Abstract

Conventional fluorescence detection is sensitive to an intricate product of the number of fluorescent emitters times the local excitation intensity. Here, we describe a method to locally quantify the excitation intensity and the number of emitters separately, enabling a clear distinction between the phenomena responsible for a given fluorescence signal. Our technique is based on harmonic excitation modulation and higher-order fluorescence demodulation. It is compatible with a wide range of observations, opening new characterization routes for fluorescence detection or imaging on complex systems, such as plasmonic substrates or scattering media.

© 2011 Optical Society of America

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References

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  1. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, 2006).
    [CrossRef]
  2. K. Aslan, I. Gryczynski, J. Malicka, E. Matveeva, J. R. Lakowicz, and C. D. Geddes, Curr. Opin. Biotechnol. 16, 55 (2005).
    [CrossRef] [PubMed]
  3. E. Fort and S. Grésillon, J. Phys. D 41, 013001 (2008).
    [CrossRef]
  4. E. Le Moal, E. Fort, S. Lévêque-Fort, F. P. Cordelières, M.-P. Fontaine-Aupart, and C. Ricolleau, Biophys. J. 92, 2150 (2007).
    [CrossRef]
  5. X. Cui, K. Tawa, H. Hori, and J. Nishii, Adv. Funct. Mater. 20, 546 (2010).
    [CrossRef]
  6. I. M. Vellekoop, A. Lagendijk, and A. P. Mosk, Nat. Photon. 4, 320 (2010).
    [CrossRef]
  7. F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
    [CrossRef] [PubMed]
  8. K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
    [CrossRef]
  9. S. Kawano, N. I. Smith, M. Yamanaka, S. Kawata, and K. Fujita, Appl. Phys. Express 4, 042401 (2011).
    [CrossRef]
  10. M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, J. Biomed. Opt. 13, 050507 (2008).
    [CrossRef] [PubMed]
  11. J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, Opt. Express 16, 3008 (2008).
    [CrossRef] [PubMed]

2011 (1)

S. Kawano, N. I. Smith, M. Yamanaka, S. Kawata, and K. Fujita, Appl. Phys. Express 4, 042401 (2011).
[CrossRef]

2010 (2)

X. Cui, K. Tawa, H. Hori, and J. Nishii, Adv. Funct. Mater. 20, 546 (2010).
[CrossRef]

I. M. Vellekoop, A. Lagendijk, and A. P. Mosk, Nat. Photon. 4, 320 (2010).
[CrossRef]

2008 (3)

E. Fort and S. Grésillon, J. Phys. D 41, 013001 (2008).
[CrossRef]

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, J. Biomed. Opt. 13, 050507 (2008).
[CrossRef] [PubMed]

J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, Opt. Express 16, 3008 (2008).
[CrossRef] [PubMed]

2007 (2)

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[CrossRef]

E. Le Moal, E. Fort, S. Lévêque-Fort, F. P. Cordelières, M.-P. Fontaine-Aupart, and C. Ricolleau, Biophys. J. 92, 2150 (2007).
[CrossRef]

2005 (2)

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef] [PubMed]

K. Aslan, I. Gryczynski, J. Malicka, E. Matveeva, J. R. Lakowicz, and C. D. Geddes, Curr. Opin. Biotechnol. 16, 55 (2005).
[CrossRef] [PubMed]

Aslan, K.

K. Aslan, I. Gryczynski, J. Malicka, E. Matveeva, J. R. Lakowicz, and C. D. Geddes, Curr. Opin. Biotechnol. 16, 55 (2005).
[CrossRef] [PubMed]

Bonod, N.

Cordelières, F. P.

E. Le Moal, E. Fort, S. Lévêque-Fort, F. P. Cordelières, M.-P. Fontaine-Aupart, and C. Ricolleau, Biophys. J. 92, 2150 (2007).
[CrossRef]

Cui, X.

X. Cui, K. Tawa, H. Hori, and J. Nishii, Adv. Funct. Mater. 20, 546 (2010).
[CrossRef]

Denk, W.

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef] [PubMed]

Dintinger, J.

Ebbesen, T. W.

Fontaine-Aupart, M.-P.

