Abstract

Ratiometric imaging is an invaluable tool for quantitative microscopy, allowing for robust detection of FRET, anisotropy, and spectral shifts of nano-scale optical probes in response to local physical and chemical variations such as local pH, ion composition, and electric potential. In this paper, we propose and demonstrate a scheme for widefield ratiometric imaging that allows for continuous tuning of the cutoff wavelength between its two spectral channels. This scheme is based on angle-tuning the image splitting dichroic beamsplitter, similar to previous works on tunable interference filters. This configuration allows for ratiometric imaging of spectrally heterogeneous samples, which require spectral tunability of the detection path in order to achieve good spectrally balanced ratiometric detection.

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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  1. B. S. Launikonis, J. Zhou, L. Royer, T. R. Shannon, G. Brum, and E. Ríos, “Confocal imaging of [Ca2 + ] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence,” J. Physiol. 567(2), 523–543 (2005).
    [Crossref]
  2. C. Manno, L. Figueroa, R. Fitts, and E. Ríos, “Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS,” J. Gen. Physiol. 141(3), 371–387 (2013).
    [Crossref]
  3. S. Farooq and J. Hohlbein, “Camera-based single-molecule FRET detection with improved time resolution,” Phys. Chem. Chem. Phys. 17(41), 27862–27872 (2015).
    [Crossref]
  4. X. Michalet, S. Weiss, and M. Jäger, “Single-molecule fluorescence studies of protein folding and conformational dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
    [Crossref]
  5. G. S. Harms, M. Sonnleitner, G. J. Schütz, H. J. Gruber, and T. Schmidt, “Single-molecule anisotropy imaging,” Biophys. J. 77(5), 2864–2870 (1999).
    [Crossref]
  6. G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260(6), 3440–3450 (1985).
  7. E. D. Wieder, H. Hang, and M. H. Fox, “Measurement of Intracellular pH Using Flow Cytometry With Carobxy-SNARF-1,” Cytometry 14(8), 916–921 (1993).
    [Crossref]
  8. A. P. Demchenko, A. S. Klymchenko, V. G. Pivovarenko, and S. Ercelen, “Ratiometric Probes: Design and Applications,” in Fluorescence Spectroscopy, Imaging and Probes. (Springer, 2002).
  9. V. Montana, D. L. Farkas, and L. M. Loew, “Dual-Wavelength Ratiometric Fluorescence Measurements of Membrane Potential,” Biochemistry 28(11), 4536–4539 (1989).
    [Crossref]
  10. S. Jayaraman, J. Biwersi, and A. S. Verkman, “Synthesis and characterization of dual-wavelength Cl−-sensitive fluorescent indicators for ratio imaging,” Am. J. Physiol. 276(3), C747–C757 (1999).
    [Crossref]
  11. H. A. MacLeod, Thin-Film Optical Filters (CRC Press, 2010).
  12. T. Erdogan, “Optical Filters for Wavelength Selection in Fluorescence Instrumentation,” Curr. Protoc. Cytom. 56(1), 1–25 (2011).
    [Crossref]
  13. P. Favreau, C. Hernandez, A. S. Lindsey, D. F. Alvarez, T. Rich, P. Prabhat, and S. J. Leavesley, “Thin-film tunable filters for hyperspectral fluorescence microscopy,” J. Biomed. Opt. 19(1), 011017 (2013).
    [Crossref]
  14. M. Lequime, “Tunable thin film filters: review and perspectives,” Proc. SPIE 5250, 302–311 (2004).
    [Crossref]
  15. O. Bar-Elli, D. Steinitz, G. Yang, R. Tenne, A. Ludwig, Y. Kuo, A. Triller, S. Weiss, and D. Oron, “Rapid Voltage Sensing with Single Nanorods via the Quantum Confined Stark Effect,” ACS Photonics 5(7), 2860–2867 (2018).
    [Crossref]
  16. T. Parasassi, G. De Stasio, A. d’Ubaldo, and E. Gratton, “Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence,” Biophys. J. 57(6), 1179–1186 (1990).
    [Crossref]
  17. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, 2006).
  18. D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, and K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2012).
    [Crossref]
  19. K. Park, Z. Deutsch, J. Li, D. Oron, and S. Weiss, “Single Molecule Quantum-Confined Stark Effect Measurements of Semiconductor Nanoparticles at Room Temperatue,” ACS Nano 6(11), 10013–10023 (2012).
    [Crossref]
  20. R. S. Bedlack, M.-d. Wei, and L. M. Loew, “Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth,” Neuron 9(3), 393–403 (1992).
    [Crossref]
  21. G. Pucihar, T. Kotnik, and D. Miklavčič, “Measuring the Induced Membrane Voltage with Di-8-ANEPPS,” J. Visualized Exp. 33, 1659 (2009).
    [Crossref]
  22. G. Pucihar, T. Kotnik, B. Valič, and D. Miklavčič, “Numerical Determination of Transmembrane Voltage Induced on Irregularly Shaped Cells,” Ann. Biomed. Eng. 34(4), 642–652 (2006).
    [Crossref]
  23. H. P. Schwan, “Electrical Properties of Tissue and Cell Suspensions,” Adv. Biol. Med. Phys. 5, 147–209 (1957).
    [Crossref]
  24. W. Y. Kao, C. E. Davis, Y. I. Kim, and J. M. Beach, “Fluorescence Emission Spectral Shift Measurements of Membrane Potential in Single Cells,” Biophys. J. 81(2), 1163–1170 (2001).
    [Crossref]
  25. A. D. Bachtel, R. A. Gray, J. M. Stohlman, E. B. Bourgeois, A. E. Pollard, and J. M. Rogers, “A Novel Approach to Dual Excitation Ratiometric Optical Mapping of Cardiac Action Potentials With Di-4-ANEPPS Using Pulsed LED Excitation,” IEEE Trans. Biomed. Eng. 58(7), 2120–2126 (2011).
    [Crossref]

