Abstract

We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization.

© 2016 Optical Society of America

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References

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  1. D. B. Murphy, Fundamentals of Light Microscopy and Electronic Imaging (Wiley-Liss, 2001).
  2. D. J. Webb and C. M. Brown, “Epi-Fluorescence Microscopy,” in Cell Imaging Techniques, D. J. Taatjes and J. Roth, eds. (Humana Press, 2012), Vol. 931, pp. 29–59.
  3. P. J. Shaw, “Comparison of widefield/deconvolution and confocal microscopy for three-dimensional imaging,” in Handbook of Biological Confocal Microscopy (Springer, 2006), pp. 453–467.
  4. D. Tseng and A. Ozcan with Hongying Zhu and O. Yaglidere, Ting-Wei Su, “Wide-field fluorescent microscopy on a cell-phone,” in (IEEE, 2011), pp. 6801–6804.
  5. J. C. Waters, “Accuracy and precision in quantitative fluorescence microscopy,” J. Cell Biol. 185(7), 1135–1148 (2009).
    [Crossref] [PubMed]
  6. J. W. Lichtman and J.-A. Conchello, “Fluorescence microscopy,” Nat. Methods 2(12), 910–919 (2005).
    [Crossref] [PubMed]
  7. C. Yang, V. Hou, L. Y. Nelson, and E. J. Seibel, “Mitigating fluorescence spectral overlap in wide-field endoscopic imaging,” J. Biomed. Opt. 18(8), 086012 (2013).
    [Crossref] [PubMed]
  8. T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003).
    [Crossref] [PubMed]
  9. B. Kraus, M. Ziegler, and H. Wolff, “Linear fluorescence unmixing in cell biological research,” Mod. Res. Educ. Top. Microsc. 2, 863–873 (2007).
  10. T. Zimmermann, “Spectral Imaging and Linear Unmixing in Light Microscopy,” in Microscopy Techniques, J. Rietdorf, ed. (Springer Berlin Heidelberg, 2005), Vol. 95, pp. 245–265.
  11. D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, “Spectral unmixing of flavin autofluorescence components in cardiac myocytes,” Biophys. J. 89(6), L55–L57 (2005).
    [Crossref] [PubMed]
  12. Y. Garini, I. T. Young, and G. McNamara, “Spectral imaging: Principles and applications,” Cytometry A 69(8), 735–747 (2006).
    [Crossref] [PubMed]
  13. R. Bishop, “Applications of fluorescence in situ hybridization (FISH) in detecting genetic aberrations of medical significance,” Biosci. Horiz. 3(1), 85–95 (2010).
    [Crossref]
  14. K. Aswani, T. Jinadasa, and C. M. Brown, “Fluorescence Microscopy Light Sources,” Micros. Today 20(04), 22–28 (2012).
    [Crossref]
  15. L. Liu, O. Yermolaieva, W. A. Johnson, F. M. Abboud, and M. J. Welsh, “Identification and function of thermosensory neurons in Drosophila larvae,” Nat. Neurosci. 6(3), 267–273 (2003).
    [Crossref] [PubMed]
  16. N. Hagen and M. W. Kudenov, “Review of snapshot spectral imaging technologies,” Opt. Eng. 52(9), 090901 (2013).
    [Crossref]
  17. N. S. White and R. J. Errington, “Fluorescence techniques for drug delivery research: theory and practice,” Adv. Drug Deliv. Rev. 57(1), 17–42 (2005).
    [Crossref] [PubMed]
  18. K. W. Dunn, M. M. Kamocka, and J. H. McDonald, “A practical guide to evaluating colocalization in biological microscopy,” Am. J. Physiol. Cell Physiol. 300(4), C723–C742 (2011).
    [Crossref] [PubMed]
  19. K. S. Park, W. J. Choi, J. B. Eom, K. S. Chang, and B. H. Lee, “High-contrast epi-fluorescence wide-field imaging of biological cells using integrating-bucket method,” Opt. Commun. 355, 427–432 (2015).
    [Crossref]
  20. W. J. Choi, S. Y. Ryu, J. K. Kim, D. U. Kim, G. H. Kim, and K. S. Chang, “High-speed thermoreflectance microscopy using charge-coupled device-based Fourier-domain filtering,” Opt. Lett. 38(18), 3581–3584 (2013).
    [Crossref] [PubMed]

