Abstract

Autofocusing is a routine technique in redressing focus drift that occurs in time-lapse microscopic image acquisition. To date, most automatic microscopes are designed on the distance detection scheme to fulfill the autofocusing operation, which may suffer from the low contrast of the reflected signal due to the refractive index mismatch at the water/glass interface. To achieve high autofocusing speed with minimal motion artifacts, we developed a compact multi-band fluorescent microscope with an electrically tunable lens (ETL) device for autofocusing. A modified searching algorithm based on equidistant scanning and curve fitting is proposed, which no longer requires a single-peak focus curve and then efficiently restrains the impact of external disturbance. This technique enables us to achieve an autofocusing time of down to 170 ms and the reproductivity of over 97%. The imaging head of the microscope has dimensions of 12 cm × 12 cm × 6 cm. This portable instrument can easily fit inside standard incubators for real-time imaging of living specimens.

© 2015 Optical Society of America

Full Article  |  PDF Article
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References

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2015 (1)

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

2014 (1)

2013 (2)

J. L. Chen, O. A. Pfäffli, F. F. Voigt, D. J. Margolis, and F. Helmchen, “Online correction of licking-induced brain motion during two-photon imaging with a tunable lens,” J. Physiol. 591(19), 4689–4698 (2013).
[Crossref] [PubMed]

F. O. Fahrbach, F. F. Voigt, B. Schmid, F. Helmchen, and J. Huisken, “Rapid 3D light-sheet microscopy with a tunable lens,” Opt. Express 21(18), 21010–21026 (2013).
[Crossref] [PubMed]

2012 (2)

C. Geisler, T. Hotz, A. Schönle, S. W. Hell, A. Munk, and A. Egner, “Drift estimation for single marker switching based imaging schemes,” Opt. Express 20(7), 7274–7289 (2012).
[Crossref] [PubMed]

R. Redondo, G. Bueno, J. C. Valdiviezo, R. Nava, G. Cristóbal, O. Déniz, M. García-Rojo, J. Salido, M. M. Fernández, J. Vidal, and B. Escalante-Ramírez, “Autofocus evaluation for brightfield microscopy pathology,” J. Biomed. Opt. 17(3), 036008 (2012).
[Crossref] [PubMed]

2011 (3)

2010 (1)

O. A. Osibote, R. Dendere, S. Krishnan, and T. S. Douglas, “Automated focusing in bright-field microscopy for tuberculosis detection,” J. Microsc. 240(2), 155–163 (2010).
[Crossref] [PubMed]

2009 (4)

M. M. Frigault, J. Lacoste, J. L. Swift, and C. M. Brown, “Live-cell microscopy - tips and tools,” J. Cell Sci. 122(6), 753–767 (2009).
[Crossref] [PubMed]

H. Landecker, “Seeing things: from microcinematography to live cell imaging,” Nat. Methods 6(10), 707–709 (2009).
[Crossref] [PubMed]

M. Humar, M. Ravnik, S. Pajk, and I. Muševič, “Electrically tunable liquid crystal optical microresonators,” Nat. Photonics 3(10), 595–600 (2009).
[Crossref]

M. Zeder and J. Pernthaler, “Multispot live-image autofocusing for high-throughput microscopy of fluorescently stained bacteria,” Cytometry A 75(9), 781–788 (2009).
[Crossref] [PubMed]

2008 (1)

2007 (1)

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(1), 15–23 (2007).
[Crossref] [PubMed]

2006 (2)

2005 (1)

M. Kreft, M. Stenovec, and R. Zorec, “Focus-drift correction in time-lapse confocal imaging,” Ann. N. Y. Acad. Sci. 1048(1), 321–330 (2005).
[Crossref] [PubMed]

1997 (1)

W. Böcker, W. Rolf, W. U. Müller, and C. Streffer, “A fast autofocus unit for fluorescence microscopy,” Phys. Med. Biol. 42(10), 1981–1992 (1997).
[Crossref] [PubMed]

1994 (1)

S. K. Nayar and Y. Nakagawa, “Shape from focus,” Pattern Anal. Machine Intell. 16(8), 824–831 (1994).
[Crossref]

Andilla, J.