2018 (1)

O. Bar-Elli, D. Steinitz, G. Yang, R. Tenne, A. Ludwig, Y. Kuo, A. Triller, S. Weiss, and D. Oron, “Rapid Voltage Sensing with Single Nanorods via the Quantum Confined Stark Effect,” ACS Photonics 5(7), 2860–2867 (2018).
[Crossref]

2015 (1)

S. Farooq and J. Hohlbein, “Camera-based single-molecule FRET detection with improved time resolution,” Phys. Chem. Chem. Phys. 17(41), 27862–27872 (2015).
[Crossref]

2013 (2)

C. Manno, L. Figueroa, R. Fitts, and E. Ríos, “Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS,” J. Gen. Physiol. 141(3), 371–387 (2013).
[Crossref]

P. Favreau, C. Hernandez, A. S. Lindsey, D. F. Alvarez, T. Rich, P. Prabhat, and S. J. Leavesley, “Thin-film tunable filters for hyperspectral fluorescence microscopy,” J. Biomed. Opt. 19(1), 011017 (2013).
[Crossref]

2012 (2)

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, and K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2012).
[Crossref]

K. Park, Z. Deutsch, J. Li, D. Oron, and S. Weiss, “Single Molecule Quantum-Confined Stark Effect Measurements of Semiconductor Nanoparticles at Room Temperatue,” ACS Nano 6(11), 10013–10023 (2012).
[Crossref]

2011 (2)

T. Erdogan, “Optical Filters for Wavelength Selection in Fluorescence Instrumentation,” Curr. Protoc. Cytom. 56(1), 1–25 (2011).
[Crossref]

A. D. Bachtel, R. A. Gray, J. M. Stohlman, E. B. Bourgeois, A. E. Pollard, and J. M. Rogers, “A Novel Approach to Dual Excitation Ratiometric Optical Mapping of Cardiac Action Potentials With Di-4-ANEPPS Using Pulsed LED Excitation,” IEEE Trans. Biomed. Eng. 58(7), 2120–2126 (2011).
[Crossref]

2009 (1)

G. Pucihar, T. Kotnik, and D. Miklavčič, “Measuring the Induced Membrane Voltage with Di-8-ANEPPS,” J. Visualized Exp. 33, 1659 (2009).
[Crossref]

2006 (2)

G. Pucihar, T. Kotnik, B. Valič, and D. Miklavčič, “Numerical Determination of Transmembrane Voltage Induced on Irregularly Shaped Cells,” Ann. Biomed. Eng. 34(4), 642–652 (2006).
[Crossref]

X. Michalet, S. Weiss, and M. Jäger, “Single-molecule fluorescence studies of protein folding and conformational dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
[Crossref]

2005 (1)

B. S. Launikonis, J. Zhou, L. Royer, T. R. Shannon, G. Brum, and E. Ríos, “Confocal imaging of [Ca2 + ] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence,” J. Physiol. 567(2), 523–543 (2005).
[Crossref]

2004 (1)

M. Lequime, “Tunable thin film filters: review and perspectives,” Proc. SPIE 5250, 302–311 (2004).
[Crossref]

2001 (1)

W. Y. Kao, C. E. Davis, Y. I. Kim, and J. M. Beach, “Fluorescence Emission Spectral Shift Measurements of Membrane Potential in Single Cells,” Biophys. J. 81(2), 1163–1170 (2001).
[Crossref]

1999 (2)

S. Jayaraman, J. Biwersi, and A. S. Verkman, “Synthesis and characterization of dual-wavelength Cl−-sensitive fluorescent indicators for ratio imaging,” Am. J. Physiol. 276(3), C747–C757 (1999).
[Crossref]

G. S. Harms, M. Sonnleitner, G. J. Schütz, H. J. Gruber, and T. Schmidt, “Single-molecule anisotropy imaging,” Biophys. J. 77(5), 2864–2870 (1999).
[Crossref]

1993 (1)

E. D. Wieder, H. Hang, and M. H. Fox, “Measurement of Intracellular pH Using Flow Cytometry With Carobxy-SNARF-1,” Cytometry 14(8), 916–921 (1993).
[Crossref]

1992 (1)

R. S. Bedlack, M.-d. Wei, and L. M. Loew, “Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth,” Neuron 9(3), 393–403 (1992).
[Crossref]

1990 (1)

T. Parasassi, G. De Stasio, A. d’Ubaldo, and E. Gratton, “Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence,” Biophys. J. 57(6), 1179–1186 (1990).
[Crossref]

1989 (1)

V. Montana, D. L. Farkas, and L. M. Loew, “Dual-Wavelength Ratiometric Fluorescence Measurements of Membrane Potential,” Biochemistry 28(11), 4536–4539 (1989).
[Crossref]

1985 (1)

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260(6), 3440–3450 (1985).

1957 (1)

H. P. Schwan, “Electrical Properties of Tissue and Cell Suspensions,” Adv. Biol. Med. Phys. 5, 147–209 (1957).
[Crossref]

Abu-Siniyeh, A.

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, and K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2012).
[Crossref]

Alvarez, D. F.

P. Favreau, C. Hernandez, A. S. Lindsey, D. F. Alvarez, T. Rich, P. Prabhat, and S. J. Leavesley, “Thin-film tunable filters for hyperspectral fluorescence microscopy,” J. Biomed. Opt. 19(1), 011017 (2013).
[Crossref]

Bachtel, A. D.

