Abstract

We present a frequency-multiplexed multi-site two-photon imaging method utilizing amplitude modulation of femtosecond laser pulses in the MHz range to tag each excitation beam and the corresponding fluorescence signals with specific frequencies. The frequency tags are generated with an interferometric scheme employing acousto-optic deflectors (AODs) to achieve precise spatial overlap of femtosecond laser pulses with periodically varying phase shift. Creating matching excitation beam patterns in each interferometer arm using multiple AOD driving frequencies, and subsequently overlapping these matching patterns, results in multiple encoded excitation beams with unique beat frequencies available for scanning. As a proof-of-concept, we demonstrate multiplexed two-photon image acquisition using test samples, and compare the performance of this approach to conventional two-photon laser scanning microscopy.

© 2017 Optical Society of America

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  7. K. P. Lillis, A. Eng, J. A. White, and J. Mertz, “Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution,” J. Neurosci. Methods 172(2), 178–184 (2008).
    [Crossref] [PubMed]
  8. A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
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    [Crossref] [PubMed]

2016 (3)

N. Ji, J. Freeman, and S. L. Smith, “Technologies for imaging neural activity in large volumes,” Nat. Neurosci. 19(9), 1154–1164 (2016).
[Crossref] [PubMed]

N. J. Sofroniew, D. Flickinger, J. King, and K. Svoboda, “A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging,” eLife 5, e14472 (2016).
[Crossref] [PubMed]

J. N. Stirman, I. T. Smith, M. W. Kudenov, and S. L. Smith, “Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain,” Nat. Biotechnol. 34(8), 857–862 (2016).
[Crossref] [PubMed]

2013 (2)

M. Ducros, Y. Goulam Houssen, J. Bradley, V. de Sars, and S. Charpak, “Encoded multisite two-photon microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(32), 13138–13143 (2013).
[Crossref] [PubMed]

E. D. Diebold, B. W. Buckley, D. R. Gossett, and B. Jalali, “Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy,” Nat. Photonics 7(10), 806–810 (2013).
[Crossref]

2012 (3)

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

S. S. Howard, A. Straub, N. Horton, D. Kobat, and C. Xu, “Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy,” Nat. Photonics 7(1), 33–37 (2012).
[Crossref] [PubMed]

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

2011 (2)

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[Crossref] [PubMed]

J. Zheng, Y. Jiang, Y. Zhang, P. Tang, A. Huang, and S. Zhuang, “Experiments and quantitative analysis of frequency division multiplexing confocal fluorescence microscopy with UV excitation,” J. Microsc. 244(2), 129–135 (2011).
[Crossref] [PubMed]

2010 (1)

2008 (2)

K. P. Lillis, A. Eng, J. A. White, and J. Mertz, “Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution,” J. Neurosci. Methods 172(2), 178–184 (2008).
[Crossref] [PubMed]

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

2007 (2)

S. Zeng, X. Lv, K. Bi, C. Zhan, D. Li, W. R. Chen, W. Xiong, S. L. Jacques, and Q. Luo, “Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging,” J. Biomed. Opt. 12(2), 024015 (2007).
[Crossref] [PubMed]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref] [PubMed]

2006 (1)

F. Wu, X. Zhang, J. Y. Cheung, K. Shi, Z. Liu, C. Luo, S. Yin, and P. Ruffin, “Frequency division multiplexed multichannel high-speed fluorescence confocal microscope,” Biophys. J. 91(6), 2290–2296 (2006).
[Crossref] [PubMed]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Arganda-Carreras, I.

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

Arisaka, K.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[Crossref] [PubMed]

Bahlmann, K.

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

Bi, K.

S. Zeng, X. Lv, K. Bi, C. Zhan, D. Li, W. R. Chen, W. Xiong, S. L. Jacques, and Q. Luo, “Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging,” J. Biomed. Opt. 12(2), 024015 (2007).
[Crossref] [PubMed]

Bradley, J.