Artigas, D.

Aviles-Espinosa, R.

Azucena, O.

Böcker, W.

W. Böcker, W. Rolf, W. U. Müller, and C. Streffer, “A fast autofocus unit for fluorescence microscopy,” Phys. Med. Biol. 42(10), 1981–1992 (1997).
[Crossref] [PubMed]

Boilot, V.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Brown, C. M.

M. M. Frigault, J. Lacoste, J. L. Swift, and C. M. Brown, “Live-cell microscopy - tips and tools,” J. Cell Sci. 122(6), 753–767 (2009).
[Crossref] [PubMed]

Bueno, G.

R. Redondo, G. Bueno, J. C. Valdiviezo, R. Nava, G. Cristóbal, O. Déniz, M. García-Rojo, J. Salido, M. M. Fernández, J. Vidal, and B. Escalante-Ramírez, “Autofocus evaluation for brightfield microscopy pathology,” J. Biomed. Opt. 17(3), 036008 (2012).
[Crossref] [PubMed]

Chen, D. C.

Chen, J. L.

J. L. Chen, O. A. Pfäffli, F. F. Voigt, D. J. Margolis, and F. Helmchen, “Online correction of licking-induced brain motion during two-photon imaging with a tunable lens,” J. Physiol. 591(19), 4689–4698 (2013).
[Crossref] [PubMed]

Cheng, S.

Cheng, Y. S. L.

Clark, J.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Corwin, A. D.

Cristóbal, G.

R. Redondo, G. Bueno, J. C. Valdiviezo, R. Nava, G. Cristóbal, O. Déniz, M. García-Rojo, J. Salido, M. M. Fernández, J. Vidal, and B. Escalante-Ramírez, “Autofocus evaluation for brightfield microscopy pathology,” J. Biomed. Opt. 17(3), 036008 (2012).
[Crossref] [PubMed]

Cuenca, R.

Dendere, R.

O. A. Osibote, R. Dendere, S. Krishnan, and T. S. Douglas, “Automated focusing in bright-field microscopy for tuberculosis detection,” J. Microsc. 240(2), 155–163 (2010).
[Crossref] [PubMed]

Déniz, O.

R. Redondo, G. Bueno, J. C. Valdiviezo, R. Nava, G. Cristóbal, O. Déniz, M. García-Rojo, J. Salido, M. M. Fernández, J. Vidal, and B. Escalante-Ramírez, “Autofocus evaluation for brightfield microscopy pathology,” J. Biomed. Opt. 17(3), 036008 (2012).
[Crossref] [PubMed]

Dixon, E. L.

Douglas, T. S.

O. A. Osibote, R. Dendere, S. Krishnan, and T. S. Douglas, “Automated focusing in bright-field microscopy for tuberculosis detection,” J. Microsc. 240(2), 155–163 (2010).
[Crossref] [PubMed]

Draviam, V. M.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Egner, A.

Escalante-Ramírez, B.

R. Redondo, G. Bueno, J. C. Valdiviezo, R. Nava, G. Cristóbal, O. Déniz, M. García-Rojo, J. Salido, M. M. Fernández, J. Vidal, and B. Escalante-Ramírez, “Autofocus evaluation for brightfield microscopy pathology,” J. Biomed. Opt. 17(3), 036008 (2012).
[Crossref] [PubMed]

Fahrbach, F. O.

Fernández, M. M.

R. Redondo, G. Bueno, J. C. Valdiviezo, R. Nava, G. Cristóbal, O. Déniz, M. García-Rojo, J. Salido, M. M. Fernández, J. Vidal, and B. Escalante-Ramírez, “Autofocus evaluation for brightfield microscopy pathology,” J. Biomed. Opt. 17(3), 036008 (2012).
[Crossref] [PubMed]

Filkins, R. J.