E. Le Moal, E. Fort, S. Lévêque-Fort, F. P. Cordelières, M.-P. Fontaine-Aupart, and C. Ricolleau, Biophys. J. 92, 2150 (2007).
[CrossRef]

Fort, E.

E. Fort and S. Grésillon, J. Phys. D 41, 013001 (2008).
[CrossRef]

E. Le Moal, E. Fort, S. Lévêque-Fort, F. P. Cordelières, M.-P. Fontaine-Aupart, and C. Ricolleau, Biophys. J. 92, 2150 (2007).
[CrossRef]

Fujita, K.

S. Kawano, N. I. Smith, M. Yamanaka, S. Kawata, and K. Fujita, Appl. Phys. Express 4, 042401 (2011).
[CrossRef]

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, J. Biomed. Opt. 13, 050507 (2008).
[CrossRef] [PubMed]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[CrossRef]

Geddes, C. D.

K. Aslan, I. Gryczynski, J. Malicka, E. Matveeva, J. R. Lakowicz, and C. D. Geddes, Curr. Opin. Biotechnol. 16, 55 (2005).
[CrossRef] [PubMed]

Gérard, D.

Grésillon, S.

E. Fort and S. Grésillon, J. Phys. D 41, 013001 (2008).
[CrossRef]

Gryczynski, I.

K. Aslan, I. Gryczynski, J. Malicka, E. Matveeva, J. R. Lakowicz, and C. D. Geddes, Curr. Opin. Biotechnol. 16, 55 (2005).
[CrossRef] [PubMed]

Helmchen, F.

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef] [PubMed]

Hori, H.

X. Cui, K. Tawa, H. Hori, and J. Nishii, Adv. Funct. Mater. 20, 546 (2010).
[CrossRef]

Kawano, S.

S. Kawano, N. I. Smith, M. Yamanaka, S. Kawata, and K. Fujita, Appl. Phys. Express 4, 042401 (2011).
[CrossRef]

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, J. Biomed. Opt. 13, 050507 (2008).
[CrossRef] [PubMed]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[CrossRef]

Kawata, S.

S. Kawano, N. I. Smith, M. Yamanaka, S. Kawata, and K. Fujita, Appl. Phys. Express 4, 042401 (2011).
[CrossRef]

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, J. Biomed. Opt. 13, 050507 (2008).
[CrossRef] [PubMed]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[CrossRef]

Kobayashi, M.

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[CrossRef]

Lagendijk, A.

I. M. Vellekoop, A. Lagendijk, and A. P. Mosk, Nat. Photon. 4, 320 (2010).
[CrossRef]

Lakowicz, J. R.

K. Aslan, I. Gryczynski, J. Malicka, E. Matveeva, J. R. Lakowicz, and C. D. Geddes, Curr. Opin. Biotechnol. 16, 55 (2005).
[CrossRef] [PubMed]

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

Le Moal, E.

E. Le Moal, E. Fort, S. Lévêque-Fort, F. P. Cordelières, M.-P. Fontaine-Aupart, and C. Ricolleau, Biophys. J. 92, 2150 (2007).
[CrossRef]

Lévêque-Fort, S.

E. Le Moal, E. Fort, S. Lévêque-Fort, F. P. Cordelières, M.-P. Fontaine-Aupart, and C. Ricolleau, Biophys. J. 92, 2150 (2007).
[CrossRef]

Mahboub, O.

Malicka, J.

K. Aslan, I. Gryczynski, J. Malicka, E. Matveeva, J. R. Lakowicz, and C. D. Geddes, Curr. Opin. Biotechnol. 16, 55 (2005).
[CrossRef] [PubMed]

Matveeva, E.

K. Aslan, I. Gryczynski, J. Malicka, E. Matveeva, J. R. Lakowicz, and C. D. Geddes, Curr. Opin. Biotechnol. 16, 55 (2005).
[CrossRef] [PubMed]

Mosk, A. P.

I. M. Vellekoop, A. Lagendijk, and A. P. Mosk, Nat. Photon. 4, 320 (2010).
[CrossRef]

Nishii, J.

X. Cui, K. Tawa, H. Hori, and J. Nishii, Adv. Funct. Mater. 20, 546 (2010).
[CrossRef]

Popov, E.

Ricolleau, C.

