Abstract

In this paper we present a method for autofocusing images of sputum smears taken from a microscope which combines the finding of the optimal focus distance with an algorithm for extending the depth of field (EDoF). Our multifocus fusion method produces an unique image where all the relevant objects of the analyzed scene are well focused, independently to their distance to the sensor. This process is computationally expensive which makes unfeasible its automation using traditional embedded processors. For this purpose a low-cost optimized implementation is proposed using limited resources embedded GPU integrated on cutting-edge NVIDIA system on chip. The extensive tests performed on different sputum smear image sets show the real-time capabilities of our implementation maintaining the quality of the output image.

© 2017 Optical Society of America

Full Article  |  PDF Article

Corrections

27 February 2017: A correction was made to the funding section.


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References

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  1. J.-M. Geusebroek, F. Cornelissen, A. W. Smeulders, and H. Geerts, “Robust autofocusing in microscopy,” Cytometry 39, 1–9 (2000).
    [Crossref] [PubMed]
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    [PubMed]
  3. A. N. Murali Subbarao and T. Choi, “Focusing Techniques,” Journal of Optical Engineering
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    [Crossref]
  5. S. Allegro, C. Chanel, and J. Jacot, “Autofocus for automated microassembly under a microscope,” Proceedings of 3rd IEEE International Conference on Image Processing1, 677–680 (1996).
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  7. S. Jin, J. Cho, K. H. Kwon, and J. W. Jeon, “A dedicated hardware architecture for real-time auto-focusing using an FPGA,” Machine Vision and Applications 21, 727–734 (2010).
    [Crossref]
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    [Crossref] [PubMed]
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  10. J. Valdiviezo, J. Hernández-Tapia, L. Mera-González, C. Toxqui-Quitl, and A. Padilla-Vivanco, “Autofocusing in microscopy systems using graphics processing units,” Proc. SPIE 8856, 88560 (2013).
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    [Crossref] [PubMed]
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  13. H. Kang, S. Lee, E. Lee, S. Kim, and T. Lee, “Real-Time GPU-Accelerated Processing and Volumetric Display for Wide-Field Laser-Scanning Optical-Resolution Photoacoustic Microscopy,” Biomed. Opt. Express 6, 4650 (2015).
    [Crossref] [PubMed]
  14. J. M. Mateos-Pérez, R. Redondo, R. Nava, J. C. Valdiviezo, G. Cristóbal, B. Escalante-Ramírez, M. J. Ruiz-Serrano, J. Pascau, and M. Desco, “Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis,” Cytometry Part A 81A, 213–221 (2012).
    [Crossref]
  15. R. Wheeler, “Extended Depth of Field,” ImageJ plugin, http://www.richardwheeler.net . Last access Nov. (2016).
  16. NVIDIA Maxwell, “ https://developer.nvidia.com/maxwell-compute-architecture . Last access Nov. 2016.”.
  17. OpenCV4Tegra, “ http://elinux.org/Jetson/Computer_Vision_Performance . Last access Nov. 2016.”.
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    [Crossref]
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    [Crossref]

2015 (2)

2013 (2)

M. Doğar, H. A. İlhan, and M. Özcan, “Real-time, auto-focusing digital holographic microscope using graphics processors,” Rev. Sci. Instrum. 84, 083704 (2013).
[Crossref] [PubMed]

J. Valdiviezo, J. Hernández-Tapia, L. Mera-González, C. Toxqui-Quitl, and A. Padilla-Vivanco, “Autofocusing in microscopy systems using graphics processing units,” Proc. SPIE 8856, 88560 (2013).

2012 (1)

J. M. Mateos-Pérez, R. Redondo, R. Nava, J. C. Valdiviezo, G. Cristóbal, B. Escalante-Ramírez, M. J. Ruiz-Serrano, J. Pascau, and M. Desco, “Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis,” Cytometry Part A 81A, 213–221 (2012).
[Crossref]

2010 (2)

C.-Y. Chen, R.-C. Hwang, and Y.-J. Chen, “A passive auto-focus camera control system,” Applied Soft Computing 10, 296–303 (2010).
[Crossref]

S. Jin, J. Cho, K. H. Kwon, and J. W. Jeon, “A dedicated hardware architecture for real-time auto-focusing using an FPGA,” Machine Vision and Applications 21, 727–734 (2010).
[Crossref]

2008 (2)

2007 (1)

2005 (1)

