Abstract

We propose a simple and compact microscope combining phase imaging with multi-color fluorescence using a standard bright-field objective. The phase image of the sample is reconstructed from a single, approximately 100 μm out-of-focus image taken under semi-coherent illumination, while fluorescence is recorded in-focus in epi-fluorescence geometry. The reproducible changes of the focus are achieved with specifically introduced chromatic aberration in the imaging system. This allows us to move the focal plane simply by changing the imaging wavelength. No mechanical movement of neither sample nor objective or any other part of the setup is therefore required to alternate between the imaging modality. Due to its small size and the absence of motorized components the microscope can easily be used inside a standard biological incubator and allows long-term imaging of cell culture in physiological conditions. A field-of-view of 1.2 mm2 allows simultaneous observation of thousands of cells with micro-meter spatial resolution in phase and multi-channel fluorescence mode. In this manuscript we characterize the system and show a time-lapse of cell culture in phase and multi-channel fluorescence recorded inside an incubator. We believe that the small dimensions, easy usage and low cost of the system make it a useful tool for biological research.

© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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References

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2019 (2)

I. de Kernier, A. Ali-Cherif, N. Rongeat, O. Cioni, S. Morales, and J. Savatier, “Large field-of-view phase and fluorescence mesoscope with microscopic resolution,” J. Biomed. Opt. 24(03), 1–9 (2019).
[Crossref]

C. Allier, L. Hervé, O. Mandula, P. Blandin, Y. Usson, J. Savatier, S. Monneret, and S. Morales, “Quantitative phase imaging of adherent mammalian cells: a comparative study,” Biomed. Opt. Express 10(6), 2768–2783 (2019).
[Crossref]

2018 (3)

2016 (1)

W. Yu, X. Tian, X. He, X. Song, L. Xue, C. Liu, and S. Wang, “Real time quantitative phase microscopy based on single-shot transport of intensity equation (sstie) method,” Appl. Phys. Lett. 109(7), 071112 (2016).
[Crossref]

2015 (1)

X. Quan, K. Nitta, O. Matoba, P. Xia, and Y. Awatsuji, “Phase and fluorescence imaging by combination of digital holographic microscopy and fluorescence microscopy,” Opt. Rev. 22(2), 349–353 (2015).
[Crossref]

2014 (1)

2013 (1)

2012 (2)

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, and J.Y. Tinevez, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

A. Greenbaum and A. Ozcan, “Maskless imaging of dense samples using pixel super-resolution based multi-height lensfree on-chip microscopy,” Opt. Express 20(3), 3129–3143 (2012).
[Crossref]

2011 (1)

2010 (1)

2006 (1)

2005 (1)

C. Mollinari, J.-P. Kleman, Y. Saoudi, S. A. Jablonski, J. Perard, T. J. Yen, and R. L. Margolis, “Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not,” Mol. Biol. Cell 16(3), 1043–1055 (2005).
[Crossref]

2004 (2)

U. Agero, L. Mesquita, B. Neves, R. Gazzinelli, and O. Mesquita, “Defocusing microscopy,” Microsc. Res. Tech. 65(3), 159–165 (2004).
[Crossref]

C. J. R. Sheppard, “Defocused transfer function for a partially coherent microscope and application to phase retrieval,” J. Opt. Soc. Am. A 21(5), 828–831 (2004).
[Crossref]

2003 (1)

U. Agero, C. H. Monken, C. Ropert, R. T. Gazzinelli, and O. N. Mesquita, “Cell surface fluctuations studied with defocusing microscopy,” Phys. Rev. E 67(5), 051904 (2003).
[Crossref]

2002 (1)

D. Paganin, S. C. Mayo, T. E. Gureyev, P. R. Miller, and S. W. Wilkins, “Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object,” J. Microsc. 206(1), 33–40 (2002).
[Crossref]

1999 (1)

P. Cloetens, W. Ludwig, J. Baruchel, D. Van Dyck, J. Van Landuyt, J. P. Guigay, and M. Schlenker, “Holotomography: Quantitative phase tomography with micrometer resolution using hard synchrotron radiation x rays,” Appl. Phys. Lett. 75(19), 2912–2914 (1999).
[Crossref]

1992 (1)

M. A. Browne, O. Akinyemi, and A. Boyde, “Confocal surface profiling utilizing chromatic aberration,” Scanning 14(3), 145–153 (1992).
[Crossref]

1985 (1)

1984 (2)

N. Streibl, “Phase imaging by the transport equation of intensity,” Opt. Commun. 49(1), 6–10 (1984).
[Crossref]

G. Molesini, G. Pedrini, P. Poggi, and F. Quercioli, “Focus-wavelength encoded optical profilometer,” Opt. Commun. 49(4), 229–233 (1984).
[Crossref]

1983 (1)

S. Sternberg, “Biomedical image processing,” Computer 16(1), 22–34 (1983).
[Crossref]

1955 (2)

G. Nomarski, “Differential microinterferometer with polarized waves,” J. Phys. Radium 16, 9S–11S (1955).

F. Zernike, “How I Discovered Phase Contrast,” Science 121(3141), 345–349 (1955).
[Crossref]

1942 (1)

F. Zernike, “Phase contrast, a new method for the microscopic observation of transparent objects,” Physica 9(7), 686–698 (1942).
[Crossref]

Agero, U.

