Abstract

The analog mean-delay (AMD) method is a new alternative method to measure the lifetime of a fluorescence molecule. Because of its powerful advantages of accurate lifetime determination, good photon economy, and a high photon detection rate, the AMD method is considered to be very suitable for high-speed confocal fluorescence lifetime imaging microscopy (FLIM). For the practical usage of the AMD method in FLIM (AMD-FLIM), detailed study on various experimental conditions and parameters that affect the precision and the accuracy of the AMD method is required. In this paper, we present the relation between the precision and accuracy of the lifetime versus iteration number in the AMD method, the best cutoff frequency of a low-pass filter used in the AMD-FLIM system for a given fluorophore, and the optimum position and width of the integration window by using Monte Carlo simulations and a series of AMD-FLIM experiments.

© 2011 Optical Society of America

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References

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  1. H. C. Gerritsen, A. Draaijer, D. J. van den Heuvel, and A. V. Agronskaia, “Fluorescence lifetime imaging in scanning microscopy,” in Handbook of Biological Confocal Microscopy, 3rd ed., J.B.Pawley, ed. (Springer, 2006).
    [CrossRef]
  2. J. Neefjes and N. P. Dantuma, “Fluorescence probes for proteolysis: tools for drug discovery,” Nat. Rev. Drug Discov. 3, 58–69 (2004).
    [CrossRef] [PubMed]
  3. R. Sanders, H. Gerritsen, A. Draaijer, P. Houpt, and Y. K. Levine, “Fluorescence lifetime imaging of free calcium in single cells,” Bioimaging 2, 131–138 (1994).
    [CrossRef]
  4. H.-J. van Manen, P. Verkuijlen, Paul Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94, L67–L69 (2008).
    [CrossRef] [PubMed]
  5. A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS One 3, 1–10 (2008).
    [CrossRef]
  6. J. M. Vroom, K. J. De Grauw, H. C. Gerritsen, D. J. Bradshaw, P. D. Marsh, G. K. Watson, J. J. Birmingham, and C. Allison, “Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy,” Appl. Environ. Microbiol. 65, 3502–3511 (1999).
    [PubMed]
  7. D. K. Nair, M. Jose, T. Kuner, W. Zuschratter, and R. Hartig, “FRET-FLIM at nanometer spectral resolution from living cells,” Opt. Express 14, 12217–12229 (2006).
    [CrossRef] [PubMed]
  8. W. Zhong, M. Wu, C. Chang, K. A. Merrick, S. D. Merajver, and M. Mycek, “Picosecond-resolution fluorescence lifetime imaging microscopy: a useful tool for sensing molecular interactions in vivo via FRET,” Opt. Express 15, 18220–18235 (2007).
    [CrossRef] [PubMed]
  9. P. J. Verveer, F. S. Wouters, A. R. Reynolds, and P. I. Bastiaens, “Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane,” Science 290, 1567–1570(2000).
    [CrossRef] [PubMed]
  10. S. Moon, Y. J. Won, and D. Y. Kim, “Analog mean-delay method for high-speed fluorescence lifetime measurement,” Opt. Express 17, 2834–2849 (2009).
    [CrossRef] [PubMed]
  11. Y. J. Won, S. Moon, W. Yang, D. U. Kim, W. T. Han, and D. Y. Kim, “High-speed confocal fluorescence lifetime imaging microscopy (FLIM) with the analog mean delay (AMD) method,” Opt. Express 19, 3396–3405 (2011).
    [CrossRef] [PubMed]
  12. A. I. Zverev, Handbook of Filter Synthesis (Wiley & Sons, 2005).
  13. Y. J. Won, S. Moon, W. T. Han, and D. Y. Kim, “Referencing techniques for the analog mean-delay method in fluorescence lifetime imaging,” J. Opt. Soc. Am. A 27, 2402–2410 (2010).
    [CrossRef]
  14. ISS, Inc., “Lifetime data of selected fluorophores,” http://www.iss.com/resources/fluorophores.html.
  15. H. C. Gerritsen, M. A. H. Assselbergs, A. V. Agronskaia, and W. G. Van Sark, “Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution,” J. Microsc. 206, 218–224 (2002).
    [CrossRef] [PubMed]
  16. A. Esposito, H. C. Gerritsen, and F. S. Wouters, “Optimizing frequency-domain fluorescence lifetime sensing for high-throughput applications: photon economy and acquisition speed,” J. Opt. Soc. Am. A 24, 3261–3273 (2007).
    [CrossRef]

2011 (1)

2010 (1)

2009 (1)

2008 (2)

H.-J. van Manen, P. Verkuijlen, Paul Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94, L67–L69 (2008).
[CrossRef] [PubMed]

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS One 3, 1–10 (2008).
[CrossRef]

2007 (2)

2006 (1)

2004 (1)

J. Neefjes and N. P. Dantuma, “Fluorescence probes for proteolysis: tools for drug discovery,” Nat. Rev. Drug Discov. 3, 58–69 (2004).
[CrossRef] [PubMed]