A. Goshtasby, “Fusion of multi-exposure images,” Image and Vision Computing 23, 611–618 (2005).
[Crossref]

2000 (1)

J.-M. Geusebroek, F. Cornelissen, A. W. Smeulders, and H. Geerts, “Robust autofocusing in microscopy,” Cytometry 39, 1–9 (2000).
[Crossref] [PubMed]

1999 (1)

K. Veropoulos, G. Learmonth, C. Campbell, B. Knight, and J. Simpson, “Automated identification of tubercle bacilli in sputum. A preliminary investigation,” Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 21, 277–282 (1999).
[PubMed]

1996 (1)

K.H Esbesen, K.H. Hjelmen, and K. Kvaal, “The AMT approach in chemometrics-first forays,” J. Chemometrics 10, 569–590 (1996).
[Crossref]

Allegro, S.

S. Allegro, C. Chanel, and J. Jacot, “Autofocus for automated microassembly under a microscope,” Proceedings of 3rd IEEE International Conference on Image Processing1, 677–680 (1996).

Bian, Z.

Campbell, C.

K. Veropoulos, G. Learmonth, C. Campbell, B. Knight, and J. Simpson, “Automated identification of tubercle bacilli in sputum. A preliminary investigation,” Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 21, 277–282 (1999).
[PubMed]

Chanel, C.

S. Allegro, C. Chanel, and J. Jacot, “Autofocus for automated microassembly under a microscope,” Proceedings of 3rd IEEE International Conference on Image Processing1, 677–680 (1996).

Chen, C.-Y.

C.-Y. Chen, R.-C. Hwang, and Y.-J. Chen, “A passive auto-focus camera control system,” Applied Soft Computing 10, 296–303 (2010).
[Crossref]

Chen, Y.-J.

C.-Y. Chen, R.-C. Hwang, and Y.-J. Chen, “A passive auto-focus camera control system,” Applied Soft Computing 10, 296–303 (2010).
[Crossref]

Cho, J.

S. Jin, J. Cho, K. H. Kwon, and J. W. Jeon, “A dedicated hardware architecture for real-time auto-focusing using an FPGA,” Machine Vision and Applications 21, 727–734 (2010).
[Crossref]

Choi, T.

A. N. Murali Subbarao and T. Choi, “Focusing Techniques,” Journal of Optical Engineering

Cornelissen, F.

J.-M. Geusebroek, F. Cornelissen, A. W. Smeulders, and H. Geerts, “Robust autofocusing in microscopy,” Cytometry 39, 1–9 (2000).
[Crossref] [PubMed]

Corwin, A.

Cristobal, G.

Cristóbal, G.

J. M. Mateos-Pérez, R. Redondo, R. Nava, J. C. Valdiviezo, G. Cristóbal, B. Escalante-Ramírez, M. J. Ruiz-Serrano, J. Pascau, and M. Desco, “Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis,” Cytometry Part A 81A, 213–221 (2012).
[Crossref]

Desco, M.

J. M. Mateos-Pérez, R. Redondo, R. Nava, J. C. Valdiviezo, G. Cristóbal, B. Escalante-Ramírez, M. J. Ruiz-Serrano, J. Pascau, and M. Desco, “Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis,” Cytometry Part A 81A, 213–221 (2012).
[Crossref]

Dixon, E.

Dogar, M.

M. Doğar, H. A. İlhan, and M. Özcan, “Real-time, auto-focusing digital holographic microscope using graphics processors,” Rev. Sci. Instrum. 84, 083704 (2013).
[Crossref] [PubMed]

Esbesen, K.H

K.H Esbesen, K.H. Hjelmen, and K. Kvaal, “The AMT approach in chemometrics-first forays,” J. Chemometrics 10, 569–590 (1996).
[Crossref]

Escalante-Ramírez, B.

J. M. Mateos-Pérez, R. Redondo, R. Nava, J. C. Valdiviezo, G. Cristóbal, B. Escalante-Ramírez, M. J. Ruiz-Serrano, J. Pascau, and M. Desco, “Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis,” Cytometry Part A 81A, 213–221 (2012).
[Crossref]

Filkins, R.

Gabarda, S.

Geerts, H.

J.-M. Geusebroek, F. Cornelissen, A. W. Smeulders, and H. Geerts, “Robust autofocusing in microscopy,” Cytometry 39, 1–9 (2000).
[Crossref] [PubMed]

Geusebroek, J.-M.

