Abstract

A simple image-based autofocusing scheme for digital microscopy is demonstrated that uses as few as two intermediate images to bring the sample into focus. The algorithm is adapted to a commercial inverted microscope and used to automate brightfield and fluorescence imaging of histopathology tissue sections.

© 2008 Optical Society of America

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  1. R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
    [CrossRef]
  2. M. G. Rojo, G. B. Garcia, C. P. Mateos, J. G. Garcia, and M. C. Vicente, "Critical comparison of 31 commercially available digital slide systems in pathology," Intl. J. Surg. Pathol. 14, 285-305 (2006)
    [CrossRef]
  3. D. L. Taylor, E. S. Woo, and K. A. Giuliano, "Real-time molecular and cellular analysis: the new frontier of drug discovery," Curr. Opin. Biotechnol. 8, 1085-1093 (2001)
  4. R. L. Camp, G. C. Chung, and D. L. Rimm, "Automated subcellular localization and quantification of protein expression in tissue microarrays," Nat. Med. 8, 1323-1327 (2002)
    [CrossRef] [PubMed]
  5. Y. Sun, S. Duthaler, and B. J. Nelson, "Autofocusing in computer microscopy: Selecting the optimal focus algorithm," Microsc. Res. Tech. 65, 139-149 (2004)
    [CrossRef] [PubMed]
  6. L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston, "Comparison of autofocus methods for automated microscopy," Cytometry 12, 195-206 (1991)
    [CrossRef] [PubMed]
  7. V. Della Mea, F. Viel, and C. A. Beltrami, "A pixel-based autofocusing technique for digital histologic and cytologic slides," Comput. Med. Imag. Grap. 29, 333-341 (2005)
    [CrossRef]
  8. Y. Liron, Y. Paran, G. Zatorsky, B. Geiger, and Z. Kam, "Laser autofocusing for high-resolution cell biological imaging," J. Microsc. 221, 145-151 (2006)
    [CrossRef] [PubMed]
  9. S. K. Nayar, and Y. Nakagawa, "Shape from focus," IEEE Trans. Pattern Anal. Machine Intell. 16, 824-831 (1994)
    [CrossRef]
  10. J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, "An automated microscope for cytologic research a preliminary evaluation," J. Histochem. Cytochem. 24, 100-111 (1976)
    [CrossRef] [PubMed]
  11. <jrn>11. X. Y. Liu, W. H. Wang, and Y. Sun, "Dynamic evaluation of autofocusing for automated microscopic analysis of blood smear and pap smear," J. Microsc. 227, 15-23(2007).</jrn>
    [CrossRef] [PubMed]

2006 (2)

M. G. Rojo, G. B. Garcia, C. P. Mateos, J. G. Garcia, and M. C. Vicente, "Critical comparison of 31 commercially available digital slide systems in pathology," Intl. J. Surg. Pathol. 14, 285-305 (2006)
[CrossRef]

Y. Liron, Y. Paran, G. Zatorsky, B. Geiger, and Z. Kam, "Laser autofocusing for high-resolution cell biological imaging," J. Microsc. 221, 145-151 (2006)
[CrossRef] [PubMed]

2005 (1)

V. Della Mea, F. Viel, and C. A. Beltrami, "A pixel-based autofocusing technique for digital histologic and cytologic slides," Comput. Med. Imag. Grap. 29, 333-341 (2005)
[CrossRef]

2004 (1)

Y. Sun, S. Duthaler, and B. J. Nelson, "Autofocusing in computer microscopy: Selecting the optimal focus algorithm," Microsc. Res. Tech. 65, 139-149 (2004)
[CrossRef] [PubMed]

2002 (1)

R. L. Camp, G. C. Chung, and D. L. Rimm, "Automated subcellular localization and quantification of protein expression in tissue microarrays," Nat. Med. 8, 1323-1327 (2002)
[CrossRef] [PubMed]

2001 (2)

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

D. L. Taylor, E. S. Woo, and K. A. Giuliano, "Real-time molecular and cellular analysis: the new frontier of drug discovery," Curr. Opin. Biotechnol. 8, 1085-1093 (2001)

1994 (1)

S. K. Nayar, and Y. Nakagawa, "Shape from focus," IEEE Trans. Pattern Anal. Machine Intell. 16, 824-831 (1994)
[CrossRef]

1991 (1)

L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston, "Comparison of autofocus methods for automated microscopy," Cytometry 12, 195-206 (1991)
[CrossRef] [PubMed]

1976 (1)

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, "An automated microscope for cytologic research a preliminary evaluation," J. Histochem. Cytochem. 24, 100-111 (1976)
[CrossRef] [PubMed]

Beltrami, C. A.