U. Agero, L. Mesquita, B. Neves, R. Gazzinelli, and O. Mesquita, “Defocusing microscopy,” Microsc. Res. Tech. 65(3), 159–165 (2004).
[Crossref]

U. Agero, C. H. Monken, C. Ropert, R. T. Gazzinelli, and O. N. Mesquita, “Cell surface fluctuations studied with defocusing microscopy,” Phys. Rev. E 67(5), 051904 (2003).
[Crossref]

Akinyemi, O.

M. A. Browne, O. Akinyemi, and A. Boyde, “Confocal surface profiling utilizing chromatic aberration,” Scanning 14(3), 145–153 (1992).
[Crossref]

Ali-Cherif, A.

I. de Kernier, A. Ali-Cherif, N. Rongeat, O. Cioni, S. Morales, and J. Savatier, “Large field-of-view phase and fluorescence mesoscope with microscopic resolution,” J. Biomed. Opt. 24(03), 1–9 (2019).
[Crossref]

Allier, C.

C. Allier, L. Hervé, O. Mandula, P. Blandin, Y. Usson, J. Savatier, S. Monneret, and S. Morales, “Quantitative phase imaging of adherent mammalian cells: a comparative study,” Biomed. Opt. Express 10(6), 2768–2783 (2019).
[Crossref]

O. Mandula, C. Allier, L. Hervé, E. Denarier, A. Fourest-Lieuvin, S. Gory-Fauré, A. Vinit, and S. Morales, “Phase from defocus,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (SPIE, 2018), pp. 112–123.

L. Herve, O. Cioni, P. Blandin, F. Navarro, M. Menneteau, T. Bordy, S. Morales, and C. Allier, “Multispectral total-variation reconstruction applied to lens-free microscopy,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (OSA, 2018), pp. 5828–5836.

Arganda-Carreras, I.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, and J.Y. Tinevez, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Asundi, A.

Awatsuji, Y.

X. Quan, K. Nitta, O. Matoba, P. Xia, and Y. Awatsuji, “Phase and fluorescence imaging by combination of digital holographic microscopy and fluorescence microscopy,” Opt. Rev. 22(2), 349–353 (2015).
[Crossref]

Badizadegan, K.

Barbastathis, G.

Baruchel, J.

P. Cloetens, W. Ludwig, J. Baruchel, D. Van Dyck, J. Van Landuyt, J. P. Guigay, and M. Schlenker, “Holotomography: Quantitative phase tomography with micrometer resolution using hard synchrotron radiation x rays,” Appl. Phys. Lett. 75(19), 2912–2914 (1999).
[Crossref]

Baumbach, T.

Blandin, P.

C. Allier, L. Hervé, O. Mandula, P. Blandin, Y. Usson, J. Savatier, S. Monneret, and S. Morales, “Quantitative phase imaging of adherent mammalian cells: a comparative study,” Biomed. Opt. Express 10(6), 2768–2783 (2019).
[Crossref]

L. Herve, O. Cioni, P. Blandin, F. Navarro, M. Menneteau, T. Bordy, S. Morales, and C. Allier, “Multispectral total-variation reconstruction applied to lens-free microscopy,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (OSA, 2018), pp. 5828–5836.

Bohic, S.

Bordy, T.

L. Herve, O. Cioni, P. Blandin, F. Navarro, M. Menneteau, T. Bordy, S. Morales, and C. Allier, “Multispectral total-variation reconstruction applied to lens-free microscopy,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (OSA, 2018), pp. 5828–5836.

Born, M.

M. Born and E. Wolf, Principles of optics, vol. 1 (Cambridge University Press, 1999).

Boyde, A.

M. A. Browne, O. Akinyemi, and A. Boyde, “Confocal surface profiling utilizing chromatic aberration,” Scanning 14(3), 145–153 (1992).
[Crossref]

Browne, M. A.

M. A. Browne, O. Akinyemi, and A. Boyde, “Confocal surface profiling utilizing chromatic aberration,” Scanning 14(3), 145–153 (1992).
[Crossref]

Chen, Q.

Chu, K.

Q. Lu, G. Liu, C. Xiao, C. Hu, S. Zhang, R. X. Xu, K. Chu, Q. Xu, and Z. J. Smith, “A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings,” PLoS One 13(3), e0194063 (2018).
[Crossref]

Cioni, O.

I. de Kernier, A. Ali-Cherif, N. Rongeat, O. Cioni, S. Morales, and J. Savatier, “Large field-of-view phase and fluorescence mesoscope with microscopic resolution,” J. Biomed. Opt. 24(03), 1–9 (2019).
[Crossref]

L. Herve, O. Cioni, P. Blandin, F. Navarro, M. Menneteau, T. Bordy, S. Morales, and C. Allier, “Multispectral total-variation reconstruction applied to lens-free microscopy,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (OSA, 2018), pp. 5828–5836.

Cloetens, P.