A. D. Bachtel, R. A. Gray, J. M. Stohlman, E. B. Bourgeois, A. E. Pollard, and J. M. Rogers, “A Novel Approach to Dual Excitation Ratiometric Optical Mapping of Cardiac Action Potentials With Di-4-ANEPPS Using Pulsed LED Excitation,” IEEE Trans. Biomed. Eng. 58(7), 2120–2126 (2011).
[Crossref]

Bar-Elli, O.

O. Bar-Elli, D. Steinitz, G. Yang, R. Tenne, A. Ludwig, Y. Kuo, A. Triller, S. Weiss, and D. Oron, “Rapid Voltage Sensing with Single Nanorods via the Quantum Confined Stark Effect,” ACS Photonics 5(7), 2860–2867 (2018).
[Crossref]

Beach, J. M.

W. Y. Kao, C. E. Davis, Y. I. Kim, and J. M. Beach, “Fluorescence Emission Spectral Shift Measurements of Membrane Potential in Single Cells,” Biophys. J. 81(2), 1163–1170 (2001).
[Crossref]

Bedlack, R. S.

R. S. Bedlack, M.-d. Wei, and L. M. Loew, “Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth,” Neuron 9(3), 393–403 (1992).
[Crossref]

Biwersi, J.

S. Jayaraman, J. Biwersi, and A. S. Verkman, “Synthesis and characterization of dual-wavelength Cl−-sensitive fluorescent indicators for ratio imaging,” Am. J. Physiol. 276(3), C747–C757 (1999).
[Crossref]

Bourgeois, E. B.

A. D. Bachtel, R. A. Gray, J. M. Stohlman, E. B. Bourgeois, A. E. Pollard, and J. M. Rogers, “A Novel Approach to Dual Excitation Ratiometric Optical Mapping of Cardiac Action Potentials With Di-4-ANEPPS Using Pulsed LED Excitation,” IEEE Trans. Biomed. Eng. 58(7), 2120–2126 (2011).
[Crossref]

Brum, G.

B. S. Launikonis, J. Zhou, L. Royer, T. R. Shannon, G. Brum, and E. Ríos, “Confocal imaging of [Ca2 + ] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence,” J. Physiol. 567(2), 523–543 (2005).
[Crossref]

d’Ubaldo, A.

T. Parasassi, G. De Stasio, A. d’Ubaldo, and E. Gratton, “Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence,” Biophys. J. 57(6), 1179–1186 (1990).
[Crossref]

Davis, C. E.

W. Y. Kao, C. E. Davis, Y. I. Kim, and J. M. Beach, “Fluorescence Emission Spectral Shift Measurements of Membrane Potential in Single Cells,” Biophys. J. 81(2), 1163–1170 (2001).
[Crossref]

De Stasio, G.

T. Parasassi, G. De Stasio, A. d’Ubaldo, and E. Gratton, “Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence,” Biophys. J. 57(6), 1179–1186 (1990).
[Crossref]

Demchenko, A. P.

A. P. Demchenko, A. S. Klymchenko, V. G. Pivovarenko, and S. Ercelen, “Ratiometric Probes: Design and Applications,” in Fluorescence Spectroscopy, Imaging and Probes. (Springer, 2002).

Deutsch, Z.

K. Park, Z. Deutsch, J. Li, D. Oron, and S. Weiss, “Single Molecule Quantum-Confined Stark Effect Measurements of Semiconductor Nanoparticles at Room Temperatue,” ACS Nano 6(11), 10013–10023 (2012).
[Crossref]

Ercelen, S.

A. P. Demchenko, A. S. Klymchenko, V. G. Pivovarenko, and S. Ercelen, “Ratiometric Probes: Design and Applications,” in Fluorescence Spectroscopy, Imaging and Probes. (Springer, 2002).

Erdogan, T.

T. Erdogan, “Optical Filters for Wavelength Selection in Fluorescence Instrumentation,” Curr. Protoc. Cytom. 56(1), 1–25 (2011).
[Crossref]

Farkas, D. L.

V. Montana, D. L. Farkas, and L. M. Loew, “Dual-Wavelength Ratiometric Fluorescence Measurements of Membrane Potential,” Biochemistry 28(11), 4536–4539 (1989).
[Crossref]

Farooq, S.

S. Farooq and J. Hohlbein, “Camera-based single-molecule FRET detection with improved time resolution,” Phys. Chem. Chem. Phys. 17(41), 27862–27872 (2015).
[Crossref]

Favreau, P.

P. Favreau, C. Hernandez, A. S. Lindsey, D. F. Alvarez, T. Rich, P. Prabhat, and S. J. Leavesley, “Thin-film tunable filters for hyperspectral fluorescence microscopy,” J. Biomed. Opt. 19(1), 011017 (2013).
[Crossref]

Figueroa, L.

C. Manno, L. Figueroa, R. Fitts, and E. Ríos, “Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS,” J. Gen. Physiol. 141(3), 371–387 (2013).
[Crossref]

Fitts, R.

C. Manno, L. Figueroa, R. Fitts, and E. Ríos, “Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS,” J. Gen. Physiol. 141(3), 371–387 (2013).
[Crossref]

Fox, M. H.

E. D. Wieder, H. Hang, and M. H. Fox, “Measurement of Intracellular pH Using Flow Cytometry With Carobxy-SNARF-1,” Cytometry 14(8), 916–921 (1993).
[Crossref]

Gaus, K.

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, and K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2012).
[Crossref]

Gratton, E.