J.-M. Geusebroek, F. Cornelissen, A. W. Smeulders, and H. Geerts, “Robust autofocusing in microscopy,” Cytometry 39, 1–9 (2000).
[Crossref] [PubMed]

Goshtasby, A.

A. Goshtasby, “Fusion of multi-exposure images,” Image and Vision Computing 23, 611–618 (2005).
[Crossref]

Guo, K.

Guojin, C.

Z. Miaofen, W. Wanqiang, C. Guojin, and L. Yongning, “Image focusing system based on FPGA,” 2010 International Symposium on Computer, Communication, Control and Automation (3CA)1, 415–418 (2010).

Heng, X.

Hernández-Tapia, J.

J. Valdiviezo, J. Hernández-Tapia, L. Mera-González, C. Toxqui-Quitl, and A. Padilla-Vivanco, “Autofocusing in microscopy systems using graphics processing units,” Proc. SPIE 8856, 88560 (2013).

Hjelmen, K.H.

K.H Esbesen, K.H. Hjelmen, and K. Kvaal, “The AMT approach in chemometrics-first forays,” J. Chemometrics 10, 569–590 (1996).
[Crossref]

Hwang, R.-C.

C.-Y. Chen, R.-C. Hwang, and Y.-J. Chen, “A passive auto-focus camera control system,” Applied Soft Computing 10, 296–303 (2010).
[Crossref]

Ilhan, H. A.

M. Doğar, H. A. İlhan, and M. Özcan, “Real-time, auto-focusing digital holographic microscope using graphics processors,” Rev. Sci. Instrum. 84, 083704 (2013).
[Crossref] [PubMed]

Ito, T.

Jacot, J.

S. Allegro, C. Chanel, and J. Jacot, “Autofocus for automated microassembly under a microscope,” Proceedings of 3rd IEEE International Conference on Image Processing1, 677–680 (1996).

Jeon, J. W.

S. Jin, J. Cho, K. H. Kwon, and J. W. Jeon, “A dedicated hardware architecture for real-time auto-focusing using an FPGA,” Machine Vision and Applications 21, 727–734 (2010).
[Crossref]

Jin, S.

S. Jin, J. Cho, K. H. Kwon, and J. W. Jeon, “A dedicated hardware architecture for real-time auto-focusing using an FPGA,” Machine Vision and Applications 21, 727–734 (2010).
[Crossref]

Kang, H.

Kenny, K.

Kim, S.

Knight, B.

K. Veropoulos, G. Learmonth, C. Campbell, B. Knight, and J. Simpson, “Automated identification of tubercle bacilli in sputum. A preliminary investigation,” Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 21, 277–282 (1999).
[PubMed]

Kvaal, K.

K.H Esbesen, K.H. Hjelmen, and K. Kvaal, “The AMT approach in chemometrics-first forays,” J. Chemometrics 10, 569–590 (1996).
[Crossref]

Kwon, K. H.

S. Jin, J. Cho, K. H. Kwon, and J. W. Jeon, “A dedicated hardware architecture for real-time auto-focusing using an FPGA,” Machine Vision and Applications 21, 727–734 (2010).
[Crossref]

Learmonth, G.

K. Veropoulos, G. Learmonth, C. Campbell, B. Knight, and J. Simpson, “Automated identification of tubercle bacilli in sputum. A preliminary investigation,” Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 21, 277–282 (1999).
[PubMed]

Lee, E.

Lee, S.

Lee, T.

Liao, J.

Mateos-Pérez, J. M.

J. M. Mateos-Pérez, R. Redondo, R. Nava, J. C. Valdiviezo, G. Cristóbal, B. Escalante-Ramírez, M. J. Ruiz-Serrano, J. Pascau, and M. Desco, “Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis,” Cytometry Part A 81A, 213–221 (2012).
[Crossref]

Mera-González, L.

J. Valdiviezo, J. Hernández-Tapia, L. Mera-González, C. Toxqui-Quitl, and A. Padilla-Vivanco, “Autofocusing in microscopy systems using graphics processing units,” Proc. SPIE 8856, 88560 (2013).

Miaofen, Z.

Z. Miaofen, W. Wanqiang, C. Guojin, and L. Yongning, “Image focusing system based on FPGA,” 2010 International Symposium on Computer, Communication, Control and Automation (3CA)1, 415–418 (2010).

Miura, J.

Nava, R.

J. M. Mateos-Pérez, R. Redondo, R. Nava, J. C. Valdiviezo, G. Cristóbal, B. Escalante-Ramírez, M. J. Ruiz-Serrano, J. Pascau, and M. Desco, “Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis,” Cytometry Part A 81A, 213–221 (2012).
[Crossref]

Özcan, M.