F. Fus, Y. Yang, A. Pacureanu, S. Bohic, and P. Cloetens, “Unsupervised solution for in-line holography phase retrieval using Bayesian inference,” Opt. Express 26(25), 32847 (2018).
[Crossref]

P. Cloetens, W. Ludwig, J. Baruchel, D. Van Dyck, J. Van Landuyt, J. P. Guigay, and M. Schlenker, “Holotomography: Quantitative phase tomography with micrometer resolution using hard synchrotron radiation x rays,” Appl. Phys. Lett. 75(19), 2912–2914 (1999).
[Crossref]

Dasari, R. R.

de Kernier, I.

I. de Kernier, A. Ali-Cherif, N. Rongeat, O. Cioni, S. Morales, and J. Savatier, “Large field-of-view phase and fluorescence mesoscope with microscopic resolution,” J. Biomed. Opt. 24(03), 1–9 (2019).
[Crossref]

Denarier, E.

O. Mandula, C. Allier, L. Hervé, E. Denarier, A. Fourest-Lieuvin, S. Gory-Fauré, A. Vinit, and S. Morales, “Phase from defocus,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (SPIE, 2018), pp. 112–123.

Faridian, A.

Feld, M. S.

Fourest-Lieuvin, A.

O. Mandula, C. Allier, L. Hervé, E. Denarier, A. Fourest-Lieuvin, S. Gory-Fauré, A. Vinit, and S. Morales, “Phase from defocus,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (SPIE, 2018), pp. 112–123.

Frise, E.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, and J.Y. Tinevez, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Fus, F.

Gao, P.

Gazzinelli, R.

U. Agero, L. Mesquita, B. Neves, R. Gazzinelli, and O. Mesquita, “Defocusing microscopy,” Microsc. Res. Tech. 65(3), 159–165 (2004).
[Crossref]

Gazzinelli, R. T.

U. Agero, C. H. Monken, C. Ropert, R. T. Gazzinelli, and O. N. Mesquita, “Cell surface fluctuations studied with defocusing microscopy,” Phys. Rev. E 67(5), 051904 (2003).
[Crossref]

Goodman, J. W.

J. W. Goodman, Introduction to Fourier optics (Roberts and Company Publishers, 2005).

Gory-Fauré, S.

O. Mandula, C. Allier, L. Hervé, E. Denarier, A. Fourest-Lieuvin, S. Gory-Fauré, A. Vinit, and S. Morales, “Phase from defocus,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (SPIE, 2018), pp. 112–123.

Greenbaum, A.

Guigay, J. P.

P. Cloetens, W. Ludwig, J. Baruchel, D. Van Dyck, J. Van Landuyt, J. P. Guigay, and M. Schlenker, “Holotomography: Quantitative phase tomography with micrometer resolution using hard synchrotron radiation x rays,” Appl. Phys. Lett. 75(19), 2912–2914 (1999).
[Crossref]

Gureyev, T. E.

D. Paganin, S. C. Mayo, T. E. Gureyev, P. R. Miller, and S. W. Wilkins, “Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object,” J. Microsc. 206(1), 33–40 (2002).
[Crossref]

Haruta, M.

He, X.

W. Yu, X. Tian, X. He, X. Song, L. Xue, C. Liu, and S. Wang, “Real time quantitative phase microscopy based on single-shot transport of intensity equation (sstie) method,” Appl. Phys. Lett. 109(7), 071112 (2016).
[Crossref]

Herve, L.

L. Herve, O. Cioni, P. Blandin, F. Navarro, M. Menneteau, T. Bordy, S. Morales, and C. Allier, “Multispectral total-variation reconstruction applied to lens-free microscopy,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (OSA, 2018), pp. 5828–5836.

Hervé, L.

C. Allier, L. Hervé, O. Mandula, P. Blandin, Y. Usson, J. Savatier, S. Monneret, and S. Morales, “Quantitative phase imaging of adherent mammalian cells: a comparative study,” Biomed. Opt. Express 10(6), 2768–2783 (2019).
[Crossref]

O. Mandula, C. Allier, L. Hervé, E. Denarier, A. Fourest-Lieuvin, S. Gory-Fauré, A. Vinit, and S. Morales, “Phase from defocus,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (SPIE, 2018), pp. 112–123.

Hofmann, R.

Hu, C.

Q. Lu, G. Liu, C. Xiao, C. Hu, S. Zhang, R. X. Xu, K. Chu, Q. Xu, and Z. J. Smith, “A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings,” PLoS One 13(3), e0194063 (2018).
[Crossref]

Jablonski, S. A.

C. Mollinari, J.-P. Kleman, Y. Saoudi, S. A. Jablonski, J. Perard, T. J. Yen, and R. L. Margolis, “Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not,” Mol. Biol. Cell 16(3), 1043–1055 (2005).
[Crossref]

Kaynig, V.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, and J.Y. Tinevez, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Kim, M. K.

M. K. Kim, Digital Holographic Microscopy (Springer, 2011).

Kimura, A.

Kleman, J.-P.

C. Mollinari, J.-P. Kleman, Y. Saoudi, S. A. Jablonski, J. Perard, T. J. Yen, and R. L. Margolis, “Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not,” Mol. Biol. Cell 16(3), 1043–1055 (2005).
[Crossref]

Körner, K.

Kou, S. S.

Kumar Singh, A.

Liu, C.