2015 (1)

K. S. Park, W. J. Choi, J. B. Eom, K. S. Chang, and B. H. Lee, “High-contrast epi-fluorescence wide-field imaging of biological cells using integrating-bucket method,” Opt. Commun. 355, 427–432 (2015).
[Crossref]

2013 (3)

W. J. Choi, S. Y. Ryu, J. K. Kim, D. U. Kim, G. H. Kim, and K. S. Chang, “High-speed thermoreflectance microscopy using charge-coupled device-based Fourier-domain filtering,” Opt. Lett. 38(18), 3581–3584 (2013).
[Crossref] [PubMed]

N. Hagen and M. W. Kudenov, “Review of snapshot spectral imaging technologies,” Opt. Eng. 52(9), 090901 (2013).
[Crossref]

C. Yang, V. Hou, L. Y. Nelson, and E. J. Seibel, “Mitigating fluorescence spectral overlap in wide-field endoscopic imaging,” J. Biomed. Opt. 18(8), 086012 (2013).
[Crossref] [PubMed]

2012 (1)

K. Aswani, T. Jinadasa, and C. M. Brown, “Fluorescence Microscopy Light Sources,” Micros. Today 20(04), 22–28 (2012).
[Crossref]

2011 (1)

K. W. Dunn, M. M. Kamocka, and J. H. McDonald, “A practical guide to evaluating colocalization in biological microscopy,” Am. J. Physiol. Cell Physiol. 300(4), C723–C742 (2011).
[Crossref] [PubMed]

2010 (1)

R. Bishop, “Applications of fluorescence in situ hybridization (FISH) in detecting genetic aberrations of medical significance,” Biosci. Horiz. 3(1), 85–95 (2010).
[Crossref]

2009 (1)

J. C. Waters, “Accuracy and precision in quantitative fluorescence microscopy,” J. Cell Biol. 185(7), 1135–1148 (2009).
[Crossref] [PubMed]

2007 (1)

B. Kraus, M. Ziegler, and H. Wolff, “Linear fluorescence unmixing in cell biological research,” Mod. Res. Educ. Top. Microsc. 2, 863–873 (2007).

2006 (1)

Y. Garini, I. T. Young, and G. McNamara, “Spectral imaging: Principles and applications,” Cytometry A 69(8), 735–747 (2006).
[Crossref] [PubMed]

2005 (3)

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, “Spectral unmixing of flavin autofluorescence components in cardiac myocytes,” Biophys. J. 89(6), L55–L57 (2005).
[Crossref] [PubMed]

J. W. Lichtman and J.-A. Conchello, “Fluorescence microscopy,” Nat. Methods 2(12), 910–919 (2005).
[Crossref] [PubMed]

N. S. White and R. J. Errington, “Fluorescence techniques for drug delivery research: theory and practice,” Adv. Drug Deliv. Rev. 57(1), 17–42 (2005).
[Crossref] [PubMed]

2003 (2)

L. Liu, O. Yermolaieva, W. A. Johnson, F. M. Abboud, and M. J. Welsh, “Identification and function of thermosensory neurons in Drosophila larvae,” Nat. Neurosci. 6(3), 267–273 (2003).
[Crossref] [PubMed]

T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003).
[Crossref] [PubMed]

Abboud, F. M.

L. Liu, O. Yermolaieva, W. A. Johnson, F. M. Abboud, and M. J. Welsh, “Identification and function of thermosensory neurons in Drosophila larvae,” Nat. Neurosci. 6(3), 267–273 (2003).
[Crossref] [PubMed]

Aswani, K.

K. Aswani, T. Jinadasa, and C. M. Brown, “Fluorescence Microscopy Light Sources,” Micros. Today 20(04), 22–28 (2012).
[Crossref]

Bishop, R.

R. Bishop, “Applications of fluorescence in situ hybridization (FISH) in detecting genetic aberrations of medical significance,” Biosci. Horiz. 3(1), 85–95 (2010).
[Crossref]

Brown, C. M.

K. Aswani, T. Jinadasa, and C. M. Brown, “Fluorescence Microscopy Light Sources,” Micros. Today 20(04), 22–28 (2012).
[Crossref]

Cagalinec, M.