M. Ducros, Y. Goulam Houssen, J. Bradley, V. de Sars, and S. Charpak, “Encoded multisite two-photon microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(32), 13138–13143 (2013).
[Crossref] [PubMed]

Buckley, B. W.

E. D. Diebold, B. W. Buckley, D. R. Gossett, and B. Jalali, “Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy,” Nat. Photonics 7(10), 806–810 (2013).
[Crossref]

Charpak, S.

M. Ducros, Y. Goulam Houssen, J. Bradley, V. de Sars, and S. Charpak, “Encoded multisite two-photon microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(32), 13138–13143 (2013).
[Crossref] [PubMed]

Chen, N.

Chen, W. R.

S. Zeng, X. Lv, K. Bi, C. Zhan, D. Li, W. R. Chen, W. Xiong, S. L. Jacques, and Q. Luo, “Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging,” J. Biomed. Opt. 12(2), 024015 (2007).
[Crossref] [PubMed]

Cheng, A.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[Crossref] [PubMed]

Cheung, J. Y.

F. Wu, X. Zhang, J. Y. Cheung, K. Shi, Z. Liu, C. Luo, S. Yin, and P. Ruffin, “Frequency division multiplexed multichannel high-speed fluorescence confocal microscope,” Biophys. J. 91(6), 2290–2296 (2006).
[Crossref] [PubMed]

Chiovini, B.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

Chong, S. P.

de Sars, V.

M. Ducros, Y. Goulam Houssen, J. Bradley, V. de Sars, and S. Charpak, “Encoded multisite two-photon microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(32), 13138–13143 (2013).
[Crossref] [PubMed]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Diebold, E. D.

E. D. Diebold, B. W. Buckley, D. R. Gossett, and B. Jalali, “Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy,” Nat. Photonics 7(10), 806–810 (2013).
[Crossref]

Donnert, G.

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref] [PubMed]

Ducros, M.

M. Ducros, Y. Goulam Houssen, J. Bradley, V. de Sars, and S. Charpak, “Encoded multisite two-photon microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(32), 13138–13143 (2013).
[Crossref] [PubMed]

Duemani Reddy, G.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

Eggeling, C.

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref] [PubMed]

Eng, A.

K. P. Lillis, A. Eng, J. A. White, and J. Mertz, “Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution,” J. Neurosci. Methods 172(2), 178–184 (2008).
[Crossref] [PubMed]

Fink, R.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

Flickinger, D.

N. J. Sofroniew, D. Flickinger, J. King, and K. Svoboda, “A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging,” eLife 5, e14472 (2016).
[Crossref] [PubMed]

Freeman, J.

N. Ji, J. Freeman, and S. L. Smith, “Technologies for imaging neural activity in large volumes,” Nat. Neurosci. 19(9), 1154–1164 (2016).
[Crossref] [PubMed]

Golshani, P.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[Crossref] [PubMed]

Gonçalves, J. T.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[Crossref] [PubMed]

Gossett, D. R.

E. D. Diebold, B. W. Buckley, D. R. Gossett, and B. Jalali, “Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy,” Nat. Photonics 7(10), 806–810 (2013).
[Crossref]

Goulam Houssen, Y.

M. Ducros, Y. Goulam Houssen, J. Bradley, V. de Sars, and S. Charpak, “Encoded multisite two-photon microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(32), 13138–13143 (2013).
[Crossref] [PubMed]

Hell, S. W.

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref] [PubMed]

Hillier, D.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

Horton, N.

S. S. Howard, A. Straub, N. Horton, D. Kobat, and C. Xu, “Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy,” Nat. Photonics 7(1), 33–37 (2012).
[Crossref] [PubMed]

Howard, S. S.

S. S. Howard, A. Straub, N. Horton, D. Kobat, and C. Xu, “Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy,” Nat. Photonics 7(1), 33–37 (2012).
[Crossref] [PubMed]

Huang, A.