M. Doğar, H. A. İlhan, and M. Özcan, “Real-time, auto-focusing digital holographic microscope using graphics processors,” Rev. Sci. Instrum. 84, 083704 (2013).
[Crossref] [PubMed]

Padilla-Vivanco, A.

J. Valdiviezo, J. Hernández-Tapia, L. Mera-González, C. Toxqui-Quitl, and A. Padilla-Vivanco, “Autofocusing in microscopy systems using graphics processing units,” Proc. SPIE 8856, 88560 (2013).

Pascau, J.

J. M. Mateos-Pérez, R. Redondo, R. Nava, J. C. Valdiviezo, G. Cristóbal, B. Escalante-Ramírez, M. J. Ruiz-Serrano, J. Pascau, and M. Desco, “Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis,” Cytometry Part A 81A, 213–221 (2012).
[Crossref]

Redondo, R.

J. M. Mateos-Pérez, R. Redondo, R. Nava, J. C. Valdiviezo, G. Cristóbal, B. Escalante-Ramírez, M. J. Ruiz-Serrano, J. Pascau, and M. Desco, “Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis,” Cytometry Part A 81A, 213–221 (2012).
[Crossref]

Ruiz-Serrano, M. J.

J. M. Mateos-Pérez, R. Redondo, R. Nava, J. C. Valdiviezo, G. Cristóbal, B. Escalante-Ramírez, M. J. Ruiz-Serrano, J. Pascau, and M. Desco, “Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis,” Cytometry Part A 81A, 213–221 (2012).
[Crossref]

Sato, Y.

Shimobaba, T.

Simpson, J.

K. Veropoulos, G. Learmonth, C. Campbell, B. Knight, and J. Simpson, “Automated identification of tubercle bacilli in sputum. A preliminary investigation,” Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 21, 277–282 (1999).
[PubMed]

Smeulders, A. W.

J.-M. Geusebroek, F. Cornelissen, A. W. Smeulders, and H. Geerts, “Robust autofocusing in microscopy,” Cytometry 39, 1–9 (2000).
[Crossref] [PubMed]

Subbarao, A. N. Murali

A. N. Murali Subbarao and T. Choi, “Focusing Techniques,” Journal of Optical Engineering

Takenouchi, M.

Tasimi, K.

Toxqui-Quitl, C.

J. Valdiviezo, J. Hernández-Tapia, L. Mera-González, C. Toxqui-Quitl, and A. Padilla-Vivanco, “Autofocusing in microscopy systems using graphics processing units,” Proc. SPIE 8856, 88560 (2013).

Valdiviezo, J.

J. Valdiviezo, J. Hernández-Tapia, L. Mera-González, C. Toxqui-Quitl, and A. Padilla-Vivanco, “Autofocusing in microscopy systems using graphics processing units,” Proc. SPIE 8856, 88560 (2013).

Valdiviezo, J. C.

J. M. Mateos-Pérez, R. Redondo, R. Nava, J. C. Valdiviezo, G. Cristóbal, B. Escalante-Ramírez, M. J. Ruiz-Serrano, J. Pascau, and M. Desco, “Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis,” Cytometry Part A 81A, 213–221 (2012).
[Crossref]

Veropoulos, K.

K. Veropoulos, G. Learmonth, C. Campbell, B. Knight, and J. Simpson, “Automated identification of tubercle bacilli in sputum. A preliminary investigation,” Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 21, 277–282 (1999).
[PubMed]

Wanqiang, W.

Z. Miaofen, W. Wanqiang, C. Guojin, and L. Yongning, “Image focusing system based on FPGA,” 2010 International Symposium on Computer, Communication, Control and Automation (3CA)1, 415–418 (2010).

Yazdanfar, S.

Yongning, L.

Z. Miaofen, W. Wanqiang, C. Guojin, and L. Yongning, “Image focusing system based on FPGA,” 2010 International Symposium on Computer, Communication, Control and Automation (3CA)1, 415–418 (2010).

Zheng, G.

Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology (1)

K. Veropoulos, G. Learmonth, C. Campbell, B. Knight, and J. Simpson, “Automated identification of tubercle bacilli in sputum. A preliminary investigation,” Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 21, 277–282 (1999).
[PubMed]

Applied Soft Computing (1)

C.-Y. Chen, R.-C. Hwang, and Y.-J. Chen, “A passive auto-focus camera control system,” Applied Soft Computing 10, 296–303 (2010).
[Crossref]

Biomed. Opt. Express (2)

Cytometry (1)

J.-M. Geusebroek, F. Cornelissen, A. W. Smeulders, and H. Geerts, “Robust autofocusing in microscopy,” Cytometry 39, 1–9 (2000).
[Crossref] [PubMed]

Cytometry Part A (1)

J. M. Mateos-Pérez, R. Redondo, R. Nava, J. C. Valdiviezo, G. Cristóbal, B. Escalante-Ramírez, M. J. Ruiz-Serrano, J. Pascau, and M. Desco, “Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis,” Cytometry Part A 81A, 213–221 (2012).
[Crossref]

Image and Vision Computing (1)

A. Goshtasby, “Fusion of multi-exposure images,” Image and Vision Computing 23, 611–618 (2005).
[Crossref]

J. Chemometrics (1)

K.H Esbesen, K.H. Hjelmen, and K. Kvaal, “The AMT approach in chemometrics-first forays,” J. Chemometrics 10, 569–590 (1996).
[Crossref]

J. Opt. Soc. Am. A (1)

Machine Vision and Applications (1)

S. Jin, J. Cho, K. H. Kwon, and J. W. Jeon, “A dedicated hardware architecture for real-time auto-focusing using an FPGA,” Machine Vision and Applications 21, 727–734 (2010).
[Crossref]

Opt. Express (2)

Proc. SPIE (1)

J. Valdiviezo, J. Hernández-Tapia, L. Mera-González, C. Toxqui-Quitl, and A. Padilla-Vivanco, “Autofocusing in microscopy systems using graphics processing units,” Proc. SPIE 8856, 88560 (2013).

Rev. Sci. Instrum. (1)

M. Doğar, H. A. İlhan, and M. Özcan, “Real-time, auto-focusing digital holographic microscope using graphics processors,” Rev. Sci. Instrum. 84, 083704 (2013).
[Crossref] [PubMed]

Other (6)

S. Allegro, C. Chanel, and J. Jacot, “Autofocus for automated microassembly under a microscope,” Proceedings of 3rd IEEE International Conference on Image Processing1, 677–680 (1996).

Z. Miaofen, W. Wanqiang, C. Guojin, and L. Yongning, “Image focusing system based on FPGA,” 2010 International Symposium on Computer, Communication, Control and Automation (3CA)1, 415–418 (2010).

A. N. Murali Subbarao and T. Choi, “Focusing Techniques,” Journal of Optical Engineering

R. Wheeler, “Extended Depth of Field,” ImageJ plugin, http://www.richardwheeler.net . Last access Nov. (2016).

NVIDIA Maxwell, “ https://developer.nvidia.com/maxwell-compute-architecture . Last access Nov. 2016.”.

OpenCV4Tegra, “ http://elinux.org/Jetson/Computer_Vision_Performance . Last access Nov. 2016.”.

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Figures (7)

Fig. 1
Fig. 1 Workflow of Multifocus Fusion method.
Fig. 2
Fig. 2 (a) Original autofocus image; (b) Example of particle cleaning performed by the top-hat algorithm with a square 9 × 9 mask.
Fig. 3
Fig. 3 Example of a 8-Stack of images obtained by a microscope (converted to 8-bit grayscale) with a stepsize of 3µm (1-8) and the final result of multifocus (9). Scale bars represent 30µm.
Fig. 4
Fig. 4 (a) The Angular Measure Spectrum produces a scale-spectrum that reflects the signal complexity on all possible scales simultaneously. The figure represents the AMT spectrum corresponding to a TB autofocus image (labeled ’original’) and defocused images with Gaussian blur (σ = 4; σ = 8); (b) Normalized focus measures of the 30 selected stacks corresponding to the autofocused and multifocus fused images. Bars represent mean values and error bars the standard deviation.
Fig. 5
Fig. 5 Execution time in seconds on Multicore and GPU baseline implementations. The values represent the average among VOL4, MDCT and TEN algorithms grouped by image size.
Fig. 6
Fig. 6 Optimized Multicore/GPU implementation.
Fig. 7
Fig. 7 Execution time for each stage, (a) Stack Reduction stage and (b) Extended Depth of Field stage. In (a) the three methods VOL4, MDCT and TEN are compared for three sizes of image. In (b) the comparison is made for the same three image sizes in (a) and four levels of fusioned images.

Metrics