Frigault, M. M.

M. M. Frigault, J. Lacoste, J. L. Swift, and C. M. Brown, “Live-cell microscopy - tips and tools,” J. Cell Sci. 122(6), 753–767 (2009).
[Crossref] [PubMed]

Fu, M.

Funahashi, A.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

García-Rojo, M.

R. Redondo, G. Bueno, J. C. Valdiviezo, R. Nava, G. Cristóbal, O. Déniz, M. García-Rojo, J. Salido, M. M. Fernández, J. Vidal, and B. Escalante-Ramírez, “Autofocus evaluation for brightfield microscopy pathology,” J. Biomed. Opt. 17(3), 036008 (2012).
[Crossref] [PubMed]

Geisler, C.

Grewe, B. F.

Hahn, K.

F. Shen, L. Hodgson, and K. Hahn, “Digital autofocus methods for automated microscopy,” Methods Enzymol. 414, 620–632 (2006).
[Crossref] [PubMed]

Hell, S. W.

Helmchen, F.

Hiraiwa, T.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Hiroi, N.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Hodgson, L.

F. Shen, L. Hodgson, and K. Hahn, “Digital autofocus methods for automated microscopy,” Methods Enzymol. 414, 620–632 (2006).
[Crossref] [PubMed]

Hotz, T.

Huisken, J.

Humar, M.

M. Humar, M. Ravnik, S. Pajk, and I. Muševič, “Electrically tunable liquid crystal optical microresonators,” Nat. Photonics 3(10), 595–600 (2009).
[Crossref]

Jabbour, J. M.

Jo, J. A.

Kenny, K. B.

Kreft, M.

M. Kreft, M. Stenovec, and R. Zorec, “Focus-drift correction in time-lapse confocal imaging,” Ann. N. Y. Acad. Sci. 1048(1), 321–330 (2005).
[Crossref] [PubMed]

Krishnan, S.

O. A. Osibote, R. Dendere, S. Krishnan, and T. S. Douglas, “Automated focusing in bright-field microscopy for tuberculosis detection,” J. Microsc. 240(2), 155–163 (2010).
[Crossref] [PubMed]

Kubby, J.

Lacoste, J.

M. M. Frigault, J. Lacoste, J. L. Swift, and C. M. Brown, “Live-cell microscopy - tips and tools,” J. Cell Sci. 122(6), 753–767 (2009).
[Crossref] [PubMed]

Landecker, H.

H. Landecker, “Seeing things: from microcinematography to live cell imaging,” Nat. Methods 6(10), 707–709 (2009).
[Crossref] [PubMed]

Lavrentovich, O. D.

Levecq, X.

Liu, X. Y.

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(1), 15–23 (2007).
[Crossref] [PubMed]

Loza-Alvarez, P.

Maitland, K. C.

Malik, B. H.

Margolis, D. J.

J. L. Chen, O. A. Pfäffli, F. F. Voigt, D. J. Margolis, and F. Helmchen, “Online correction of licking-induced brain motion during two-photon imaging with a tunable lens,” J. Physiol. 591(19), 4689–4698 (2013).
[Crossref] [PubMed]

Müller, W. U.

W. Böcker, W. Rolf, W. U. Müller, and C. Streffer, “A fast autofocus unit for fluorescence microscopy,” Phys. Med. Biol. 42(10), 1981–1992 (1997).
[Crossref] [PubMed]

Munk, A.

Muševic, I.

M. Humar, M. Ravnik, S. Pajk, and I. Muševič, “Electrically tunable liquid crystal optical microresonators,” Nat. Photonics 3(10), 595–600 (2009).
[Crossref]

Nakagawa, Y.

S. K. Nayar and Y. Nakagawa, “Shape from focus,” Pattern Anal. Machine Intell. 16(8), 824–831 (1994).
[Crossref]

Nakai, Y.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Nava, R.