T. Parasassi, G. De Stasio, A. d’Ubaldo, and E. Gratton, “Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence,” Biophys. J. 57(6), 1179–1186 (1990).
[Crossref]

Gray, R. A.

A. D. Bachtel, R. A. Gray, J. M. Stohlman, E. B. Bourgeois, A. E. Pollard, and J. M. Rogers, “A Novel Approach to Dual Excitation Ratiometric Optical Mapping of Cardiac Action Potentials With Di-4-ANEPPS Using Pulsed LED Excitation,” IEEE Trans. Biomed. Eng. 58(7), 2120–2126 (2011).
[Crossref]

Gruber, H. J.

G. S. Harms, M. Sonnleitner, G. J. Schütz, H. J. Gruber, and T. Schmidt, “Single-molecule anisotropy imaging,” Biophys. J. 77(5), 2864–2870 (1999).
[Crossref]

Grynkiewicz, G.

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260(6), 3440–3450 (1985).

Hang, H.

E. D. Wieder, H. Hang, and M. H. Fox, “Measurement of Intracellular pH Using Flow Cytometry With Carobxy-SNARF-1,” Cytometry 14(8), 916–921 (1993).
[Crossref]

Harms, G. S.

G. S. Harms, M. Sonnleitner, G. J. Schütz, H. J. Gruber, and T. Schmidt, “Single-molecule anisotropy imaging,” Biophys. J. 77(5), 2864–2870 (1999).
[Crossref]

Hernandez, C.

P. Favreau, C. Hernandez, A. S. Lindsey, D. F. Alvarez, T. Rich, P. Prabhat, and S. J. Leavesley, “Thin-film tunable filters for hyperspectral fluorescence microscopy,” J. Biomed. Opt. 19(1), 011017 (2013).
[Crossref]

Hohlbein, J.

S. Farooq and J. Hohlbein, “Camera-based single-molecule FRET detection with improved time resolution,” Phys. Chem. Chem. Phys. 17(41), 27862–27872 (2015).
[Crossref]

Jäger, M.

X. Michalet, S. Weiss, and M. Jäger, “Single-molecule fluorescence studies of protein folding and conformational dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
[Crossref]

Jayaraman, S.

S. Jayaraman, J. Biwersi, and A. S. Verkman, “Synthesis and characterization of dual-wavelength Cl−-sensitive fluorescent indicators for ratio imaging,” Am. J. Physiol. 276(3), C747–C757 (1999).
[Crossref]

Kao, W. Y.

W. Y. Kao, C. E. Davis, Y. I. Kim, and J. M. Beach, “Fluorescence Emission Spectral Shift Measurements of Membrane Potential in Single Cells,” Biophys. J. 81(2), 1163–1170 (2001).
[Crossref]

Kim, Y. I.

W. Y. Kao, C. E. Davis, Y. I. Kim, and J. M. Beach, “Fluorescence Emission Spectral Shift Measurements of Membrane Potential in Single Cells,” Biophys. J. 81(2), 1163–1170 (2001).
[Crossref]

Klymchenko, A. S.

A. P. Demchenko, A. S. Klymchenko, V. G. Pivovarenko, and S. Ercelen, “Ratiometric Probes: Design and Applications,” in Fluorescence Spectroscopy, Imaging and Probes. (Springer, 2002).

Kotnik, T.

G. Pucihar, T. Kotnik, and D. Miklavčič, “Measuring the Induced Membrane Voltage with Di-8-ANEPPS,” J. Visualized Exp. 33, 1659 (2009).
[Crossref]

G. Pucihar, T. Kotnik, B. Valič, and D. Miklavčič, “Numerical Determination of Transmembrane Voltage Induced on Irregularly Shaped Cells,” Ann. Biomed. Eng. 34(4), 642–652 (2006).
[Crossref]

Kuo, Y.

O. Bar-Elli, D. Steinitz, G. Yang, R. Tenne, A. Ludwig, Y. Kuo, A. Triller, S. Weiss, and D. Oron, “Rapid Voltage Sensing with Single Nanorods via the Quantum Confined Stark Effect,” ACS Photonics 5(7), 2860–2867 (2018).
[Crossref]

Lakowicz, J. R.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, 2006).

Launikonis, B. S.

B. S. Launikonis, J. Zhou, L. Royer, T. R. Shannon, G. Brum, and E. Ríos, “Confocal imaging of [Ca2 + ] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence,” J. Physiol. 567(2), 523–543 (2005).
[Crossref]

Leavesley, S. J.

P. Favreau, C. Hernandez, A. S. Lindsey, D. F. Alvarez, T. Rich, P. Prabhat, and S. J. Leavesley, “Thin-film tunable filters for hyperspectral fluorescence microscopy,” J. Biomed. Opt. 19(1), 011017 (2013).
[Crossref]

Lequime, M.

M. Lequime, “Tunable thin film filters: review and perspectives,” Proc. SPIE 5250, 302–311 (2004).
[Crossref]

Li, J.

K. Park, Z. Deutsch, J. Li, D. Oron, and S. Weiss, “Single Molecule Quantum-Confined Stark Effect Measurements of Semiconductor Nanoparticles at Room Temperatue,” ACS Nano 6(11), 10013–10023 (2012).
[Crossref]

Lindsey, A. S.

P. Favreau, C. Hernandez, A. S. Lindsey, D. F. Alvarez, T. Rich, P. Prabhat, and S. J. Leavesley, “Thin-film tunable filters for hyperspectral fluorescence microscopy,” J. Biomed. Opt. 19(1), 011017 (2013).
[Crossref]

Loew, L. M.