V. Della Mea, F. Viel, and C. A. Beltrami, "A pixel-based autofocusing technique for digital histologic and cytologic slides," Comput. Med. Imag. Grap. 29, 333-341 (2005)
[CrossRef]

Bhattacharyya, A. K.

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

Brenner, J. F.

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, "An automated microscope for cytologic research a preliminary evaluation," J. Histochem. Cytochem. 24, 100-111 (1976)
[CrossRef] [PubMed]

Camp, R. L.

R. L. Camp, G. C. Chung, and D. L. Rimm, "Automated subcellular localization and quantification of protein expression in tissue microarrays," Nat. Med. 8, 1323-1327 (2002)
[CrossRef] [PubMed]

Chung, G. C.

R. L. Camp, G. C. Chung, and D. L. Rimm, "Automated subcellular localization and quantification of protein expression in tissue microarrays," Nat. Med. 8, 1323-1327 (2002)
[CrossRef] [PubMed]

Cook, K.

L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston, "Comparison of autofocus methods for automated microscopy," Cytometry 12, 195-206 (1991)
[CrossRef] [PubMed]

Culp, K.

L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston, "Comparison of autofocus methods for automated microscopy," Cytometry 12, 195-206 (1991)
[CrossRef] [PubMed]

Davis, J. R.

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

Della Mea, V.

V. Della Mea, F. Viel, and C. A. Beltrami, "A pixel-based autofocusing technique for digital histologic and cytologic slides," Comput. Med. Imag. Grap. 29, 333-341 (2005)
[CrossRef]

Descour, M. R.

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

Dew, B. S.

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, "An automated microscope for cytologic research a preliminary evaluation," J. Histochem. Cytochem. 24, 100-111 (1976)
[CrossRef] [PubMed]

Dunn, B. E.

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

Duthaler, S.

Y. Sun, S. Duthaler, and B. J. Nelson, "Autofocusing in computer microscopy: Selecting the optimal focus algorithm," Microsc. Res. Tech. 65, 139-149 (2004)
[CrossRef] [PubMed]

Firestone, L.

L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston, "Comparison of autofocus methods for automated microscopy," Cytometry 12, 195-206 (1991)
[CrossRef] [PubMed]

Garcia, G. B.

M. G. Rojo, G. B. Garcia, C. P. Mateos, J. G. Garcia, and M. C. Vicente, "Critical comparison of 31 commercially available digital slide systems in pathology," Intl. J. Surg. Pathol. 14, 285-305 (2006)
[CrossRef]

Garcia, J. G.

M. G. Rojo, G. B. Garcia, C. P. Mateos, J. G. Garcia, and M. C. Vicente, "Critical comparison of 31 commercially available digital slide systems in pathology," Intl. J. Surg. Pathol. 14, 285-305 (2006)
[CrossRef]

Geiger, B.

Y. Liron, Y. Paran, G. Zatorsky, B. Geiger, and Z. Kam, "Laser autofocusing for high-resolution cell biological imaging," J. Microsc. 221, 145-151 (2006)
[CrossRef] [PubMed]

Giuliano, K. A.

D. L. Taylor, E. S. Woo, and K. A. Giuliano, "Real-time molecular and cellular analysis: the new frontier of drug discovery," Curr. Opin. Biotechnol. 8, 1085-1093 (2001)

Graham, A. R.

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

Horton, J. B.

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, "An automated microscope for cytologic research a preliminary evaluation," J. Histochem. Cytochem. 24, 100-111 (1976)
[CrossRef] [PubMed]

Kam, Z.

Y. Liron, Y. Paran, G. Zatorsky, B. Geiger, and Z. Kam, "Laser autofocusing for high-resolution cell biological imaging," J. Microsc. 221, 145-151 (2006)
[CrossRef] [PubMed]

Kayser, K.

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

King, T.

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, "An automated microscope for cytologic research a preliminary evaluation," J. Histochem. Cytochem. 24, 100-111 (1976)
[CrossRef] [PubMed]

Krupinski, E. A.

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

Liang, C.

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

Liron, Y.

Y. Liron, Y. Paran, G. Zatorsky, B. Geiger, and Z. Kam, "Laser autofocusing for high-resolution cell biological imaging," J. Microsc. 221, 145-151 (2006)
[CrossRef] [PubMed]

Mateos, C. P.

M. G. Rojo, G. B. Garcia, C. P. Mateos, J. G. Garcia, and M. C. Vicente, "Critical comparison of 31 commercially available digital slide systems in pathology," Intl. J. Surg. Pathol. 14, 285-305 (2006)
[CrossRef]

Nakagawa, Y.