W. Yu, X. Tian, X. He, X. Song, L. Xue, C. Liu, and S. Wang, “Real time quantitative phase microscopy based on single-shot transport of intensity equation (sstie) method,” Appl. Phys. Lett. 109(7), 071112 (2016).
[Crossref]

Liu, G.

Q. Lu, G. Liu, C. Xiao, C. Hu, S. Zhang, R. X. Xu, K. Chu, Q. Xu, and Z. J. Smith, “A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings,” PLoS One 13(3), e0194063 (2018).
[Crossref]

Longair, M.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, and J.Y. Tinevez, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Lu, Q.

Q. Lu, G. Liu, C. Xiao, C. Hu, S. Zhang, R. X. Xu, K. Chu, Q. Xu, and Z. J. Smith, “A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings,” PLoS One 13(3), e0194063 (2018).
[Crossref]

Ludwig, W.

P. Cloetens, W. Ludwig, J. Baruchel, D. Van Dyck, J. Van Landuyt, J. P. Guigay, and M. Schlenker, “Holotomography: Quantitative phase tomography with micrometer resolution using hard synchrotron radiation x rays,” Appl. Phys. Lett. 75(19), 2912–2914 (1999).
[Crossref]

Mandula, O.

C. Allier, L. Hervé, O. Mandula, P. Blandin, Y. Usson, J. Savatier, S. Monneret, and S. Morales, “Quantitative phase imaging of adherent mammalian cells: a comparative study,” Biomed. Opt. Express 10(6), 2768–2783 (2019).
[Crossref]

O. Mandula, C. Allier, L. Hervé, E. Denarier, A. Fourest-Lieuvin, S. Gory-Fauré, A. Vinit, and S. Morales, “Phase from defocus,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (SPIE, 2018), pp. 112–123.

Margolis, R. L.

C. Mollinari, J.-P. Kleman, Y. Saoudi, S. A. Jablonski, J. Perard, T. J. Yen, and R. L. Margolis, “Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not,” Mol. Biol. Cell 16(3), 1043–1055 (2005).
[Crossref]

Matoba, O.

X. Quan, K. Nitta, O. Matoba, P. Xia, and Y. Awatsuji, “Phase and fluorescence imaging by combination of digital holographic microscopy and fluorescence microscopy,” Opt. Rev. 22(2), 349–353 (2015).
[Crossref]

Mayo, S. C.

D. Paganin, S. C. Mayo, T. E. Gureyev, P. R. Miller, and S. W. Wilkins, “Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object,” J. Microsc. 206(1), 33–40 (2002).
[Crossref]

Menneteau, M.

L. Herve, O. Cioni, P. Blandin, F. Navarro, M. Menneteau, T. Bordy, S. Morales, and C. Allier, “Multispectral total-variation reconstruction applied to lens-free microscopy,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (OSA, 2018), pp. 5828–5836.

Mesquita, L.

U. Agero, L. Mesquita, B. Neves, R. Gazzinelli, and O. Mesquita, “Defocusing microscopy,” Microsc. Res. Tech. 65(3), 159–165 (2004).
[Crossref]

Mesquita, O.

U. Agero, L. Mesquita, B. Neves, R. Gazzinelli, and O. Mesquita, “Defocusing microscopy,” Microsc. Res. Tech. 65(3), 159–165 (2004).
[Crossref]

Mesquita, O. N.

U. Agero, C. H. Monken, C. Ropert, R. T. Gazzinelli, and O. N. Mesquita, “Cell surface fluctuations studied with defocusing microscopy,” Phys. Rev. E 67(5), 051904 (2003).
[Crossref]

Miller, P. R.

D. Paganin, S. C. Mayo, T. E. Gureyev, P. R. Miller, and S. W. Wilkins, “Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object,” J. Microsc. 206(1), 33–40 (2002).
[Crossref]

Molesini, G.

G. Molesini, G. Pedrini, P. Poggi, and F. Quercioli, “Focus-wavelength encoded optical profilometer,” Opt. Commun. 49(4), 229–233 (1984).
[Crossref]

Mollinari, C.

C. Mollinari, J.-P. Kleman, Y. Saoudi, S. A. Jablonski, J. Perard, T. J. Yen, and R. L. Margolis, “Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not,” Mol. Biol. Cell 16(3), 1043–1055 (2005).
[Crossref]

Monken, C. H.

U. Agero, C. H. Monken, C. Ropert, R. T. Gazzinelli, and O. N. Mesquita, “Cell surface fluctuations studied with defocusing microscopy,” Phys. Rev. E 67(5), 051904 (2003).
[Crossref]

Monneret, S.

Moosmann, J.

Morales, S.

I. de Kernier, A. Ali-Cherif, N. Rongeat, O. Cioni, S. Morales, and J. Savatier, “Large field-of-view phase and fluorescence mesoscope with microscopic resolution,” J. Biomed. Opt. 24(03), 1–9 (2019).
[Crossref]

C. Allier, L. Hervé, O. Mandula, P. Blandin, Y. Usson, J. Savatier, S. Monneret, and S. Morales, “Quantitative phase imaging of adherent mammalian cells: a comparative study,” Biomed. Opt. Express 10(6), 2768–2783 (2019).
[Crossref]

L. Herve, O. Cioni, P. Blandin, F. Navarro, M. Menneteau, T. Bordy, S. Morales, and C. Allier, “Multispectral total-variation reconstruction applied to lens-free microscopy,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (OSA, 2018), pp. 5828–5836.