R. S. Bedlack, M.-d. Wei, and L. M. Loew, “Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth,” Neuron 9(3), 393–403 (1992).
[Crossref]

V. Montana, D. L. Farkas, and L. M. Loew, “Dual-Wavelength Ratiometric Fluorescence Measurements of Membrane Potential,” Biochemistry 28(11), 4536–4539 (1989).
[Crossref]

Ludwig, A.

O. Bar-Elli, D. Steinitz, G. Yang, R. Tenne, A. Ludwig, Y. Kuo, A. Triller, S. Weiss, and D. Oron, “Rapid Voltage Sensing with Single Nanorods via the Quantum Confined Stark Effect,” ACS Photonics 5(7), 2860–2867 (2018).
[Crossref]

MacLeod, H. A.

H. A. MacLeod, Thin-Film Optical Filters (CRC Press, 2010).

Magenau, A.

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, and K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2012).
[Crossref]

Manno, C.

C. Manno, L. Figueroa, R. Fitts, and E. Ríos, “Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS,” J. Gen. Physiol. 141(3), 371–387 (2013).
[Crossref]

Michalet, X.

X. Michalet, S. Weiss, and M. Jäger, “Single-molecule fluorescence studies of protein folding and conformational dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
[Crossref]

Miklavcic, D.

G. Pucihar, T. Kotnik, and D. Miklavčič, “Measuring the Induced Membrane Voltage with Di-8-ANEPPS,” J. Visualized Exp. 33, 1659 (2009).
[Crossref]

G. Pucihar, T. Kotnik, B. Valič, and D. Miklavčič, “Numerical Determination of Transmembrane Voltage Induced on Irregularly Shaped Cells,” Ann. Biomed. Eng. 34(4), 642–652 (2006).
[Crossref]

Montana, V.

V. Montana, D. L. Farkas, and L. M. Loew, “Dual-Wavelength Ratiometric Fluorescence Measurements of Membrane Potential,” Biochemistry 28(11), 4536–4539 (1989).
[Crossref]

Oron, D.

O. Bar-Elli, D. Steinitz, G. Yang, R. Tenne, A. Ludwig, Y. Kuo, A. Triller, S. Weiss, and D. Oron, “Rapid Voltage Sensing with Single Nanorods via the Quantum Confined Stark Effect,” ACS Photonics 5(7), 2860–2867 (2018).
[Crossref]

K. Park, Z. Deutsch, J. Li, D. Oron, and S. Weiss, “Single Molecule Quantum-Confined Stark Effect Measurements of Semiconductor Nanoparticles at Room Temperatue,” ACS Nano 6(11), 10013–10023 (2012).
[Crossref]

Owen, D. M.

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, and K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2012).
[Crossref]

Parasassi, T.

T. Parasassi, G. De Stasio, A. d’Ubaldo, and E. Gratton, “Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence,” Biophys. J. 57(6), 1179–1186 (1990).
[Crossref]

Park, K.

K. Park, Z. Deutsch, J. Li, D. Oron, and S. Weiss, “Single Molecule Quantum-Confined Stark Effect Measurements of Semiconductor Nanoparticles at Room Temperatue,” ACS Nano 6(11), 10013–10023 (2012).
[Crossref]

Pivovarenko, V. G.

A. P. Demchenko, A. S. Klymchenko, V. G. Pivovarenko, and S. Ercelen, “Ratiometric Probes: Design and Applications,” in Fluorescence Spectroscopy, Imaging and Probes. (Springer, 2002).

Poenie, M.

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260(6), 3440–3450 (1985).

Pollard, A. E.

A. D. Bachtel, R. A. Gray, J. M. Stohlman, E. B. Bourgeois, A. E. Pollard, and J. M. Rogers, “A Novel Approach to Dual Excitation Ratiometric Optical Mapping of Cardiac Action Potentials With Di-4-ANEPPS Using Pulsed LED Excitation,” IEEE Trans. Biomed. Eng. 58(7), 2120–2126 (2011).
[Crossref]

Prabhat, P.

P. Favreau, C. Hernandez, A. S. Lindsey, D. F. Alvarez, T. Rich, P. Prabhat, and S. J. Leavesley, “Thin-film tunable filters for hyperspectral fluorescence microscopy,” J. Biomed. Opt. 19(1), 011017 (2013).
[Crossref]

Pucihar, G.

G. Pucihar, T. Kotnik, and D. Miklavčič, “Measuring the Induced Membrane Voltage with Di-8-ANEPPS,” J. Visualized Exp. 33, 1659 (2009).
[Crossref]

G. Pucihar, T. Kotnik, B. Valič, and D. Miklavčič, “Numerical Determination of Transmembrane Voltage Induced on Irregularly Shaped Cells,” Ann. Biomed. Eng. 34(4), 642–652 (2006).
[Crossref]

Rentero, C.

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, and K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2012).
[Crossref]

Rich, T.

P. Favreau, C. Hernandez, A. S. Lindsey, D. F. Alvarez, T. Rich, P. Prabhat, and S. J. Leavesley, “Thin-film tunable filters for hyperspectral fluorescence microscopy,” J. Biomed. Opt. 19(1), 011017 (2013).
[Crossref]

Ríos, E.

C. Manno, L. Figueroa, R. Fitts, and E. Ríos, “Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS,” J. Gen. Physiol. 141(3), 371–387 (2013).
[Crossref]

B. S. Launikonis, J. Zhou, L. Royer, T. R. Shannon, G. Brum, and E. Ríos, “Confocal imaging of [Ca2 + ] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence,” J. Physiol. 567(2), 523–543 (2005).
[Crossref]

Rogers, J. M.

