Abstract

We present a series of experiments that demonstrate a super-sensitive chemical imaging technique based on multiphoton frequency-domain fluorescence lifetime imaging microscopy (MPM-FD-FLIM) that shows a 2× improvement in imaging speed compared to the theoretical limit of conventional MPM-FD-FLIM. Additionally, this technique produces unprecedented sensitivity over a large range of fluorescence lifetimes. These results are achieved through simple modifications to data analysis in a conventional MPM-FD-FLIM microscope and are based on an analytical model describing the signal-to-noise ratio (SNR) of a MPM-FD-FLIM system [J. Opt. Soc. Am. A 33, B1 (2016)]. Here we experimentally validate this model.

© 2016 Optical Society of America

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References

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    [Crossref]
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    [Crossref]
  4. S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods 7, 755–759 (2010).
    [Crossref]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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2016 (1)

2015 (1)

A. A. Khan, S. K. Fullerton-Shirey, and S. S. Howard, “Easily prepared ruthenium-complex nanomicelle probes for two-photon quantitative imaging of oxygen in aqueous media,” RSC Adv. 5, 291–300 (2015).
[Crossref]

2013 (2)

2012 (1)

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

2011 (1)

J. Lecoq, A. Parpaleix, E. Roussakis, M. Ducros, Y. G. Houssen, S. A. Vinogradov, and S. Charpak, “Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels,” Nat. Med. 17, 893–898 (2011).
[Crossref] [PubMed]

2010 (3)

2007 (2)

2005 (1)

G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15, 805–815 (2005).
[Crossref] [PubMed]

2003 (2)

J. Philip and K. Carlsson, “Theoretical investigation of the signal-to-noise ratio in fluorescence lifetime imaging,” J. Opt. Soc. Am. A 20, 368–379 (2003).
[Crossref]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369–1377 (2003).
[Crossref] [PubMed]

1990 (1)

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

Anand, P.

Anand, U.

Arai, K.

S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods 7, 755–759 (2010).
[Crossref]

Arlt, J.

Bahlmann, K.

Becker, W.

Benham, C.

Benninger, R. K. P.

Beule, P. A. A. D.

Boas, D. A.

M. A. Yaseen, S. Sakadžić, W. Wu, W. Becker, K. A. Kasischke, and D. A. Boas, “In vivo imaging of cerebral energy metabolism with two-photon fluorescence lifetime microscopy of NADH,” Biomed. Opt. Express 4, 307–321 (2013).
[Crossref] [PubMed]

S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods 7, 755–759 (2010).
[Crossref]

Buehler, C.

Buts, A.

Carlsson, K.

Charbon, E.

Charpak, S.

J. Lecoq, A. Parpaleix, E. Roussakis, M. Ducros, Y. G. Houssen, S. A. Vinogradov, and S. Charpak, “Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels,” Nat. Med. 17, 893–898 (2011).
[Crossref] [PubMed]

Clegg, R. M.

G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15, 805–815 (2005).
[Crossref] [PubMed]

Davis, D. M.

Denk, W.

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

Devor, A.

S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods 7, 755–759 (2010).
[Crossref]

Ducros, M.

J. Lecoq, A. Parpaleix, E. Roussakis, M. Ducros, Y. G. Houssen, S. A. Vinogradov, and S. Charpak, “Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels,” Nat. Med. 17, 893–898 (2011).
[Crossref] [PubMed]

Dunn, A. K.

Dunsby, C.

Estrada, A. D.

Fantini, S.

French, P. M. W.

Fullerton-Shirey, S. K.

A. A. Khan, S. K. Fullerton-Shirey, and S. S. Howard, “Easily prepared ruthenium-complex nanomicelle probes for two-photon quantitative imaging of oxygen in aqueous media,” RSC Adv. 5, 291–300 (2015).
[Crossref]

Heffer, E. L.

Henderson, R.

Hoover, E. E.

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics 7, 93–101 (2013).
[Crossref] [PubMed]

Horton, N. G.

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

Houssen, Y. G.

J. Lecoq, A. Parpaleix, E. Roussakis, M. Ducros, Y. G. Houssen, S. A. Vinogradov, and S. Charpak, “Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels,” Nat. Med. 17, 893–898 (2011).
[Crossref] [PubMed]

Howard, S. S.

Y. Zhang, A. A. Khan, G. D. Vigil, and S. S. Howard, “Investigation of signal-to-noise ratio in frequency-domain multiphoton fluorescence lifetime imaging microscopy,” J. Opt. Soc. Am. A 33, B1–B11 (2016).
[Crossref]

A. A. Khan, S. K. Fullerton-Shirey, and S. S. Howard, “Easily prepared ruthenium-complex nanomicelle probes for two-photon quantitative imaging of oxygen in aqueous media,” RSC Adv. 5, 291–300 (2015).
[Crossref]

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

Kasischke, K. A.

Khan, A. A.

Y. Zhang, A. A. Khan, G. D. Vigil, and S. S. Howard, “Investigation of signal-to-noise ratio in frequency-domain multiphoton fluorescence lifetime imaging microscopy,” J. Opt. Soc. Am. A 33, B1–B11 (2016).
[Crossref]

A. A. Khan, S. K. Fullerton-Shirey, and S. S. Howard, “Easily prepared ruthenium-complex nanomicelle probes for two-photon quantitative imaging of oxygen in aqueous media,” RSC Adv. 5, 291–300 (2015).
[Crossref]

Kim, K. H.

Kobat, D.