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, “Spectral unmixing of flavin autofluorescence components in cardiac myocytes,” Biophys. J. 89(6), L55–L57 (2005).
[Crossref] [PubMed]

Chang, K. S.

K. S. Park, W. J. Choi, J. B. Eom, K. S. Chang, and B. H. Lee, “High-contrast epi-fluorescence wide-field imaging of biological cells using integrating-bucket method,” Opt. Commun. 355, 427–432 (2015).
[Crossref]

W. J. Choi, S. Y. Ryu, J. K. Kim, D. U. Kim, G. H. Kim, and K. S. Chang, “High-speed thermoreflectance microscopy using charge-coupled device-based Fourier-domain filtering,” Opt. Lett. 38(18), 3581–3584 (2013).
[Crossref] [PubMed]

Choi, W. J.

K. S. Park, W. J. Choi, J. B. Eom, K. S. Chang, and B. H. Lee, “High-contrast epi-fluorescence wide-field imaging of biological cells using integrating-bucket method,” Opt. Commun. 355, 427–432 (2015).
[Crossref]

W. J. Choi, S. Y. Ryu, J. K. Kim, D. U. Kim, G. H. Kim, and K. S. Chang, “High-speed thermoreflectance microscopy using charge-coupled device-based Fourier-domain filtering,” Opt. Lett. 38(18), 3581–3584 (2013).
[Crossref] [PubMed]

Chorvat, D.

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, “Spectral unmixing of flavin autofluorescence components in cardiac myocytes,” Biophys. J. 89(6), L55–L57 (2005).
[Crossref] [PubMed]

Chorvatova, A.

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, “Spectral unmixing of flavin autofluorescence components in cardiac myocytes,” Biophys. J. 89(6), L55–L57 (2005).
[Crossref] [PubMed]

Conchello, J.-A.

J. W. Lichtman and J.-A. Conchello, “Fluorescence microscopy,” Nat. Methods 2(12), 910–919 (2005).
[Crossref] [PubMed]

Dunn, K. W.

K. W. Dunn, M. M. Kamocka, and J. H. McDonald, “A practical guide to evaluating colocalization in biological microscopy,” Am. J. Physiol. Cell Physiol. 300(4), C723–C742 (2011).
[Crossref] [PubMed]

Eom, J. B.

K. S. Park, W. J. Choi, J. B. Eom, K. S. Chang, and B. H. Lee, “High-contrast epi-fluorescence wide-field imaging of biological cells using integrating-bucket method,” Opt. Commun. 355, 427–432 (2015).
[Crossref]

Errington, R. J.

N. S. White and R. J. Errington, “Fluorescence techniques for drug delivery research: theory and practice,” Adv. Drug Deliv. Rev. 57(1), 17–42 (2005).
[Crossref] [PubMed]

Garini, Y.

Y. Garini, I. T. Young, and G. McNamara, “Spectral imaging: Principles and applications,” Cytometry A 69(8), 735–747 (2006).
[Crossref] [PubMed]

Hagen, N.

N. Hagen and M. W. Kudenov, “Review of snapshot spectral imaging technologies,” Opt. Eng. 52(9), 090901 (2013).
[Crossref]

Hou, V.

C. Yang, V. Hou, L. Y. Nelson, and E. J. Seibel, “Mitigating fluorescence spectral overlap in wide-field endoscopic imaging,” J. Biomed. Opt. 18(8), 086012 (2013).
[Crossref] [PubMed]

Jinadasa, T.

K. Aswani, T. Jinadasa, and C. M. Brown, “Fluorescence Microscopy Light Sources,” Micros. Today 20(04), 22–28 (2012).
[Crossref]

Johnson, W. A.

L. Liu, O. Yermolaieva, W. A. Johnson, F. M. Abboud, and M. J. Welsh, “Identification and function of thermosensory neurons in Drosophila larvae,” Nat. Neurosci. 6(3), 267–273 (2003).
[Crossref] [PubMed]

Kamocka, M. M.

K. W. Dunn, M. M. Kamocka, and J. H. McDonald, “A practical guide to evaluating colocalization in biological microscopy,” Am. J. Physiol. Cell Physiol. 300(4), C723–C742 (2011).
[Crossref] [PubMed]

Kim, D. U.

Kim, G. H.

Kim, J. K.

Kirchnerova, J.