E. Le Moal, E. Fort, S. Lévêque-Fort, F. P. Cordelières, M.-P. Fontaine-Aupart, and C. Ricolleau, Biophys. J. 92, 2150 (2007).
[CrossRef]

Rigneault, H.

Smith, N. I.

S. Kawano, N. I. Smith, M. Yamanaka, S. Kawata, and K. Fujita, Appl. Phys. Express 4, 042401 (2011).
[CrossRef]

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, J. Biomed. Opt. 13, 050507 (2008).
[CrossRef] [PubMed]

Tawa, K.

X. Cui, K. Tawa, H. Hori, and J. Nishii, Adv. Funct. Mater. 20, 546 (2010).
[CrossRef]

Vellekoop, I. M.

I. M. Vellekoop, A. Lagendijk, and A. P. Mosk, Nat. Photon. 4, 320 (2010).
[CrossRef]

Wenger, J.

Yamanaka, M.

S. Kawano, N. I. Smith, M. Yamanaka, S. Kawata, and K. Fujita, Appl. Phys. Express 4, 042401 (2011).
[CrossRef]

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, J. Biomed. Opt. 13, 050507 (2008).
[CrossRef] [PubMed]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[CrossRef]

Adv. Funct. Mater. (1)

X. Cui, K. Tawa, H. Hori, and J. Nishii, Adv. Funct. Mater. 20, 546 (2010).
[CrossRef]

Appl. Phys. Express (1)

S. Kawano, N. I. Smith, M. Yamanaka, S. Kawata, and K. Fujita, Appl. Phys. Express 4, 042401 (2011).
[CrossRef]

Biophys. J. (1)

E. Le Moal, E. Fort, S. Lévêque-Fort, F. P. Cordelières, M.-P. Fontaine-Aupart, and C. Ricolleau, Biophys. J. 92, 2150 (2007).
[CrossRef]

Curr. Opin. Biotechnol. (1)

K. Aslan, I. Gryczynski, J. Malicka, E. Matveeva, J. R. Lakowicz, and C. D. Geddes, Curr. Opin. Biotechnol. 16, 55 (2005).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, J. Biomed. Opt. 13, 050507 (2008).
[CrossRef] [PubMed]

J. Phys. D (1)

E. Fort and S. Grésillon, J. Phys. D 41, 013001 (2008).
[CrossRef]

Nat. Methods (1)

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef] [PubMed]

Nat. Photon. (1)

I. M. Vellekoop, A. Lagendijk, and A. P. Mosk, Nat. Photon. 4, 320 (2010).
[CrossRef]

Opt. Express (1)

Phys. Rev. Lett. (1)

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[CrossRef]

Other (1)

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

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Figures (2)

Fig. 1
Fig. 1

(a) Experimental setup. (b) Demodulated fluorescence signal at f = 5 kHz and 2 f = 10 kHz . Dashed lines display numerical fits results according to Eqs. (4, 5).

Fig. 2
Fig. 2

(a) Sketch of the sample configuration to test the capacity to distinguish the contributions from excitation intensity and number of emitters. Experimental results for the demodulated fluorescence signals at (b)  f = 5 kHz and (c)  2 f = 10 kHz . All experiments are performed with Alexa Fluor 647 dyes diluted in a pure water solution. Excitation powers of 100, 140, and 200 μW correspond to intensities of 45, 63, and 90 kW / cm 2 , respectively. (d) Represents the ratio ρ = F 2 f / F f , which is only sensitive to the excitation intensity, while (e) represents the ratio F f / ρ , which quantifies the number of emitters.

Equations (7)

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I e ( t ) = I avg ( 1 + α cos ( 2 π f t ) ) ,
F ( t ) = N κ ϕ σ I e ( t ) 1 + I e ( t ) / I sat ,
F ( t ) = F 0 + F f cos ( 2 π f t ) + F 2 f cos ( 4 π f t ) +
F f = N Γ α I avg / I sat ( 1 + I avg / I sat ) 2 ,
F 2 f = N Γ 2 α 2 ( I avg / I sat ) 2 ( 1 + I avg / I sat ) 3 .
ρ = F 2 f F f = α 2 I avg I avg + I sat .
F f ρ = F f 2 F 2 f = 2 N Γ 1 1 + I avg / I sat .

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