A. D. Bachtel, R. A. Gray, J. M. Stohlman, E. B. Bourgeois, A. E. Pollard, and J. M. Rogers, “A Novel Approach to Dual Excitation Ratiometric Optical Mapping of Cardiac Action Potentials With Di-4-ANEPPS Using Pulsed LED Excitation,” IEEE Trans. Biomed. Eng. 58(7), 2120–2126 (2011).
[Crossref]

Royer, L.

B. S. Launikonis, J. Zhou, L. Royer, T. R. Shannon, G. Brum, and E. Ríos, “Confocal imaging of [Ca2 + ] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence,” J. Physiol. 567(2), 523–543 (2005).
[Crossref]

Schmidt, T.

G. S. Harms, M. Sonnleitner, G. J. Schütz, H. J. Gruber, and T. Schmidt, “Single-molecule anisotropy imaging,” Biophys. J. 77(5), 2864–2870 (1999).
[Crossref]

Schütz, G. J.

G. S. Harms, M. Sonnleitner, G. J. Schütz, H. J. Gruber, and T. Schmidt, “Single-molecule anisotropy imaging,” Biophys. J. 77(5), 2864–2870 (1999).
[Crossref]

Schwan, H. P.

H. P. Schwan, “Electrical Properties of Tissue and Cell Suspensions,” Adv. Biol. Med. Phys. 5, 147–209 (1957).
[Crossref]

Shannon, T. R.

B. S. Launikonis, J. Zhou, L. Royer, T. R. Shannon, G. Brum, and E. Ríos, “Confocal imaging of [Ca2 + ] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence,” J. Physiol. 567(2), 523–543 (2005).
[Crossref]

Sonnleitner, M.

G. S. Harms, M. Sonnleitner, G. J. Schütz, H. J. Gruber, and T. Schmidt, “Single-molecule anisotropy imaging,” Biophys. J. 77(5), 2864–2870 (1999).
[Crossref]

Steinitz, D.

O. Bar-Elli, D. Steinitz, G. Yang, R. Tenne, A. Ludwig, Y. Kuo, A. Triller, S. Weiss, and D. Oron, “Rapid Voltage Sensing with Single Nanorods via the Quantum Confined Stark Effect,” ACS Photonics 5(7), 2860–2867 (2018).
[Crossref]

Stohlman, J. M.

A. D. Bachtel, R. A. Gray, J. M. Stohlman, E. B. Bourgeois, A. E. Pollard, and J. M. Rogers, “A Novel Approach to Dual Excitation Ratiometric Optical Mapping of Cardiac Action Potentials With Di-4-ANEPPS Using Pulsed LED Excitation,” IEEE Trans. Biomed. Eng. 58(7), 2120–2126 (2011).
[Crossref]

Tenne, R.

O. Bar-Elli, D. Steinitz, G. Yang, R. Tenne, A. Ludwig, Y. Kuo, A. Triller, S. Weiss, and D. Oron, “Rapid Voltage Sensing with Single Nanorods via the Quantum Confined Stark Effect,” ACS Photonics 5(7), 2860–2867 (2018).
[Crossref]

Triller, A.

O. Bar-Elli, D. Steinitz, G. Yang, R. Tenne, A. Ludwig, Y. Kuo, A. Triller, S. Weiss, and D. Oron, “Rapid Voltage Sensing with Single Nanorods via the Quantum Confined Stark Effect,” ACS Photonics 5(7), 2860–2867 (2018).
[Crossref]

Tsien, R. Y.

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260(6), 3440–3450 (1985).

Valic, B.

G. Pucihar, T. Kotnik, B. Valič, and D. Miklavčič, “Numerical Determination of Transmembrane Voltage Induced on Irregularly Shaped Cells,” Ann. Biomed. Eng. 34(4), 642–652 (2006).
[Crossref]

Verkman, A. S.

S. Jayaraman, J. Biwersi, and A. S. Verkman, “Synthesis and characterization of dual-wavelength Cl−-sensitive fluorescent indicators for ratio imaging,” Am. J. Physiol. 276(3), C747–C757 (1999).
[Crossref]

Wei, M.-d.

R. S. Bedlack, M.-d. Wei, and L. M. Loew, “Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth,” Neuron 9(3), 393–403 (1992).
[Crossref]

Weiss, S.

O. Bar-Elli, D. Steinitz, G. Yang, R. Tenne, A. Ludwig, Y. Kuo, A. Triller, S. Weiss, and D. Oron, “Rapid Voltage Sensing with Single Nanorods via the Quantum Confined Stark Effect,” ACS Photonics 5(7), 2860–2867 (2018).
[Crossref]

K. Park, Z. Deutsch, J. Li, D. Oron, and S. Weiss, “Single Molecule Quantum-Confined Stark Effect Measurements of Semiconductor Nanoparticles at Room Temperatue,” ACS Nano 6(11), 10013–10023 (2012).
[Crossref]

X. Michalet, S. Weiss, and M. Jäger, “Single-molecule fluorescence studies of protein folding and conformational dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
[Crossref]

Wieder, E. D.

E. D. Wieder, H. Hang, and M. H. Fox, “Measurement of Intracellular pH Using Flow Cytometry With Carobxy-SNARF-1,” Cytometry 14(8), 916–921 (1993).
[Crossref]

Yang, G.

O. Bar-Elli, D. Steinitz, G. Yang, R. Tenne, A. Ludwig, Y. Kuo, A. Triller, S. Weiss, and D. Oron, “Rapid Voltage Sensing with Single Nanorods via the Quantum Confined Stark Effect,” ACS Photonics 5(7), 2860–2867 (2018).
[Crossref]

Zhou, J.