R. Redondo, G. Bueno, J. C. Valdiviezo, R. Nava, G. Cristóbal, O. Déniz, M. García-Rojo, J. Salido, M. M. Fernández, J. Vidal, and B. Escalante-Ramírez, “Autofocus evaluation for brightfield microscopy pathology,” J. Biomed. Opt. 17(3), 036008 (2012).
[Crossref] [PubMed]

Nayar, S. K.

S. K. Nayar and Y. Nakagawa, “Shape from focus,” Pattern Anal. Machine Intell. 16(8), 824–831 (1994).
[Crossref]

Nieto, M.

Nonaka, S.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Oku, H.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Olarte, O. E.

Olsovsky, C.

Osibote, O. A.

O. A. Osibote, R. Dendere, S. Krishnan, and T. S. Douglas, “Automated focusing in bright-field microscopy for tuberculosis detection,” J. Microsc. 240(2), 155–163 (2010).
[Crossref] [PubMed]

Ozeki, M.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Pajk, S.

M. Humar, M. Ravnik, S. Pajk, and I. Muševič, “Electrically tunable liquid crystal optical microresonators,” Nat. Photonics 3(10), 595–600 (2009).
[Crossref]

Pernthaler, J.

M. Zeder and J. Pernthaler, “Multispot live-image autofocusing for high-throughput microscopy of fluorescently stained bacteria,” Cytometry A 75(9), 781–788 (2009).
[Crossref] [PubMed]

Pfäffli, O. A.

J. L. Chen, O. A. Pfäffli, F. F. Voigt, D. J. Margolis, and F. Helmchen, “Online correction of licking-induced brain motion during two-photon imaging with a tunable lens,” J. Physiol. 591(19), 4689–4698 (2013).
[Crossref] [PubMed]

Pishnyak, O.

Porcar-Guezenec, R.

Ravnik, M.

M. Humar, M. Ravnik, S. Pajk, and I. Muševič, “Electrically tunable liquid crystal optical microresonators,” Nat. Photonics 3(10), 595–600 (2009).
[Crossref]

Redondo, R.

R. Redondo, G. Bueno, J. C. Valdiviezo, R. Nava, G. Cristóbal, O. Déniz, M. García-Rojo, J. Salido, M. M. Fernández, J. Vidal, and B. Escalante-Ramírez, “Autofocus evaluation for brightfield microscopy pathology,” J. Biomed. Opt. 17(3), 036008 (2012).
[Crossref] [PubMed]

Rolf, W.

W. Böcker, W. Rolf, W. U. Müller, and C. Streffer, “A fast autofocus unit for fluorescence microscopy,” Phys. Med. Biol. 42(10), 1981–1992 (1997).
[Crossref] [PubMed]

Salido, J.

R. Redondo, G. Bueno, J. C. Valdiviezo, R. Nava, G. Cristóbal, O. Déniz, M. García-Rojo, J. Salido, M. M. Fernández, J. Vidal, and B. Escalante-Ramírez, “Autofocus evaluation for brightfield microscopy pathology,” J. Biomed. Opt. 17(3), 036008 (2012).
[Crossref] [PubMed]

Sato, S.

Schmid, B.

Schönle, A.

Shen, F.

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[Crossref] [PubMed]

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M. Kreft, M. Stenovec, and R. Zorec, “Focus-drift correction in time-lapse confocal imaging,” Ann. N. Y. Acad. Sci. 1048(1), 321–330 (2005).
[Crossref] [PubMed]

Streffer, C.

W. Böcker, W. Rolf, W. U. Müller, and C. Streffer, “A fast autofocus unit for fluorescence microscopy,” Phys. Med. Biol. 42(10), 1981–1992 (1997).
[Crossref] [PubMed]

Sun, Y.