J. Zheng, Y. Jiang, Y. Zhang, P. Tang, A. Huang, and S. Zhuang, “Experiments and quantitative analysis of frequency division multiplexing confocal fluorescence microscopy with UV excitation,” J. Microsc. 244(2), 129–135 (2011).
[Crossref] [PubMed]

Jacques, S. L.

S. Zeng, X. Lv, K. Bi, C. Zhan, D. Li, W. R. Chen, W. Xiong, S. L. Jacques, and Q. Luo, “Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging,” J. Biomed. Opt. 12(2), 024015 (2007).
[Crossref] [PubMed]

Jalali, B.

E. D. Diebold, B. W. Buckley, D. R. Gossett, and B. Jalali, “Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy,” Nat. Photonics 7(10), 806–810 (2013).
[Crossref]

Ji, N.

N. Ji, J. Freeman, and S. L. Smith, “Technologies for imaging neural activity in large volumes,” Nat. Neurosci. 19(9), 1154–1164 (2016).
[Crossref] [PubMed]

Jiang, Y.

J. Zheng, Y. Jiang, Y. Zhang, P. Tang, A. Huang, and S. Zhuang, “Experiments and quantitative analysis of frequency division multiplexing confocal fluorescence microscopy with UV excitation,” J. Microsc. 244(2), 129–135 (2011).
[Crossref] [PubMed]

Kadiri, L. R.

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

Kaszás, A.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

Katona, G.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

Kelleher, K.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

Kim, Y.

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

King, J.

N. J. Sofroniew, D. Flickinger, J. King, and K. Svoboda, “A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging,” eLife 5, e14472 (2016).
[Crossref] [PubMed]

Kobat, D.

S. S. Howard, A. Straub, N. Horton, D. Kobat, and C. Xu, “Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy,” Nat. Photonics 7(1), 33–37 (2012).
[Crossref] [PubMed]

Kudenov, M. W.

J. N. Stirman, I. T. Smith, M. W. Kudenov, and S. L. Smith, “Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain,” Nat. Biotechnol. 34(8), 857–862 (2016).
[Crossref] [PubMed]

Li, D.

S. Zeng, X. Lv, K. Bi, C. Zhan, D. Li, W. R. Chen, W. Xiong, S. L. Jacques, and Q. Luo, “Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging,” J. Biomed. Opt. 12(2), 024015 (2007).
[Crossref] [PubMed]

Lillis, K. P.

K. P. Lillis, A. Eng, J. A. White, and J. Mertz, “Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution,” J. Neurosci. Methods 172(2), 178–184 (2008).
[Crossref] [PubMed]

Liu, Z.

F. Wu, X. Zhang, J. Y. Cheung, K. Shi, Z. Liu, C. Luo, S. Yin, and P. Ruffin, “Frequency division multiplexed multichannel high-speed fluorescence confocal microscope,” Biophys. J. 91(6), 2290–2296 (2006).
[Crossref] [PubMed]

Luo, C.

F. Wu, X. Zhang, J. Y. Cheung, K. Shi, Z. Liu, C. Luo, S. Yin, and P. Ruffin, “Frequency division multiplexed multichannel high-speed fluorescence confocal microscope,” Biophys. J. 91(6), 2290–2296 (2006).
[Crossref] [PubMed]

Luo, Q.

S. Zeng, X. Lv, K. Bi, C. Zhan, D. Li, W. R. Chen, W. Xiong, S. L. Jacques, and Q. Luo, “Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging,” J. Biomed. Opt. 12(2), 024015 (2007).
[Crossref] [PubMed]

Lv, X.

S. Zeng, X. Lv, K. Bi, C. Zhan, D. Li, W. R. Chen, W. Xiong, S. L. Jacques, and Q. Luo, “Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging,” J. Biomed. Opt. 12(2), 024015 (2007).
[Crossref] [PubMed]

Maák, P.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

Mertz, J.

K. P. Lillis, A. Eng, J. A. White, and J. Mertz, “Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution,” J. Neurosci. Methods 172(2), 178–184 (2008).
[Crossref] [PubMed]

Osten, P.