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, “Spectral unmixing of flavin autofluorescence components in cardiac myocytes,” Biophys. J. 89(6), L55–L57 (2005).
[Crossref] [PubMed]

Kraus, B.

B. Kraus, M. Ziegler, and H. Wolff, “Linear fluorescence unmixing in cell biological research,” Mod. Res. Educ. Top. Microsc. 2, 863–873 (2007).

Kudenov, M. W.

N. Hagen and M. W. Kudenov, “Review of snapshot spectral imaging technologies,” Opt. Eng. 52(9), 090901 (2013).
[Crossref]

Lee, B. H.

K. S. Park, W. J. Choi, J. B. Eom, K. S. Chang, and B. H. Lee, “High-contrast epi-fluorescence wide-field imaging of biological cells using integrating-bucket method,” Opt. Commun. 355, 427–432 (2015).
[Crossref]

Lichtman, J. W.

J. W. Lichtman and J.-A. Conchello, “Fluorescence microscopy,” Nat. Methods 2(12), 910–919 (2005).
[Crossref] [PubMed]

Liu, L.

L. Liu, O. Yermolaieva, W. A. Johnson, F. M. Abboud, and M. J. Welsh, “Identification and function of thermosensory neurons in Drosophila larvae,” Nat. Neurosci. 6(3), 267–273 (2003).
[Crossref] [PubMed]

Mateasik, A.

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, “Spectral unmixing of flavin autofluorescence components in cardiac myocytes,” Biophys. J. 89(6), L55–L57 (2005).
[Crossref] [PubMed]

McDonald, J. H.

K. W. Dunn, M. M. Kamocka, and J. H. McDonald, “A practical guide to evaluating colocalization in biological microscopy,” Am. J. Physiol. Cell Physiol. 300(4), C723–C742 (2011).
[Crossref] [PubMed]

McNamara, G.

Y. Garini, I. T. Young, and G. McNamara, “Spectral imaging: Principles and applications,” Cytometry A 69(8), 735–747 (2006).
[Crossref] [PubMed]

Nelson, L. Y.

C. Yang, V. Hou, L. Y. Nelson, and E. J. Seibel, “Mitigating fluorescence spectral overlap in wide-field endoscopic imaging,” J. Biomed. Opt. 18(8), 086012 (2013).
[Crossref] [PubMed]

Park, K. S.

K. S. Park, W. J. Choi, J. B. Eom, K. S. Chang, and B. H. Lee, “High-contrast epi-fluorescence wide-field imaging of biological cells using integrating-bucket method,” Opt. Commun. 355, 427–432 (2015).
[Crossref]

Pepperkok, R.

T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003).
[Crossref] [PubMed]

Rietdorf, J.

T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003).
[Crossref] [PubMed]

Ryu, S. Y.

Seibel, E. J.

C. Yang, V. Hou, L. Y. Nelson, and E. J. Seibel, “Mitigating fluorescence spectral overlap in wide-field endoscopic imaging,” J. Biomed. Opt. 18(8), 086012 (2013).
[Crossref] [PubMed]

Smolka, J.

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, “Spectral unmixing of flavin autofluorescence components in cardiac myocytes,” Biophys. J. 89(6), L55–L57 (2005).
[Crossref] [PubMed]

Waters, J. C.

J. C. Waters, “Accuracy and precision in quantitative fluorescence microscopy,” J. Cell Biol. 185(7), 1135–1148 (2009).
[Crossref] [PubMed]

Welsh, M. J.

L. Liu, O. Yermolaieva, W. A. Johnson, F. M. Abboud, and M. J. Welsh, “Identification and function of thermosensory neurons in Drosophila larvae,” Nat. Neurosci. 6(3), 267–273 (2003).
[Crossref] [PubMed]

White, N. S.

N. S. White and R. J. Errington, “Fluorescence techniques for drug delivery research: theory and practice,” Adv. Drug Deliv. Rev. 57(1), 17–42 (2005).
[Crossref] [PubMed]

Wolff, H.

B. Kraus, M. Ziegler, and H. Wolff, “Linear fluorescence unmixing in cell biological research,” Mod. Res. Educ. Top. Microsc. 2, 863–873 (2007).

Yang, C.

C. Yang, V. Hou, L. Y. Nelson, and E. J. Seibel, “Mitigating fluorescence spectral overlap in wide-field endoscopic imaging,” J. Biomed. Opt. 18(8), 086012 (2013).
[Crossref] [PubMed]

Yermolaieva, O.