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(1), 15–23 (2007).
[Crossref] [PubMed]

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M. M. Frigault, J. Lacoste, J. L. Swift, and C. M. Brown, “Live-cell microscopy - tips and tools,” J. Cell Sci. 122(6), 753–767 (2009).
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Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Taniguchi, A.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Tanimoto, R.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
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Tasimi, K.

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Vidal, J.

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Wang, W. H.

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(1), 15–23 (2007).
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M. Zeder and J. Pernthaler, “Multispot live-image autofocusing for high-throughput microscopy of fluorescently stained bacteria,” Cytometry A 75(9), 781–788 (2009).
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Ann. N. Y. Acad. Sci. (1)

M. Kreft, M. Stenovec, and R. Zorec, “Focus-drift correction in time-lapse confocal imaging,” Ann. N. Y. Acad. Sci. 1048(1), 321–330 (2005).
[Crossref] [PubMed]

Appl. Opt. (1)

Biomed. Opt. Express (3)

Cytometry A (1)

M. Zeder and J. Pernthaler, “Multispot live-image autofocusing for high-throughput microscopy of fluorescently stained bacteria,” Cytometry A 75(9), 781–788 (2009).
[Crossref] [PubMed]

J. Biomed. Opt. (1)

R. Redondo, G. Bueno, J. C. Valdiviezo, R. Nava, G. Cristóbal, O. Déniz, M. García-Rojo, J. Salido, M. M. Fernández, J. Vidal, and B. Escalante-Ramírez, “Autofocus evaluation for brightfield microscopy pathology,” J. Biomed. Opt. 17(3), 036008 (2012).
[Crossref] [PubMed]

J. Cell Sci. (1)

M. M. Frigault, J. Lacoste, J. L. Swift, and C. M. Brown, “Live-cell microscopy - tips and tools,” J. Cell Sci. 122(6), 753–767 (2009).
[Crossref] [PubMed]

J. Microsc. (2)

O. A. Osibote, R. Dendere, S. Krishnan, and T. S. Douglas, “Automated focusing in bright-field microscopy for tuberculosis detection,” J. Microsc. 240(2), 155–163 (2010).
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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(1), 15–23 (2007).
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J. Physiol. (1)

J. L. Chen, O. A. Pfäffli, F. F. Voigt, D. J. Margolis, and F. Helmchen, “Online correction of licking-induced brain motion during two-photon imaging with a tunable lens,” J. Physiol. 591(19), 4689–4698 (2013).
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Methods Enzymol. (1)

F. Shen, L. Hodgson, and K. Hahn, “Digital autofocus methods for automated microscopy,” Methods Enzymol. 414, 620–632 (2006).
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Nat. Methods (1)

H. Landecker, “Seeing things: from microcinematography to live cell imaging,” Nat. Methods 6(10), 707–709 (2009).
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Opt. Express (3)

Opt. Lett. (1)

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[Crossref]

Phys. Med. Biol. (1)

W. Böcker, W. Rolf, W. U. Müller, and C. Streffer, “A fast autofocus unit for fluorescence microscopy,” Phys. Med. Biol. 42(10), 1981–1992 (1997).
[Crossref] [PubMed]

Rev. Sci. Instrum. (1)

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Other (5)

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Supplementary Material (1)

NameDescription
» Visualization 1: MOV (5521 KB)      Main interface of the autofocusing software

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Figures (12)

Fig. 1
Fig. 1

Schematic of the compact multi-band fluorescent microscope with an ETL for autofocusing. (a) Light path diagram of the microscope imaging head. F1: excitation filter, F2: emission filter, ETL: electrically tunable lens, OL: offset lens, MO: microscope objective, M1 and M2: mirrors, TL: tube lens, DM1 and DM2: dichroic mirrors. (b) and (c) are the diagrams of the setup in upright and inverted geometries, respectively. (d) Photograph of the microscope instrument with an upright arrangement.