S. K. Nayar, and Y. Nakagawa, "Shape from focus," IEEE Trans. Pattern Anal. Machine Intell. 16, 824-831 (1994)
[CrossRef]

Nayar, S. K.

S. K. Nayar, and Y. Nakagawa, "Shape from focus," IEEE Trans. Pattern Anal. Machine Intell. 16, 824-831 (1994)
[CrossRef]

Nelson, B. J.

Y. Sun, S. Duthaler, and B. J. Nelson, "Autofocusing in computer microscopy: Selecting the optimal focus algorithm," Microsc. Res. Tech. 65, 139-149 (2004)
[CrossRef] [PubMed]

Neurath, P. W.

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, "An automated microscope for cytologic research a preliminary evaluation," J. Histochem. Cytochem. 24, 100-111 (1976)
[CrossRef] [PubMed]

Paran, Y.

Y. Liron, Y. Paran, G. Zatorsky, B. Geiger, and Z. Kam, "Laser autofocusing for high-resolution cell biological imaging," J. Microsc. 221, 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, 195-206 (1991)
[CrossRef] [PubMed]

Richter, L.

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

Rimm, D. L.

R. L. Camp, G. C. Chung, and D. L. Rimm, "Automated subcellular localization and quantification of protein expression in tissue microarrays," Nat. Med. 8, 1323-1327 (2002)
[CrossRef] [PubMed]

Rojo, M. G.

M. G. Rojo, G. B. Garcia, C. P. Mateos, J. G. Garcia, and M. C. Vicente, "Critical comparison of 31 commercially available digital slide systems in pathology," Intl. J. Surg. Pathol. 14, 285-305 (2006)
[CrossRef]

Scott, K. M.

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

Selles, W. D.

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, "An automated microscope for cytologic research a preliminary evaluation," J. Histochem. Cytochem. 24, 100-111 (1976)
[CrossRef] [PubMed]

Sun, Y.

Y. Sun, S. Duthaler, and B. J. Nelson, "Autofocusing in computer microscopy: Selecting the optimal focus algorithm," Microsc. Res. Tech. 65, 139-149 (2004)
[CrossRef] [PubMed]

Szymus, J.

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

Talsania, N.

L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston, "Comparison of autofocus methods for automated microscopy," Cytometry 12, 195-206 (1991)
[CrossRef] [PubMed]

Taylor, D. L.

D. L. Taylor, E. S. Woo, and K. A. Giuliano, "Real-time molecular and cellular analysis: the new frontier of drug discovery," Curr. Opin. Biotechnol. 8, 1085-1093 (2001)

Vicente, M. C.

M. G. Rojo, G. B. Garcia, C. P. Mateos, J. G. Garcia, and M. C. Vicente, "Critical comparison of 31 commercially available digital slide systems in pathology," Intl. J. Surg. Pathol. 14, 285-305 (2006)
[CrossRef]

Viel, F.

V. Della Mea, F. Viel, and C. A. Beltrami, "A pixel-based autofocusing technique for digital histologic and cytologic slides," Comput. Med. Imag. Grap. 29, 333-341 (2005)
[CrossRef]

Weinstein, R. S.

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

Woo, E. S.

D. L. Taylor, E. S. Woo, and K. A. Giuliano, "Real-time molecular and cellular analysis: the new frontier of drug discovery," Curr. Opin. Biotechnol. 8, 1085-1093 (2001)

Zatorsky, G.

Y. Liron, Y. Paran, G. Zatorsky, B. Geiger, and Z. Kam, "Laser autofocusing for high-resolution cell biological imaging," J. Microsc. 221, 145-151 (2006)
[CrossRef] [PubMed]

Comput. Med. Imag. Grap. (1)

V. Della Mea, F. Viel, and C. A. Beltrami, "A pixel-based autofocusing technique for digital histologic and cytologic slides," Comput. Med. Imag. Grap. 29, 333-341 (2005)
[CrossRef]

Curr. Opin. Biotechnol. (1)

D. L. Taylor, E. S. Woo, and K. A. Giuliano, "Real-time molecular and cellular analysis: the new frontier of drug discovery," Curr. Opin. Biotechnol. 8, 1085-1093 (2001)

Cytometry (1)

L. Firestone, K. Cook, K. Culp, N. Talsania, and K. Preston, "Comparison of autofocus methods for automated microscopy," Cytometry 12, 195-206 (1991)
[CrossRef] [PubMed]

Hum. Pathol. (1)