O. Mandula, C. Allier, L. Hervé, E. Denarier, A. Fourest-Lieuvin, S. Gory-Fauré, A. Vinit, and S. Morales, “Phase from defocus,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (SPIE, 2018), pp. 112–123.

Naik, D.

Navarro, F.

L. Herve, O. Cioni, P. Blandin, F. Navarro, M. Menneteau, T. Bordy, S. Morales, and C. Allier, “Multispectral total-variation reconstruction applied to lens-free microscopy,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (OSA, 2018), pp. 5828–5836.

Neves, B.

U. Agero, L. Mesquita, B. Neves, R. Gazzinelli, and O. Mesquita, “Defocusing microscopy,” Microsc. Res. Tech. 65(3), 159–165 (2004).
[Crossref]

Nitta, K.

X. Quan, K. Nitta, O. Matoba, P. Xia, and Y. Awatsuji, “Phase and fluorescence imaging by combination of digital holographic microscopy and fluorescence microscopy,” Opt. Rev. 22(2), 349–353 (2015).
[Crossref]

Noda, T.

Nomarski, G.

G. Nomarski, “Differential microinterferometer with polarized waves,” J. Phys. Radium 16, 9S–11S (1955).

Ohta, J.

Osten, W.

Ozcan, A.

Pacureanu, A.

Paganin, D.

D. Paganin, S. C. Mayo, T. E. Gureyev, P. R. Miller, and S. W. Wilkins, “Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object,” J. Microsc. 206(1), 33–40 (2002).
[Crossref]

Park, Y.

Pedrini, G.

W. Osten, A. Faridian, P. Gao, K. Körner, D. Naik, G. Pedrini, A. Kumar Singh, M. Takeda, and M. Wilke, “Recent advances in digital holography,” Appl. Opt. 53(27), G44–63 (2014).
[Crossref]

G. Molesini, G. Pedrini, P. Poggi, and F. Quercioli, “Focus-wavelength encoded optical profilometer,” Opt. Commun. 49(4), 229–233 (1984).
[Crossref]

Perard, J.

C. Mollinari, J.-P. Kleman, Y. Saoudi, S. A. Jablonski, J. Perard, T. J. Yen, and R. L. Margolis, “Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not,” Mol. Biol. Cell 16(3), 1043–1055 (2005).
[Crossref]

Pietzsch, T.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, and J.Y. Tinevez, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Poggi, P.

G. Molesini, G. Pedrini, P. Poggi, and F. Quercioli, “Focus-wavelength encoded optical profilometer,” Opt. Commun. 49(4), 229–233 (1984).
[Crossref]

Popescu, G.

Preibisch, S.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, and J.Y. Tinevez, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Qu, W.

Quan, X.

X. Quan, K. Nitta, O. Matoba, P. Xia, and Y. Awatsuji, “Phase and fluorescence imaging by combination of digital holographic microscopy and fluorescence microscopy,” Opt. Rev. 22(2), 349–353 (2015).
[Crossref]

Quercioli, F.

G. Molesini, G. Pedrini, P. Poggi, and F. Quercioli, “Focus-wavelength encoded optical profilometer,” Opt. Commun. 49(4), 229–233 (1984).
[Crossref]

Rongeat, N.

I. de Kernier, A. Ali-Cherif, N. Rongeat, O. Cioni, S. Morales, and J. Savatier, “Large field-of-view phase and fluorescence mesoscope with microscopic resolution,” J. Biomed. Opt. 24(03), 1–9 (2019).
[Crossref]

Ropert, C.

U. Agero, C. H. Monken, C. Ropert, R. T. Gazzinelli, and O. N. Mesquita, “Cell surface fluctuations studied with defocusing microscopy,” Phys. Rev. E 67(5), 051904 (2003).
[Crossref]

Rueden, C.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, and J.Y. Tinevez, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Saalfeld, S.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, and J.Y. Tinevez, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Saoudi, Y.

C. Mollinari, J.-P. Kleman, Y. Saoudi, S. A. Jablonski, J. Perard, T. J. Yen, and R. L. Margolis, “Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not,” Mol. Biol. Cell 16(3), 1043–1055 (2005).
[Crossref]

Sasagawa, K.

Savatier, J.

I. de Kernier, A. Ali-Cherif, N. Rongeat, O. Cioni, S. Morales, and J. Savatier, “Large field-of-view phase and fluorescence mesoscope with microscopic resolution,” J. Biomed. Opt. 24(03), 1–9 (2019).
[Crossref]

C. Allier, L. Hervé, O. Mandula, P. Blandin, Y. Usson, J. Savatier, S. Monneret, and S. Morales, “Quantitative phase imaging of adherent mammalian cells: a comparative study,” Biomed. Opt. Express 10(6), 2768–2783 (2019).
[Crossref]

Schindelin, J.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, and J.Y. Tinevez, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Schlenker, M.

