Montalto, “The accuracy of dynamic predictive autofocusing for whole slide imaging,” J. Pathol. Inform. 2(1), 38 (2011).
[Crossref] [PubMed]

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]

Pantanowitz, L.

L. Pantanowitz, J. H. Sinard, W. H. Henricks, L. A. Fatheree, A. B. Carter, L. Contis, B. A. Beckwith, A. J. Evans, A. Lal, A. V. Parwani, and College of American Pathologists Pathology and Laboratory Quality Center, “Validating whole slide imaging for diagnostic purposes in pathology: guideline from the College of American Pathologists Pathology and Laboratory Quality Center,” Arch. Pathol. Lab. Med. 137(12), 1710–1722 (2013).
[Crossref] [PubMed]

Parwani, A. V.

L. Pantanowitz, J. H. Sinard, W. H. Henricks, L. A. Fatheree, A. B. Carter, L. Contis, B. A. Beckwith, A. J. Evans, A. Lal, A. V. Parwani, and College of American Pathologists Pathology and Laboratory Quality Center, “Validating whole slide imaging for diagnostic purposes in pathology: guideline from the College of American Pathologists Pathology and Laboratory Quality Center,” Arch. Pathol. Lab. Med. 137(12), 1710–1722 (2013).
[Crossref] [PubMed]

J. R. Gilbertson, J. Ho, L. Anthony, D. M. Jukic, Y. Yagi, and A. V. Parwani, “Primary histologic diagnosis using automated whole slide imaging: a validation study,” BMC Clin. Pathol. 6(1), 4 (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]

Sinard, J. H.

L. Pantanowitz, J. H. Sinard, W. H. Henricks, L. A. Fatheree, A. B. Carter, L. Contis, B. A. Beckwith, A. J. Evans, A. Lal, A. V. Parwani, and College of American Pathologists Pathology and Laboratory Quality Center, “Validating whole slide imaging for diagnostic purposes in pathology: guideline from the College of American Pathologists Pathology and Laboratory Quality Center,” Arch. Pathol. Lab. Med. 137(12), 1710–1722 (2013).
[Crossref] [PubMed]

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.

Yagi, Y.

J. R. Gilbertson, J. Ho, L. Anthony, D. M. Jukic, Y. Yagi, and A. V. Parwani, “Primary histologic diagnosis using automated whole slide imaging: a validation study,” BMC Clin. Pathol. 6(1), 4 (2006).
[Crossref] [PubMed]

Yazdanfar, S.

Zheng, G.

Arch. Pathol. Lab. Med. (1)

L. Pantanowitz, J. H. Sinard, W. H. Henricks, L. A. Fatheree, A. B. Carter, L. Contis, B. A. Beckwith, A. J. Evans, A. Lal, A. V. Parwani, and College of American Pathologists Pathology and Laboratory Quality Center, “Validating whole slide imaging for diagnostic purposes in pathology: guideline from the College of American Pathologists Pathology and Laboratory Quality Center,” Arch. Pathol. Lab. Med. 137(12), 1710–1722 (2013).
[Crossref] [PubMed]

Biomed. Opt. Express (1)

BMC Clin. Pathol. (1)

J. R. Gilbertson, J. Ho, L. Anthony, D. M. Jukic, Y. Yagi, and A. V. Parwani, “Primary histologic diagnosis using automated whole slide imaging: a validation study,” BMC Clin. Pathol. 6(1), 4 (2006).
[Crossref] [PubMed]

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]

J. Pathol. Inform. (2)

R. R. McKay, V. A. Baxi, and M. C. Montalto, “The accuracy of dynamic predictive autofocusing for whole slide imaging,” J. Pathol. Inform. 2(1), 38 (2011).
[Crossref] [PubMed]

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]

Micron (1)

Y. Fan, Y. Gal, and A. P. Bradley, “An algorithm for microscopic specimen delineation and focus candidate selection,” Micron 66, 51–62 (2014).
[Crossref] [PubMed]

Opt. Express (1)

Other (1)

B. D. Lucas and T. Kanade, “An iterative image registration technique with an application to stereo vision,” in IJCAI, 1981), 674–679.

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Figures (4)

Fig. 1
Fig. 1

The single-frame autofocusing scheme. (a) The microscope setup, where the autofocusing module is attached at the epi-illumination arm. (b) The working principle of the single-frame autofocusing scheme. The captured image from the autofocusing camera contains two copies of the object and we can recover the defocus distance based on the translation shift between the two copies.

Fig. 2
Fig. 2

The procedures for recovering the translation shift from a single captured image z[x]. (a1) The captured image z[x] from the autofocusing camera. (a2) The Fourier power spectrum of the captured image (we took the log scale to better visualize the fringe pattern). (a3) The autocorrelation function R(z[x]), which can be computed by taking the inverse Fourier transform of (a2). (a4) The line trace of (a3) and the locations of the peaks. (b) The condition for resolving the first-order peaks.

Fig. 3
Fig. 3

The autofocusing performance of our scheme. (a) Achieving a sub-pixel accuracy of the translational shift estimation. (b) The focusing error on 5 samples and 1550 different tiles. (c) Summary of the autofocusing performance. We used a 10-point Brenner gradient method to determine the ground truth position. The average focusing error is ~0.11 DOF, ~3 folds better than the conventional image-contrast-based method.

Fig. 4
Fig. 4

The fluorescence images of a breast cancer (top) and an unstained mouse kidney section (bottom). The full images can be found from http://gigapan.com/profiles/SmartImagingLab.

Equations (2)

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R(z[ x ]) = R( s[ x ] ) * R( h[ x ] ) = R( s[ x ] ) *(2δ[ x ] + δ[ x x 0 ] + δ[ x + x 0 ]),
x 0 · f cutoff > 1.56,

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