L. Liu, O. Yermolaieva, W. A. Johnson, F. M. Abboud, and M. J. Welsh, “Identification and function of thermosensory neurons in Drosophila larvae,” Nat. Neurosci. 6(3), 267–273 (2003).
[Crossref] [PubMed]

Young, I. T.

Y. Garini, I. T. Young, and G. McNamara, “Spectral imaging: Principles and applications,” Cytometry A 69(8), 735–747 (2006).
[Crossref] [PubMed]

Ziegler, M.

B. Kraus, M. Ziegler, and H. Wolff, “Linear fluorescence unmixing in cell biological research,” Mod. Res. Educ. Top. Microsc. 2, 863–873 (2007).

Zimmermann, T.

T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003).
[Crossref] [PubMed]

Adv. Drug Deliv. Rev. (1)

N. S. White and R. J. Errington, “Fluorescence techniques for drug delivery research: theory and practice,” Adv. Drug Deliv. Rev. 57(1), 17–42 (2005).
[Crossref] [PubMed]

Am. J. Physiol. Cell Physiol. (1)

K. W. Dunn, M. M. Kamocka, and J. H. McDonald, “A practical guide to evaluating colocalization in biological microscopy,” Am. J. Physiol. Cell Physiol. 300(4), C723–C742 (2011).
[Crossref] [PubMed]

Biophys. J. (1)

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, “Spectral unmixing of flavin autofluorescence components in cardiac myocytes,” Biophys. J. 89(6), L55–L57 (2005).
[Crossref] [PubMed]

Biosci. Horiz. (1)

R. Bishop, “Applications of fluorescence in situ hybridization (FISH) in detecting genetic aberrations of medical significance,” Biosci. Horiz. 3(1), 85–95 (2010).
[Crossref]

Cytometry A (1)

Y. Garini, I. T. Young, and G. McNamara, “Spectral imaging: Principles and applications,” Cytometry A 69(8), 735–747 (2006).
[Crossref] [PubMed]

FEBS Lett. (1)

T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003).
[Crossref] [PubMed]

J. Biomed. Opt. (1)

C. Yang, V. Hou, L. Y. Nelson, and E. J. Seibel, “Mitigating fluorescence spectral overlap in wide-field endoscopic imaging,” J. Biomed. Opt. 18(8), 086012 (2013).
[Crossref] [PubMed]

J. Cell Biol. (1)

J. C. Waters, “Accuracy and precision in quantitative fluorescence microscopy,” J. Cell Biol. 185(7), 1135–1148 (2009).
[Crossref] [PubMed]

Micros. Today (1)

K. Aswani, T. Jinadasa, and C. M. Brown, “Fluorescence Microscopy Light Sources,” Micros. Today 20(04), 22–28 (2012).
[Crossref]

Mod. Res. Educ. Top. Microsc. (1)

B. Kraus, M. Ziegler, and H. Wolff, “Linear fluorescence unmixing in cell biological research,” Mod. Res. Educ. Top. Microsc. 2, 863–873 (2007).

Nat. Methods (1)

J. W. Lichtman and J.-A. Conchello, “Fluorescence microscopy,” Nat. Methods 2(12), 910–919 (2005).
[Crossref] [PubMed]

Nat. Neurosci. (1)

L. Liu, O. Yermolaieva, W. A. Johnson, F. M. Abboud, and M. J. Welsh, “Identification and function of thermosensory neurons in Drosophila larvae,” Nat. Neurosci. 6(3), 267–273 (2003).
[Crossref] [PubMed]

Opt. Commun. (1)

K. S. Park, W. J. Choi, J. B. Eom, K. S. Chang, and B. H. Lee, “High-contrast epi-fluorescence wide-field imaging of biological cells using integrating-bucket method,” Opt. Commun. 355, 427–432 (2015).
[Crossref]

Opt. Eng. (1)

N. Hagen and M. W. Kudenov, “Review of snapshot spectral imaging technologies,” Opt. Eng. 52(9), 090901 (2013).
[Crossref]

Opt. Lett. (1)

Other (5)

T. Zimmermann, “Spectral Imaging and Linear Unmixing in Light Microscopy,” in Microscopy Techniques, J. Rietdorf, ed. (Springer Berlin Heidelberg, 2005), Vol. 95, pp. 245–265.