Fig. 2
Fig. 2

Photographs of the fiber-coupled output 4-wavelength LEDs array light source. (a) The 4-wavelength LEDs array module. (b) Fiber-coupled output LEDs head mounted on a 65 mm long heatsink. (c) Power supply and controller of the LED package. (d) SMA fiber collimator connecting to the input port of microscope.

Fig. 3
Fig. 3

Principle illustration of the ETL [21]. (a) An intuitional view of the structure of the ETL. Container wrapped by the polymer is filled with specially-produced optical fluid. (b) A photograph of the ETL. (c) Illustration of shifting focal plane (FP) by adjusting the control current (Ii). Different from the axial scanning control mechanism by using stages, this configuration avoids motion artifacts and the shifting range varies with the magnification and numerical aperture of the objective.

Fig. 4
Fig. 4

Characterization of variation tendency of focal position and relative magnification during tuning the control current. (a) Measured focal position of the 10 × objective lens as a function of the ETL control current. (b) Measured focal position of the 40 × objective lens as a function of the ETL control current. (c) Measured relative magnification as a function of the ETL control current.

Fig. 5
Fig. 5

Profile of an ideal focus curve. Focus values are calculated with the images captured at different axial positions. The focus curve reaches the maximum at the focal plane and decreases rapidly and monotonously with defocusing.

Fig. 6
Fig. 6

Calibration of the focus curve to compensate the nonlinear relationship between the focal position and the control current of ETL. (a) The original focus curve. (b) Nonlinear relationship between the control current and the relative axial position. (c) Calibrated focus curve obtained by mapping the relationship of control current and the focus value.

Fig. 7
Fig. 7

Measured multi-peaks focus curve. Multiple peaks caused by multi-layer specimen and jitters caused by system instability are the main reasons for local maximums, which probably lead to a mistaken convergence of the searching algorithm.

Fig. 8
Fig. 8

Flowchart of the promoted searching scheme. The whole process could be divided into two stages. In the first stage, raw images are captured at even-spaced control currents and corresponding focus values are calculated. Maximal point and its four neighbors are preserved for later use. In the second stage, the five points are assigned into two groups. F(I) in the flow chart presents the function in Eq. (4) and a, b, and z0 are the corresponding unknown coefficients. By fitting the measured points to F(I), the unknown coefficients in F(I) can be determined. Further, via solving their stationary points of the determined functions, we can get the maximum points of the fitted curves IM1 and IM2. Arithmetic mean value of the two is set as the final control current.

Fig. 9
Fig. 9

Color-fused fluorescent images of multi-dye labeled specimens. (a) The mouse kidney cells excited by the wavelengths of 405 and 470 nm successively, observed with the 10 × objective. (b) The BPAE cells excited by the wavelengths of 405, 470 and 565 nm successively, observed with the 40 × objective.

Fig. 10
Fig. 10

Main interface of the autofocusing software (see Visualization 1 for demonstration).

Fig. 11
Fig. 11

Time-sequence chart of the autofocusing process based on the camera’s time shaft. Camera works in the trigger mode and is triggered periodly. One autofocusing process contains nine steps, and each step includes three events, i.e., camera exposure, FV calculation and ETL settling.

Fig. 12
Fig. 12

Statistic histogram of the final control currents for the reproductivity calibration. The whole test can be treated as an alternate process of autofocusing and defocusing between FP_A and FP_B. ΔIDOF here represents the difference in current that is used to tune a range of DOF around the center focal point. Reproductivity is defined as the percentage of the control current falling into the range of ΔIDOF.

Tables (1)

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Table 1 Classification of autofocusing algorithms

Equations (5)

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z 10× =1.12× 10 5 I 2 +8.59× 10 3 I,
z 40× =4.10× 10 7 I 2 +5.56× 10 4 I,
f(z)=a e (z z 0 ) 2 2 b 2 ,
F(I)=a e (p I 2 +qI z 0 ) 2 2 b 2 ,
DOF= λn N A 2 + 2nP M 2 (NA) ,

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