P. Cloetens, W. Ludwig, J. Baruchel, D. Van Dyck, J. Van Landuyt, J. P. Guigay, and M. Schlenker, “Holotomography: Quantitative phase tomography with micrometer resolution using hard synchrotron radiation x rays,” Appl. Phys. Lett. 75(19), 2912–2914 (1999).
[Crossref]

Schmid, B.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, and J.Y. Tinevez, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Sheppard, C. J.

Sheppard, C. J. R.

Smith, Z. J.

Q. Lu, G. Liu, C. Xiao, C. Hu, S. Zhang, R. X. Xu, K. Chu, Q. Xu, and Z. J. Smith, “A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings,” PLoS One 13(3), e0194063 (2018).
[Crossref]

Song, X.

W. Yu, X. Tian, X. He, X. Song, L. Xue, C. Liu, and S. Wang, “Real time quantitative phase microscopy based on single-shot transport of intensity equation (sstie) method,” Appl. Phys. Lett. 109(7), 071112 (2016).
[Crossref]

Sternberg, S.

S. Sternberg, “Biomedical image processing,” Computer 16(1), 22–34 (1983).
[Crossref]

Streibl, N.

N. Streibl, “Phase imaging by the transport equation of intensity,” Opt. Commun. 49(1), 6–10 (1984).
[Crossref]

Takeda, M.

Teague, M. R.

Tian, X.

W. Yu, X. Tian, X. He, X. Song, L. Xue, C. Liu, and S. Wang, “Real time quantitative phase microscopy based on single-shot transport of intensity equation (sstie) method,” Appl. Phys. Lett. 109(7), 071112 (2016).
[Crossref]

Tinevez, J.Y.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, and J.Y. Tinevez, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Tokuda, T.

Usson, Y.

Van Dyck, D.

P. Cloetens, W. Ludwig, J. Baruchel, D. Van Dyck, J. Van Landuyt, J. P. Guigay, and M. Schlenker, “Holotomography: Quantitative phase tomography with micrometer resolution using hard synchrotron radiation x rays,” Appl. Phys. Lett. 75(19), 2912–2914 (1999).
[Crossref]

Van Landuyt, J.

P. Cloetens, W. Ludwig, J. Baruchel, D. Van Dyck, J. Van Landuyt, J. P. Guigay, and M. Schlenker, “Holotomography: Quantitative phase tomography with micrometer resolution using hard synchrotron radiation x rays,” Appl. Phys. Lett. 75(19), 2912–2914 (1999).
[Crossref]

Vinit, A.

O. Mandula, C. Allier, L. Hervé, E. Denarier, A. Fourest-Lieuvin, S. Gory-Fauré, A. Vinit, and S. Morales, “Phase from defocus,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (SPIE, 2018), pp. 112–123.

Waller, L.

Wang, S.

W. Yu, X. Tian, X. He, X. Song, L. Xue, C. Liu, and S. Wang, “Real time quantitative phase microscopy based on single-shot transport of intensity equation (sstie) method,” Appl. Phys. Lett. 109(7), 071112 (2016).
[Crossref]

Wilke, M.

Wilkins, S. W.

D. Paganin, S. C. Mayo, T. E. Gureyev, P. R. Miller, and S. W. Wilkins, “Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object,” J. Microsc. 206(1), 33–40 (2002).
[Crossref]

Wolf, E.

M. Born and E. Wolf, Principles of optics, vol. 1 (Cambridge University Press, 1999).

Xia, P.

X. Quan, K. Nitta, O. Matoba, P. Xia, and Y. Awatsuji, “Phase and fluorescence imaging by combination of digital holographic microscopy and fluorescence microscopy,” Opt. Rev. 22(2), 349–353 (2015).
[Crossref]

Xiao, C.

Q. Lu, G. Liu, C. Xiao, C. Hu, S. Zhang, R. X. Xu, K. Chu, Q. Xu, and Z. J. Smith, “A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings,” PLoS One 13(3), e0194063 (2018).
[Crossref]

Xu, Q.

Q. Lu, G. Liu, C. Xiao, C. Hu, S. Zhang, R. X. Xu, K. Chu, Q. Xu, and Z. J. Smith, “A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings,” PLoS One 13(3), e0194063 (2018).
[Crossref]

Xu, R. X.

Q. Lu, G. Liu, C. Xiao, C. Hu, S. Zhang, R. X. Xu, K. Chu, Q. Xu, and Z. J. Smith, “A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings,” PLoS One 13(3), e0194063 (2018).
[Crossref]

Xue, L.

W. Yu, X. Tian, X. He, X. Song, L. Xue, C. Liu, and S. Wang, “Real time quantitative phase microscopy based on single-shot transport of intensity equation (sstie) method,” Appl. Phys. Lett. 109(7), 071112 (2016).
[Crossref]

Yang, Y.

Yen, T. J.

C. Mollinari, J.-P. Kleman, Y. Saoudi, S. A. Jablonski, J. Perard, T. J. Yen, and R. L. Margolis, “Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not,” Mol. Biol. Cell 16(3), 1043–1055 (2005).
[Crossref]

Yu, W.