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

Kumar, S.

Lanigan, P. M. P.

Lecoq, J.

J. Lecoq, A. Parpaleix, E. Roussakis, M. Ducros, Y. G. Houssen, S. A. Vinogradov, and S. Charpak, “Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels,” Nat. Med. 17, 893–898 (2011).
[Crossref] [PubMed]

Lee, W.-C. A.

Li, D.-U.

Lo, E. H.

S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods 7, 755–759 (2010).
[Crossref]

Mandeville, E. T.

S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods 7, 755–759 (2010).
[Crossref]

Naylor, A.

Nedivi, E.

Neil, M. A. A.

Owen, D. M.

Parpaleix, A.

J. Lecoq, A. Parpaleix, E. Roussakis, M. Ducros, Y. G. Houssen, S. A. Vinogradov, and S. Charpak, “Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels,” Nat. Med. 17, 893–898 (2011).
[Crossref] [PubMed]

Philip, J.

Ragan, T.

Redford, G. I.

G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15, 805–815 (2005).
[Crossref] [PubMed]

Richardson, J.

Roussakis, E.

J. Lecoq, A. Parpaleix, E. Roussakis, M. Ducros, Y. G. Houssen, S. A. Vinogradov, and S. Charpak, “Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels,” Nat. Med. 17, 893–898 (2011).
[Crossref] [PubMed]

S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods 7, 755–759 (2010).
[Crossref]

Ruvinskaya, S.

S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods 7, 755–759 (2010).
[Crossref]

Sakadzic, S.

S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods 7, 755–759 (2010).
[Crossref]

Sakadžic, S.

So, P. T. C.

Squier, J. A.

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics 7, 93–101 (2013).
[Crossref] [PubMed]

Srinivasan, V. J.

S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods 7, 755–759 (2010).
[Crossref]

Stoppa, D.

Straub, A.

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

Strickler, J. H.

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

Vigil, G. D.

Vinogradov, S. A.

J. Lecoq, A. Parpaleix, E. Roussakis, M. Ducros, Y. G. Houssen, S. A. Vinogradov, and S. Charpak, “Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels,” Nat. Med. 17, 893–898 (2011).
[Crossref] [PubMed]

S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods 7, 755–759 (2010).
[Crossref]

Walker, R.

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369–1377 (2003).
[Crossref] [PubMed]

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

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369–1377 (2003).
[Crossref] [PubMed]

Wu, W.

Xu, C.

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

Yaseen, M. A.

M. A. Yaseen, S. Sakadžić, W. Wu, W. Becker, K. A. Kasischke, and D. A. Boas, “In vivo imaging of cerebral energy metabolism with two-photon fluorescence lifetime microscopy of NADH,” Biomed. Opt. Express 4, 307–321 (2013).
[Crossref] [PubMed]

S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods 7, 755–759 (2010).
[Crossref]

Zhang, Y.

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369–1377 (2003).
[Crossref] [PubMed]

Biomed. Opt. Express (1)

J. Fluoresc. (1)

G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15, 805–815 (2005).
[Crossref] [PubMed]

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

Nat. Biotechnol. (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369–1377 (2003).
[Crossref] [PubMed]

Nat. Med. (1)

J. Lecoq, A. Parpaleix, E. Roussakis, M. Ducros, Y. G. Houssen, S. A. Vinogradov, and S. Charpak, “Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels,” Nat. Med. 17, 893–898 (2011).
[Crossref] [PubMed]

Nat. Methods (1)

S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods 7, 755–759 (2010).
[Crossref]

Nat. Photonics (2)

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

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics 7, 93–101 (2013).
[Crossref] [PubMed]

Opt. Express (4)

RSC Adv. (1)

A. A. Khan, S. K. Fullerton-Shirey, and S. S. Howard, “Easily prepared ruthenium-complex nanomicelle probes for two-photon quantitative imaging of oxygen in aqueous media,” RSC Adv. 5, 291–300 (2015).
[Crossref]

Science (1)

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

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Figures (3)

Fig. 1
Fig. 1 MPM-FD-FLIM experimental setup.
Fig. 2
Fig. 2 (a) Representative experimental lifetime temporal plots (left) and histograms (right) for the 1ω, 2ω, and DC&1ω methods. (b) Experimental (symbols) and analytical (curves, from [13]) values of F versus modulation frequency for the 1.6 μs [Ru(dpp)3]2+ nanomi-celle sample. Inset: zoomed scale.
Fig. 3
Fig. 3 (a) Measured (symbols) and linear fit (lines) lifetime relative error versus integration time. Dashed green line: theoretical limit for one-photon sinusoidal FD-FLIM. (b) Lifetime standard deviation versus fluorescence lifetime for a constant modulation frequency of 62.5 kHz. Inset: zoomed view, clearly showing the expanded DC&1ω frequency range.

Equations (3)

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

a k = 1 T 0 T e 2 ( t ) exp ( i k ω t ) d t , d k = 1 T 0 T p ( t ) exp ( i k ω t ) d t , k = 0 , ± 1 , ± 2 , .
G 0 = m 2 + 2 2 , G 1 = m ω τ 1 + ω 2 τ 2 , G 2 = m 2 4 1 1 + 4 ω 2 τ 2 , S 0 = 0 , S 1 = m 1 1 + ω 2 τ 2 , S 2 = m 2 4 2 ω τ 1 + 4 ω 2 τ 2 .
τ = 1 ω 2 m m 2 + 2 G 0 S 1 1 .

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