B. S. Launikonis, J. Zhou, L. Royer, T. R. Shannon, G. Brum, and E. Ríos, “Confocal imaging of [Ca2 + ] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence,” J. Physiol. 567(2), 523–543 (2005).
[Crossref]

ACS Nano (1)

K. Park, Z. Deutsch, J. Li, D. Oron, and S. Weiss, “Single Molecule Quantum-Confined Stark Effect Measurements of Semiconductor Nanoparticles at Room Temperatue,” ACS Nano 6(11), 10013–10023 (2012).
[Crossref]

ACS Photonics (1)

O. Bar-Elli, D. Steinitz, G. Yang, R. Tenne, A. Ludwig, Y. Kuo, A. Triller, S. Weiss, and D. Oron, “Rapid Voltage Sensing with Single Nanorods via the Quantum Confined Stark Effect,” ACS Photonics 5(7), 2860–2867 (2018).
[Crossref]

Adv. Biol. Med. Phys. (1)

H. P. Schwan, “Electrical Properties of Tissue and Cell Suspensions,” Adv. Biol. Med. Phys. 5, 147–209 (1957).
[Crossref]

Am. J. Physiol. (1)

S. Jayaraman, J. Biwersi, and A. S. Verkman, “Synthesis and characterization of dual-wavelength Cl−-sensitive fluorescent indicators for ratio imaging,” Am. J. Physiol. 276(3), C747–C757 (1999).
[Crossref]

Ann. Biomed. Eng. (1)

G. Pucihar, T. Kotnik, B. Valič, and D. Miklavčič, “Numerical Determination of Transmembrane Voltage Induced on Irregularly Shaped Cells,” Ann. Biomed. Eng. 34(4), 642–652 (2006).
[Crossref]

Biochemistry (1)

V. Montana, D. L. Farkas, and L. M. Loew, “Dual-Wavelength Ratiometric Fluorescence Measurements of Membrane Potential,” Biochemistry 28(11), 4536–4539 (1989).
[Crossref]

Biophys. J. (3)

T. Parasassi, G. De Stasio, A. d’Ubaldo, and E. Gratton, “Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence,” Biophys. J. 57(6), 1179–1186 (1990).
[Crossref]

G. S. Harms, M. Sonnleitner, G. J. Schütz, H. J. Gruber, and T. Schmidt, “Single-molecule anisotropy imaging,” Biophys. J. 77(5), 2864–2870 (1999).
[Crossref]

W. Y. Kao, C. E. Davis, Y. I. Kim, and J. M. Beach, “Fluorescence Emission Spectral Shift Measurements of Membrane Potential in Single Cells,” Biophys. J. 81(2), 1163–1170 (2001).
[Crossref]

Chem. Rev. (1)

X. Michalet, S. Weiss, and M. Jäger, “Single-molecule fluorescence studies of protein folding and conformational dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
[Crossref]

Curr. Protoc. Cytom. (1)

T. Erdogan, “Optical Filters for Wavelength Selection in Fluorescence Instrumentation,” Curr. Protoc. Cytom. 56(1), 1–25 (2011).
[Crossref]

Cytometry (1)

E. D. Wieder, H. Hang, and M. H. Fox, “Measurement of Intracellular pH Using Flow Cytometry With Carobxy-SNARF-1,” Cytometry 14(8), 916–921 (1993).
[Crossref]

IEEE Trans. Biomed. Eng. (1)

A. D. Bachtel, R. A. Gray, J. M. Stohlman, E. B. Bourgeois, A. E. Pollard, and J. M. Rogers, “A Novel Approach to Dual Excitation Ratiometric Optical Mapping of Cardiac Action Potentials With Di-4-ANEPPS Using Pulsed LED Excitation,” IEEE Trans. Biomed. Eng. 58(7), 2120–2126 (2011).
[Crossref]

J. Biol. Chem. (1)

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260(6), 3440–3450 (1985).

J. Biomed. Opt. (1)

P. Favreau, C. Hernandez, A. S. Lindsey, D. F. Alvarez, T. Rich, P. Prabhat, and S. J. Leavesley, “Thin-film tunable filters for hyperspectral fluorescence microscopy,” J. Biomed. Opt. 19(1), 011017 (2013).
[Crossref]

J. Gen. Physiol. (1)

C. Manno, L. Figueroa, R. Fitts, and E. Ríos, “Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS,” J. Gen. Physiol. 141(3), 371–387 (2013).
[Crossref]

J. Physiol. (1)

B. S. Launikonis, J. Zhou, L. Royer, T. R. Shannon, G. Brum, and E. Ríos, “Confocal imaging of [Ca2 + ] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence,” J. Physiol. 567(2), 523–543 (2005).
[Crossref]

J. Visualized Exp. (1)

G. Pucihar, T. Kotnik, and D. Miklavčič, “Measuring the Induced Membrane Voltage with Di-8-ANEPPS,” J. Visualized Exp. 33, 1659 (2009).
[Crossref]

Nat. Protoc. (1)

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, and K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2012).
[Crossref]

Neuron (1)

R. S. Bedlack, M.-d. Wei, and L. M. Loew, “Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth,” Neuron 9(3), 393–403 (1992).
[Crossref]

Phys. Chem. Chem. Phys. (1)

S. Farooq and J. Hohlbein, “Camera-based single-molecule FRET detection with improved time resolution,” Phys. Chem. Chem. Phys. 17(41), 27862–27872 (2015).
[Crossref]

Proc. SPIE (1)

M. Lequime, “Tunable thin film filters: review and perspectives,” Proc. SPIE 5250, 302–311 (2004).
[Crossref]

Other (3)

H. A. MacLeod, Thin-Film Optical Filters (CRC Press, 2010).

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, 2006).