D. B. Murphy, Fundamentals of Light Microscopy and Electronic Imaging (Wiley-Liss, 2001).

D. J. Webb and C. M. Brown, “Epi-Fluorescence Microscopy,” in Cell Imaging Techniques, D. J. Taatjes and J. Roth, eds. (Humana Press, 2012), Vol. 931, pp. 29–59.

P. J. Shaw, “Comparison of widefield/deconvolution and confocal microscopy for three-dimensional imaging,” in Handbook of Biological Confocal Microscopy (Springer, 2006), pp. 453–467.

D. Tseng and A. Ozcan with Hongying Zhu and O. Yaglidere, Ting-Wei Su, “Wide-field fluorescent microscopy on a cell-phone,” in (IEEE, 2011), pp. 6801–6804.

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Figures (7)

Fig. 1
Fig. 1

Multiplexing scheme for multicolor imaging. Intensities of each laser are modulated with the same frequency but different phase. Fluorescence signals generated by excitation sources with different phase delays are multiplexed in the phase domain.

Fig. 2
Fig. 2

Digital image processing procedure for multiplexing the individual fluorophores using the phase image.

Fig. 3
Fig. 3

A schematic of the experimental set-up. All excitation lasers are sinusoidally modulated with the same frequency and different time delays. The imaging camera is phase-locked to the laser-intensity modulation. B: blue laser; G: green laser; R: red laser; DAQ: data acquisition board; MO: microscope objective; Multi-DM: Multi-dichroic mirror; Multi-NF: Multi-notch filter; L1, L2, and L3: lenses

Fig. 4
Fig. 4

Fluorescence excitation and emission spectra of two different polystyrene beads. Bars indicate the spectra of the two excitation sources used in the experiment.

Fig. 5
Fig. 5

Fluorescence images of two different fluorescent polystyrene beads (yellow-green and orange) mixed on a slide glass. (a) Four-bucket image taken with 488-nm laser illumination. (b) Four-bucket image taken with 532-nm laser illumination. (c) Four-bucket image taken with simultaneous illumination of both the 488-nm laser and 532-nm laser. (d) Orange and (e) yellow-green polystyrene bead images separated from (c) by applying the phase image. (f) Merged image of (d) and (e). The white bar indicates a scale of 100 µm.

Fig. 6
Fig. 6

Each organelle of a triple-labeled sample was captured using three separate single-band filter sets in sequence (Brightline 680/42 nm, 600/37 nm, and 514/30 nm from Semrock Inc.). The nucleus, mitochondria, and actin of the Hela cells were stained with TO-PRO-3 Iodide, MitoTracker Red CMXRos, and Alexa Fluo 488 Phalloidin, respectively. (a) Fluorescence image taken using a single bandpass filter with 635-nm laser illumination. (b) Fluorescence image taken with 532-nm laser illumination. (c) Fluorescence imag with 488-nm laser illumination. (d) Merged image of (a), (b) and (c). (a-c) are acquired using single bandpass filters and (e-g) are acquired using a multi-notch filter with four-bucket detection. (h) Merged image of (e), (f) and (g). (i) Normalized line intensity profiles indicated at the white dashed line in (a) and (e). (j) Phase image obtained under simultaneous irradiation of the 488-, 532-, 635-nm lasers. The white bar indicateds a scale of 40 µm.

Fig. 7
Fig. 7

Calculated signal-to-noise ratio and contrast ratio of the conventional, four-bucket and phase images.

Equations (7)

Equations on this page are rendered with MathJax. Learn more.

I(x,y,t)= I dc (x,y)+ΔI(x,y)sin(2πft+ ϕ 0 ),
I 1 (x,y,t)= 0 T/4 I(x,y,t)dt ,
I 2 (x,y,t)= T/4 T/2 I(x,y,t)dt ,
I 3 (x,y,t)= T/2 3T/4 I(x,y,t)dt ,
I 4 (x,y,t)= 3T/4 T I(x,y,t)dt .
ΔI(x,y)= π T 2 ( I 1 I 3 ) 2 + ( I 2 I 4 ) 2 ,
ϕ 0 = tan 1 ( I 1 I 3 ( I 2 + I 4 ) I 1 I 3 + I 2 I 4 ).

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