R. S. Weinstein, M. R. Descour, C. Liang, A. K. Bhattacharyya, A. R. Graham, J. R. Davis, K. M. Scott, L. Richter, E. A. Krupinski, J. Szymus, K. Kayser, and B. E. Dunn, "Telepathology overview: From concept to implementation," Hum. Pathol. 32, 1283-1299 (2001)
[CrossRef]

IEEE Trans. Pattern Anal. Machine Intell. (1)

S. K. Nayar, and Y. Nakagawa, "Shape from focus," IEEE Trans. Pattern Anal. Machine Intell. 16, 824-831 (1994)
[CrossRef]

Intl. J. Surg. Pathol. (1)

M. G. Rojo, G. B. Garcia, C. P. Mateos, J. G. Garcia, and M. C. Vicente, "Critical comparison of 31 commercially available digital slide systems in pathology," Intl. J. Surg. Pathol. 14, 285-305 (2006)
[CrossRef]

J. Histochem. Cytochem. (1)

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, "An automated microscope for cytologic research a preliminary evaluation," J. Histochem. Cytochem. 24, 100-111 (1976)
[CrossRef] [PubMed]

J. Microsc. (1)

Y. Liron, Y. Paran, G. Zatorsky, B. Geiger, and Z. Kam, "Laser autofocusing for high-resolution cell biological imaging," J. Microsc. 221, 145-151 (2006)
[CrossRef] [PubMed]

Microsc. Res. Tech. (1)

Y. Sun, S. Duthaler, and B. J. Nelson, "Autofocusing in computer microscopy: Selecting the optimal focus algorithm," Microsc. Res. Tech. 65, 139-149 (2004)
[CrossRef] [PubMed]

Nat. Med. (1)

R. L. Camp, G. C. Chung, and D. L. Rimm, "Automated subcellular localization and quantification of protein expression in tissue microarrays," Nat. Med. 8, 1323-1327 (2002)
[CrossRef] [PubMed]

Other (1)

<jrn>11. X. Y. Liu, W. H. Wang, and Y. Sun, "Dynamic evaluation of autofocusing for automated microscopic analysis of blood smear and pap smear," J. Microsc. 227, 15-23(2007).</jrn>
[CrossRef] [PubMed]

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Figures (6)

Fig. 1.
Fig. 1.

Comparison of autofocusing figures-of-merit, calculated from an image stack through focus, and normalized by the peak. The Brenner gradient [10] is acutely sensitive to focus, monotonically decreasing and symmetric about the peak, and contains no prominent local maxima outside of the peak.

Fig. 2.
Fig. 2.

(A). A Lorentzian fit of the Brenner gradient as a function of depth indicates that the sample was located 3.53 um from optimal focus. (B). Using only three images from the image stack in (A), the reciprocal of the Brenner gradient is used to calculate the focus using a parabolic fit, which resulted in a focus position of 3.99 um. The difference of 0.46 um is less than the depth-of-focus of the objective used. Insets indicate raw images (grayscale) at the positions designated by the arrows. The Brenner gradient at each position corresponds to the sum of the image data in the black-and-white images.

Fig. 3.
Fig. 3.

The algorithm exhibits single-shot convergence. Following the first image, starting ~10 µm out of focus, the autofocusing algorithm was applied four times, as described above using three auxiliary images each time. When repeatedly applied at the same position, it brings the sample into focus on the first attempt, with negligible z-axis motion in subsequent attempts. There is also negligible change in image quality after the sample comes into focus. A plot of the Brenner gradient versus image number shows little change following the initial attempt.

Fig. 4.
Fig. 4.

Automated acquisition of a histopathology image mosaic, comprised of 441 images (13.65×13.65 mm2). Each step in magnification is by a factor of four.

Fig. 5.
Fig. 5.

Autofocusing is independent of tissue type and staining, as indicated by A) breast cancer H&E section, B) colon H&E section with high dye uptake, C) colon H&E section labeled only with eosin, D) immunoperoxidase labelled pancreatic beta cells (DAB with hematoxylin counterstain), and E) a portion of H&E stained tissue micro-array (TMA). The high magnification inset in A) and TMA portions indicated by arrows show examples of resolvable nuclear texture.

Fig. 6.
Fig. 6.

Autofocusing performed on fluorescently labeled colon samples: A) Cy3 labeled b-catenin, B) nuclear labeling with DAPI, C) dual labeling of smooth muscle actin (Cy3, red) and nuclei (DAPI, green).

Equations (5)

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C = s max s min s max + s min
V = 1 μ i = 1 N j = 1 M [ s ( i , j ) μ ] 2
E = k L p k log ( p k )
B = i = 1 N j = 1 M [ s ( i , j ) s ( i + m , j ) ] 2
f ( z ) = α β + ( z z 0 ) 2

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