W. Yu, X. Tian, X. He, X. Song, L. Xue, C. Liu, and S. Wang, “Real time quantitative phase microscopy based on single-shot transport of intensity equation (sstie) method,” Appl. Phys. Lett. 109(7), 071112 (2016).
[Crossref]

Zernike, F.

F. Zernike, “How I Discovered Phase Contrast,” Science 121(3141), 345–349 (1955).
[Crossref]

F. Zernike, “Phase contrast, a new method for the microscopic observation of transparent objects,” Physica 9(7), 686–698 (1942).
[Crossref]

Zhang, S.

Q. Lu, G. Liu, C. Xiao, C. Hu, S. Zhang, R. X. Xu, K. Chu, Q. Xu, and Z. J. Smith, “A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings,” PLoS One 13(3), e0194063 (2018).
[Crossref]

Zuo, C.

Appl. Opt. (1)

Appl. Phys. Lett. (2)

P. Cloetens, W. Ludwig, J. Baruchel, D. Van Dyck, J. Van Landuyt, J. P. Guigay, and M. Schlenker, “Holotomography: Quantitative phase tomography with micrometer resolution using hard synchrotron radiation x rays,” Appl. Phys. Lett. 75(19), 2912–2914 (1999).
[Crossref]

W. Yu, X. Tian, X. He, X. Song, L. Xue, C. Liu, and S. Wang, “Real time quantitative phase microscopy based on single-shot transport of intensity equation (sstie) method,” Appl. Phys. Lett. 109(7), 071112 (2016).
[Crossref]

Biomed. Opt. Express (2)

Computer (1)

S. Sternberg, “Biomedical image processing,” Computer 16(1), 22–34 (1983).
[Crossref]

J. Biomed. Opt. (1)

I. de Kernier, A. Ali-Cherif, N. Rongeat, O. Cioni, S. Morales, and J. Savatier, “Large field-of-view phase and fluorescence mesoscope with microscopic resolution,” J. Biomed. Opt. 24(03), 1–9 (2019).
[Crossref]

J. Microsc. (1)

D. Paganin, S. C. Mayo, T. E. Gureyev, P. R. Miller, and S. W. Wilkins, “Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object,” J. Microsc. 206(1), 33–40 (2002).
[Crossref]

J. Opt. Soc. Am. A (2)

J. Phys. Radium (1)

G. Nomarski, “Differential microinterferometer with polarized waves,” J. Phys. Radium 16, 9S–11S (1955).

Microsc. Res. Tech. (1)

U. Agero, L. Mesquita, B. Neves, R. Gazzinelli, and O. Mesquita, “Defocusing microscopy,” Microsc. Res. Tech. 65(3), 159–165 (2004).
[Crossref]

Mol. Biol. Cell (1)

C. Mollinari, J.-P. Kleman, Y. Saoudi, S. A. Jablonski, J. Perard, T. J. Yen, and R. L. Margolis, “Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not,” Mol. Biol. Cell 16(3), 1043–1055 (2005).
[Crossref]

Nat. Methods (1)

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, and J.Y. Tinevez, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Opt. Commun. (2)

G. Molesini, G. Pedrini, P. Poggi, and F. Quercioli, “Focus-wavelength encoded optical profilometer,” Opt. Commun. 49(4), 229–233 (1984).
[Crossref]

N. Streibl, “Phase imaging by the transport equation of intensity,” Opt. Commun. 49(1), 6–10 (1984).
[Crossref]

Opt. Express (6)

Opt. Rev. (1)

X. Quan, K. Nitta, O. Matoba, P. Xia, and Y. Awatsuji, “Phase and fluorescence imaging by combination of digital holographic microscopy and fluorescence microscopy,” Opt. Rev. 22(2), 349–353 (2015).
[Crossref]

Phys. Rev. E (1)

U. Agero, C. H. Monken, C. Ropert, R. T. Gazzinelli, and O. N. Mesquita, “Cell surface fluctuations studied with defocusing microscopy,” Phys. Rev. E 67(5), 051904 (2003).
[Crossref]

Physica (1)

F. Zernike, “Phase contrast, a new method for the microscopic observation of transparent objects,” Physica 9(7), 686–698 (1942).
[Crossref]

PLoS One (1)

Q. Lu, G. Liu, C. Xiao, C. Hu, S. Zhang, R. X. Xu, K. Chu, Q. Xu, and Z. J. Smith, “A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings,” PLoS One 13(3), e0194063 (2018).
[Crossref]

Scanning (1)

M. A. Browne, O. Akinyemi, and A. Boyde, “Confocal surface profiling utilizing chromatic aberration,” Scanning 14(3), 145–153 (1992).
[Crossref]

Science (1)

F. Zernike, “How I Discovered Phase Contrast,” Science 121(3141), 345–349 (1955).
[Crossref]

Other (9)

O. Mandula, C. Allier, L. Hervé, E. Denarier, A. Fourest-Lieuvin, S. Gory-Fauré, A. Vinit, and S. Morales, “Phase from defocus,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (SPIE, 2018), pp. 112–123.