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

Portera-Cailliau, C.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[Crossref] [PubMed]

Ragan, T.

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

Roska, B.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

Rózsa, B.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

Ruffin, P.

F. Wu, X. Zhang, J. Y. Cheung, K. Shi, Z. Liu, C. Luo, S. Yin, and P. Ruffin, “Frequency division multiplexed multichannel high-speed fluorescence confocal microscope,” Biophys. J. 91(6), 2290–2296 (2006).
[Crossref] [PubMed]

Saggau, P.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

Seung, H. S.

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

Sheppard, C. J.

Shi, K.

F. Wu, X. Zhang, J. Y. Cheung, K. Shi, Z. Liu, C. Luo, S. Yin, and P. Ruffin, “Frequency division multiplexed multichannel high-speed fluorescence confocal microscope,” Biophys. J. 91(6), 2290–2296 (2006).
[Crossref] [PubMed]

Smith, I. T.

J. N. Stirman, I. T. Smith, M. W. Kudenov, and S. L. Smith, “Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain,” Nat. Biotechnol. 34(8), 857–862 (2016).
[Crossref] [PubMed]

Smith, S. L.

J. N. Stirman, I. T. Smith, M. W. Kudenov, and S. L. Smith, “Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain,” Nat. Biotechnol. 34(8), 857–862 (2016).
[Crossref] [PubMed]

N. Ji, J. Freeman, and S. L. Smith, “Technologies for imaging neural activity in large volumes,” Nat. Neurosci. 19(9), 1154–1164 (2016).
[Crossref] [PubMed]

Sofroniew, N. J.

N. J. Sofroniew, D. Flickinger, J. King, and K. Svoboda, “A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging,” eLife 5, e14472 (2016).
[Crossref] [PubMed]

Stirman, J. N.

J. N. Stirman, I. T. Smith, M. W. Kudenov, and S. L. Smith, “Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain,” Nat. Biotechnol. 34(8), 857–862 (2016).
[Crossref] [PubMed]

Straub, A.

S. S. Howard, A. Straub, N. Horton, D. Kobat, and C. Xu, “Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy,” Nat. Photonics 7(1), 33–37 (2012).
[Crossref] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Sutin, J.

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

Svoboda, K.

N. J. Sofroniew, D. Flickinger, J. King, and K. Svoboda, “A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging,” eLife 5, e14472 (2016).
[Crossref] [PubMed]

Szalay, G.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

Tang, P.

J. Zheng, Y. Jiang, Y. Zhang, P. Tang, A. Huang, and S. Zhuang, “Experiments and quantitative analysis of frequency division multiplexing confocal fluorescence microscopy with UV excitation,” J. Microsc. 244(2), 129–135 (2011).
[Crossref] [PubMed]

Taranda, J.

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

Venkataraju, K. U.

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

Veress, M.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

Vizi, E. S.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

White, J. A.

K. P. Lillis, A. Eng, J. A. White, and J. Mertz, “Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution,” J. Neurosci. Methods 172(2), 178–184 (2008).
[Crossref] [PubMed]

Wong, C. H.

Wong, K. F.

Wu, F.

F. Wu, X. Zhang, J. Y. Cheung, K. Shi, Z. Liu, C. Luo, S. Yin, and P. Ruffin, “Frequency division multiplexed multichannel high-speed fluorescence confocal microscope,” Biophys. J. 91(6), 2290–2296 (2006).
[Crossref] [PubMed]

Xiong, W.

S. Zeng, X. Lv, K. Bi, C. Zhan, D. Li, W. R. Chen, W. Xiong, S. L. Jacques, and Q. Luo, “Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging,” J. Biomed. Opt. 12(2), 024015 (2007).
[Crossref] [PubMed]

Xu, C.

S. S. Howard, A. Straub, N. Horton, D. Kobat, and C. Xu, “Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy,” Nat. Photonics 7(1), 33–37 (2012).
[Crossref] [PubMed]

Yin, S.