A. P. Demchenko, A. S. Klymchenko, V. G. Pivovarenko, and S. Ercelen, “Ratiometric Probes: Design and Applications,” in Fluorescence Spectroscopy, Imaging and Probes. (Springer, 2002).

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Figures (6)

Fig. 1.
Fig. 1. Optical set-up for spectrally balanced ratiometric widefield imaging. The image is split by a dichroic beamsplitter placed on a motorized rotation stage. The rotation stage holds both the image splitting dichroic beamsplitter, and a silver mirror, thus the dichroic-mirror pair act as a retroreflector, allowing for angle-tuning the dichroic beamsplitter without considerable misalignment of the reflected image.
Fig. 2.
Fig. 2. Transmission spectra characterization of two commercial filters: (i) Di03-R594 (Semrock), a longpass dichroic beamsplitter, and (ii) TLP01-628 (Semrock), a tunable longpass filter. (a) shows the transmission spectra of the filters at different angles of incidence. (b) and (c) show the cutoff wavelength and edge width vs. the angle of incidence, respectively. Since the dichroic beamsplitter (i) is designed to work optimally at 45° angle of incidence, as opposed to the angle-tunable filter (ii), considerable polarization splitting and additional transmission bands near the cutoff wavelength appear when tuning the dichroic beamsplitter to higher angles of incidence.
Fig. 3.
Fig. 3. Widefield fluorescence imaging of quantum dots at different angles of incidence (cutoff wavelengths) of the dichroic beamsplitter. CdSe/ZnS quantum dots (QSP-600, Ocean Nanotech) were diluted and dispersed on a glass coverslip, and imaged with an 100x objective (UPLSAPO100XO, Olympus) while angle-tuning dichroic beamsplitter (i). Images from the two cameras were superimposed into a single image with a small displacement, and false colored with red and green to distinguish between the transmitted and reflected channels, respectively. At the right of each image is the one-pixel line cross section for an individual particle, indicated by the yellow box, at both spectral channels, illustrating tuning of the spectral splitting between the two cameras. Scale bar: 1 µm.
Fig. 4.
Fig. 4. Theoretical ratiometric shift dependence upon the dichroic edge filter. Assuming a $\sigma = 10\, nm$ emission spectrum width which shifts its peak from 598 nm to 602 nm, as shown in (a), we use equations (1–2) to calculate the ratiometric shift $\Delta R$ as function of (b) the edge filter cutoff wavelength (assuming a constant edge width of ${\sigma _{DC}} = 10\, nm$), and (c) the dichroic edge width ${\sigma _{DC}}$ (assuming a constant edge cutoff wavelength of ${\lambda _{DC}} = 600\, nm$). Figure (d) is an alternative representation of figure (b), in which we convert the cutoff wavelength to the ratiometric value of the initial spectral state. The corresponding values of two specific cutoff wavelengths, 600 nm (black) and 580 nm (red), are shown in figures (a), and (b, d).
Fig. 5.
Fig. 5. Ratiometric measurement of QCSE in a semiconductor nanorod. (a) The emission fluorescence of a single nanorod under a square wave modulated electric field (f = 5 Hz; Emax=300 kV/cm). The cameras were recording at a 20 Hz frame rate, and synchronized to the applied field. Red-shaded areas correspond to Emax periods, and white areas indicate zero field. The emission intensity was spectrally split by angle-tuning the dichroic beamsplitter (i), providing a spectrally-balanced detection between the transmitted image (red line) and reflected image (green line). (b) and (c) show the total emission intensity and ratiometric value between the two channels, respectively. Although there is correlation between the total fluorescence emission intensity and the applied voltage, the spectral shift due to QCSE is clearly demonstrated along the whole trace via the ratiometric measurement.
Fig. 6.
Fig. 6. Ratiometric imaging of HEK 293 cells stained with di-8-ANEPPS upon pulsed electric polarization. (a) Ratiometric images of a stained HEK 293 cell at different angles of incidence (cutoff wavelengths) of the tunable filter (ii). Imaging was performed with a 60x objective (PLAPON60XO, Olympus). Images from the two cameras were superimposed into a single image, and false colored with red and green corresponding to the transmitted and reflected channels, respectively. Scale bar: 5 µm. The cells were placed between platinum electrodes and ten pulses of 80 V, each 100 ms in duration, were applied every one second. Three imaging conditions are shown: (b) and (c) were performed under 470 nm excitation at 45° (590 nm) and 40° (600 nm) angles of incidence, respectively, and (d) was performed under 440 nm excitation at 40° (600 nm) angle of incidence. The cameras were recording at a 20 Hz frame rate, and synchronized to the applied field. Red-shaded areas correspond to durations in which the external field was applied, and white areas indicate a duration of 100 ms before each pulse, in which no electric field was applied. The top panel of (b-d) show the total detected fluorescence emission from the half of the cell closer to the positive electrode, for both the transmitted (red line) and reflected (green line) images. The middle and bottom panels of (b-d) show the total emission intensity and ratiometric value between the two channels, respectively.

Equations (2)

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

F T = exp [ ( λ λ 0 2 σ ) 2 ] × ( 1 + exp [ ( λ λ D C σ D C ) ] ) 1 ,
F R = exp [ ( λ λ 0 2 σ ) 2 ] × ( 1 + exp [ ( λ λ D C σ D C ) ] ) 1 ,

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