J. W. Goodman, Introduction to Fourier optics (Roberts and Company Publishers, 2005).

L. Herve, O. Cioni, P. Blandin, F. Navarro, M. Menneteau, T. Bordy, S. Morales, and C. Allier, “Multispectral total-variation reconstruction applied to lens-free microscopy,” in Quantitative Phase Imaging IV, vol. 10503G. Popescu and Y. Park, eds., International Society for Optics and Photonics (OSA, 2018), pp. 5828–5836.

https://www.essenbioscience.com/en/products/incucyte .

https://www.thermofisher.com/order/catalog/product/AMF7000 .

http://phasicscorp.com/sectors/life-science .

M. K. Kim, Digital Holographic Microscopy (Springer, 2011).

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Supplementary Material (1)

NameDescription
» Visualization 1       Time-lapse recording of triple stained Hela cell culture in combined phase contrast (gray) and fluorescence (color) image. Hoechst stain marking cell nucleus is shown in blue, eGFP marking alpha-tubulin structures in green and non-specific pmCherry .

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Figures (8)

Fig. 1.
Fig. 1. (a) A demonstration of a non-absorbing (phase) sample (3 DIV hippocampal neurons) at different levels of defocus. The cells are almost invisible when brought in-focus (middle). (b) Our reconstruction of the in-focus phase-contrast image from a single, $100\,\rm {\mu m}$ defocused image taken under semi-coherent illumination.
Fig. 2.
Fig. 2. a) Measured chromatic displacement of the focus. The theoretical depth of field of the system ($10\,\rm {\mu m}$) is shown as a red band. While the system is approximately achromatic in the range $\lambda =500-600\,\rm {nm}$ (green to red), it is strongly chromatic at $\lambda =420\,\rm {nm}$ (violet). b) Multi-band fluorescent filter set with a phase (defocus) imaging filter at $\lambda =420\,\rm {nm}$ passing through the blue emission band.
Fig. 3.
Fig. 3. System in fluorescence (a-b) and phase (c) mode. Note the defocus due to chromatic aberration in (c). L1 blue and green LEDs for fluorescence excitation, L2 blue LED for defocused imaging in transmission mode with a narrow band filter IF. OF optical fibre, CL collimator lens, EX excitation filter, D dichroic, EM emission filter, O objective, SP sample plane, CMOS camera. (d,e) Regions of interests (ROIs) of in-focus green and red fluorescence, respectively. (f) Corresponding ROI of raw out-of-focus data. Scale bar $50\,\rm {\mu m}$. (g) The system installed in an incubator.
Fig. 4.
Fig. 4. ROI of a snapshot from 40h recording of triple stained HeLa cell culture (Blue - Hoechst staining cell nucleus, Green - GFP tubulin, Red - mCherry). Raw defocused images taken in transmission mode are numerically reconstructed to yield an in-focus phase-contrast image of the sample. This can be combined with the corresponding in-focus epi-fluorescence data (dashed box) producing a phase (gray) and fluorescence (RGB) combined image (right).
Fig. 5.
Fig. 5. USAF absorption (a-c) and phase (d-f) resolution target imaged with our system. a) Full field-of-view of absorption resolution target imaged in-focus, b) central ROI (red box in a)). We can resolve group 8, element 5 with line width of $1.23\,\rm {\mu m}$. c) line profile over group 8 (red line in b)). d) Reconstructed phase image of phase resolution target (silica slide with $300\,\rm {nm}$ thick engraving of the USAF resolution target) taken at $100\,\rm {\mu m}$ defocus. e) Central region (red box in d)) showing groups 6, 7 and 8. f) $100\,\rm {\mu m}$ defocused image (raw data) of a region shown in e).
Fig. 6.
Fig. 6. Hela cells culture in combined phase contrast (gray) and fluorescenceimage. Hoechst stain marking cell nucleus is shown in blue, eGFP marking $\alpha$-tubulin structures in green and non-specific pmCherry in red. Whole field of view ($3\,\rm {mm}^2$) with circular region ($1.2\,\rm {mm}^2$) with acceptable flat field correction (white circle). There is $\sim 700$ cells contained in the circular region. ROI shown with red dashed box at 5-hour intervals is shown in Fig. 7(a).
Fig. 7.
Fig. 7. Time-lapse of triple stained HeLa cells. Images taken every 10 mins over 40 hours. a) Selected ROI from Fig. 6 shown at 5-hour intervals (see also Visualization 1). b) A small ROI of reconstructed phase image. Cells in division show phase wrapping (red arrows).
Fig. 8.
Fig. 8. a) Phase contrast image (gray) with overlayed fluorescence (green) of Micrococcus luteus marked with SYTO9 in mixture with $2\,\rm {\mu m}$ polystyrene beads. Insets show the local displacement between phase and fluorescence image. Reconstructed phase contrast image of ROI marked with red box is shown in b), corresponding fluorescence image is shown in c) with arrows highlighting several Micrococcus clusters. The structure of the clusters, the dimers (red), tetramers (cyan) and multimers (green) of cocci, is at the resolution limit of our system. Two beads above and at the focal plane are highlighted with a red and green box, respectively.

Equations (3)

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A z ( r ) = A 0 ( r ) h z ( r ) ,
h z ( r ) = 1 i λ z exp ( i π r 2 λ z ) .
ϵ ( A 0 ) = c 1 R 2 d r | A 0 ( r ) | + c 2 Ω d r | A 0 ( r ) | 2 ,

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