F. Wu, X. Zhang, J. Y. Cheung, K. Shi, Z. Liu, C. Luo, S. Yin, and P. Ruffin, “Frequency division multiplexed multichannel high-speed fluorescence confocal microscope,” Biophys. J. 91(6), 2290–2296 (2006).
[Crossref] [PubMed]

Zeng, S.

S. Zeng, X. Lv, K. Bi, C. Zhan, D. Li, W. R. Chen, W. Xiong, S. L. Jacques, and Q. Luo, “Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging,” J. Biomed. Opt. 12(2), 024015 (2007).
[Crossref] [PubMed]

Zhan, C.

S. Zeng, X. Lv, K. Bi, C. Zhan, D. Li, W. R. Chen, W. Xiong, S. L. Jacques, and Q. Luo, “Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging,” J. Biomed. Opt. 12(2), 024015 (2007).
[Crossref] [PubMed]

Zhang, X.

F. Wu, X. Zhang, J. Y. Cheung, K. Shi, Z. Liu, C. Luo, S. Yin, and P. Ruffin, “Frequency division multiplexed multichannel high-speed fluorescence confocal microscope,” Biophys. J. 91(6), 2290–2296 (2006).
[Crossref] [PubMed]

Zhang, Y.

J. Zheng, Y. Jiang, Y. Zhang, P. Tang, A. Huang, and S. Zhuang, “Experiments and quantitative analysis of frequency division multiplexing confocal fluorescence microscopy with UV excitation,” J. Microsc. 244(2), 129–135 (2011).
[Crossref] [PubMed]

Zheng, J.

J. Zheng, Y. Jiang, Y. Zhang, P. Tang, A. Huang, and S. Zhuang, “Experiments and quantitative analysis of frequency division multiplexing confocal fluorescence microscopy with UV excitation,” J. Microsc. 244(2), 129–135 (2011).
[Crossref] [PubMed]

Zhuang, S.

J. Zheng, Y. Jiang, Y. Zhang, P. Tang, A. Huang, and S. Zhuang, “Experiments and quantitative analysis of frequency division multiplexing confocal fluorescence microscopy with UV excitation,” J. Microsc. 244(2), 129–135 (2011).
[Crossref] [PubMed]

Biomed. Opt. Express (1)

Biophys. J. (1)

F. Wu, X. Zhang, J. Y. Cheung, K. Shi, Z. Liu, C. Luo, S. Yin, and P. Ruffin, “Frequency division multiplexed multichannel high-speed fluorescence confocal microscope,” Biophys. J. 91(6), 2290–2296 (2006).
[Crossref] [PubMed]

eLife (1)

N. J. Sofroniew, D. Flickinger, J. King, and K. Svoboda, “A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging,” eLife 5, e14472 (2016).
[Crossref] [PubMed]

J. Biomed. Opt. (1)

S. Zeng, X. Lv, K. Bi, C. Zhan, D. Li, W. R. Chen, W. Xiong, S. L. Jacques, and Q. Luo, “Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging,” J. Biomed. Opt. 12(2), 024015 (2007).
[Crossref] [PubMed]

J. Microsc. (1)

J. Zheng, Y. Jiang, Y. Zhang, P. Tang, A. Huang, and S. Zhuang, “Experiments and quantitative analysis of frequency division multiplexing confocal fluorescence microscopy with UV excitation,” J. Microsc. 244(2), 129–135 (2011).
[Crossref] [PubMed]

J. Neurosci. Methods (1)

K. P. Lillis, A. Eng, J. A. White, and J. Mertz, “Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution,” J. Neurosci. Methods 172(2), 178–184 (2008).
[Crossref] [PubMed]

Nat. Biotechnol. (1)

J. N. Stirman, I. T. Smith, M. W. Kudenov, and S. L. Smith, “Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain,” Nat. Biotechnol. 34(8), 857–862 (2016).
[Crossref] [PubMed]

Nat. Methods (4)

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[Crossref] [PubMed]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref] [PubMed]

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

Nat. Neurosci. (2)

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

N. Ji, J. Freeman, and S. L. Smith, “Technologies for imaging neural activity in large volumes,” Nat. Neurosci. 19(9), 1154–1164 (2016).
[Crossref] [PubMed]

Nat. Photonics (2)

S. S. Howard, A. Straub, N. Horton, D. Kobat, and C. Xu, “Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy,” Nat. Photonics 7(1), 33–37 (2012).
[Crossref] [PubMed]

E. D. Diebold, B. W. Buckley, D. R. Gossett, and B. Jalali, “Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy,” Nat. Photonics 7(10), 806–810 (2013).
[Crossref]

Proc. Natl. Acad. Sci. U.S.A. (1)

M. Ducros, Y. Goulam Houssen, J. Bradley, V. de Sars, and S. Charpak, “Encoded multisite two-photon microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(32), 13138–13143 (2013).
[Crossref] [PubMed]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Other (1)

R. J. Cotton, E. Froudarakis, P. Storer, P. Saggau, and A. S. Tolias, “Three-dimensional mapping of microcircuit correlation structure,” Front. Neural Circuits 7, 151–1-13 (2013).
[Crossref]

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Figures (6)

Fig. 1
Fig. 1

Diagram of experimental setup for frequency-multiplexed two-photon imaging. L is lens, NPBS is non-polarizing beam splitter, AOD is acousto-optic deflector, BS is beam stop, PM is periscopic mirror, DM is dichroic mirror, OBJ is objective, PMT is photomultiplier, ω is light frequency, Ω1 and Ω2 are acoustic frequencies. Lens pair L1-L2 is 2.4 × telescope, L3-L4 are 1:1 relay lenses.

Fig. 2
Fig. 2

(a) Measured femtosecond pulse train intensity of a single beam. (b) Amplitude-modulated pulse train created by overlapping two beams with acoustically-shifted frequencies. The sampling rate is 1 GHz.

Fig. 3
Fig. 3

(a) Frequency spectra of two excitation pulse trains. (b) Two-photon fluorescence signal from a microsphere amplitude-modulated at 10 MHz frequency. The sampling rate is 100 MHz. (c) Frequency spectra of emission signals from both channels. Second order harmonics are indicated by arrows.

Fig. 4
Fig. 4

(a,b) Demultiplexed images of 2 µm fluorescent beads with 10 MHz (a) and 8 MHz (b) excitation. (c,d) Corresponding background-corrected images. (e) Background correction image created by averaging images corresponding to 2, 3, 4, and 5 MHz frequencies. Note the intensity scale bar difference. (f) Brightfield image of the same beads.

Fig. 5
Fig. 5

SNR comparison of conventional and amplitude-modulated two-photon detection according to C1 and C2 criteria. SNR values computed from digitally synthesized waveforms containing 80 samples. The plots corresponding to 1 and 4 frequency-encoded channels are shown.

Fig. 6
Fig. 6

(a) Comparison of a conventional two-photon and a frequency-encoded image. Pixel dwell time equals 1μs. Image intensity scale is normalized per maximum pixel value. The scale bar is 20 μm. (b) Recorded waveforms corresponding to the brightest pixels in the images from (a). Waveforms are offset for clarity. The sampling rate is 250 MHz. (c) SNR comparison between frequency-encoded and conventional two-photon imaging. Computational methods are explained in the main text. (d) Comparison of mean values and standard deviations computed from selected ROIs in (a) corresponding to 4 and 10 MHz decoding frequencies.

Equations (4)

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I fl (t) I 0 2 3 8 [ 1+ 4 3 cos( ΔΩt )+ 1 3 cos( 2ΔΩt ) ],
SN R C1 = 2 7 T A i 2 A 1 + A N ={ A i = A j ,j[1..N] }= 2 7 T A i N ,
SN R Conv = T A i N .
SN R C2 = 16 21 T A i N .

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