2002 (1)

H. C. Gerritsen, M. A. H. Assselbergs, A. V. Agronskaia, and W. G. Van Sark, “Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution,” J. Microsc. 206, 218–224 (2002).
[CrossRef] [PubMed]

2000 (1)

P. J. Verveer, F. S. Wouters, A. R. Reynolds, and P. I. Bastiaens, “Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane,” Science 290, 1567–1570(2000).
[CrossRef] [PubMed]

1999 (1)

J. M. Vroom, K. J. De Grauw, H. C. Gerritsen, D. J. Bradshaw, P. D. Marsh, G. K. Watson, J. J. Birmingham, and C. Allison, “Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy,” Appl. Environ. Microbiol. 65, 3502–3511 (1999).
[PubMed]

1994 (1)

R. Sanders, H. Gerritsen, A. Draaijer, P. Houpt, and Y. K. Levine, “Fluorescence lifetime imaging of free calcium in single cells,” Bioimaging 2, 131–138 (1994).
[CrossRef]

Agronskaia, A. V.

H. C. Gerritsen, M. A. H. Assselbergs, A. V. Agronskaia, and W. G. Van Sark, “Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution,” J. Microsc. 206, 218–224 (2002).
[CrossRef] [PubMed]

H. C. Gerritsen, A. Draaijer, D. J. van den Heuvel, and A. V. Agronskaia, “Fluorescence lifetime imaging in scanning microscopy,” in Handbook of Biological Confocal Microscopy, 3rd ed., J.B.Pawley, ed. (Springer, 2006).
[CrossRef]

Allison, C.

J. M. Vroom, K. J. De Grauw, H. C. Gerritsen, D. J. Bradshaw, P. D. Marsh, G. K. Watson, J. J. Birmingham, and C. Allison, “Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy,” Appl. Environ. Microbiol. 65, 3502–3511 (1999).
[PubMed]

Assselbergs, M. A. H.

H. C. Gerritsen, M. A. H. Assselbergs, A. V. Agronskaia, and W. G. Van Sark, “Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution,” J. Microsc. 206, 218–224 (2002).
[CrossRef] [PubMed]

Bannister, L. H.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS One 3, 1–10 (2008).
[CrossRef]

Bastiaens, P. I.

P. J. Verveer, F. S. Wouters, A. R. Reynolds, and P. I. Bastiaens, “Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane,” Science 290, 1567–1570(2000).
[CrossRef] [PubMed]

Birmingham, J. J.

J. M. Vroom, K. J. De Grauw, H. C. Gerritsen, D. J. Bradshaw, P. D. Marsh, G. K. Watson, J. J. Birmingham, and C. Allison, “Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy,” Appl. Environ. Microbiol. 65, 3502–3511 (1999).
[PubMed]

Bradshaw, D. J.

J. M. Vroom, K. J. De Grauw, H. C. Gerritsen, D. J. Bradshaw, P. D. Marsh, G. K. Watson, J. J. Birmingham, and C. Allison, “Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy,” Appl. Environ. Microbiol. 65, 3502–3511 (1999).
[PubMed]

Chang, C.

Dantuma, N. P.

J. Neefjes and N. P. Dantuma, “Fluorescence probes for proteolysis: tools for drug discovery,” Nat. Rev. Drug Discov. 3, 58–69 (2004).
[CrossRef] [PubMed]

De Grauw, K. J.

J. M. Vroom, K. J. De Grauw, H. C. Gerritsen, D. J. Bradshaw, P. D. Marsh, G. K. Watson, J. J. Birmingham, and C. Allison, “Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy,” Appl. Environ. Microbiol. 65, 3502–3511 (1999).
[PubMed]

Draaijer, A.

R. Sanders, H. Gerritsen, A. Draaijer, P. Houpt, and Y. K. Levine, “Fluorescence lifetime imaging of free calcium in single cells,” Bioimaging 2, 131–138 (1994).
[CrossRef]

H. C. Gerritsen, A. Draaijer, D. J. van den Heuvel, and A. V. Agronskaia, “Fluorescence lifetime imaging in scanning microscopy,” in Handbook of Biological Confocal Microscopy, 3rd ed., J.B.Pawley, ed. (Springer, 2006).
[CrossRef]

Esposito, A.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS One 3, 1–10 (2008).
[CrossRef]

A. Esposito, H. C. Gerritsen, and F. S. Wouters, “Optimizing frequency-domain fluorescence lifetime sensing for high-throughput applications: photon economy and acquisition speed,” J. Opt. Soc. Am. A 24, 3261–3273 (2007).
[CrossRef]

Gerritsen, H.

R. Sanders, H. Gerritsen, A. Draaijer, P. Houpt, and Y. K. Levine, “Fluorescence lifetime imaging of free calcium in single cells,” Bioimaging 2, 131–138 (1994).
[CrossRef]

Gerritsen, H. C.

A. Esposito, H. C. Gerritsen, and F. S. Wouters, “Optimizing frequency-domain fluorescence lifetime sensing for high-throughput applications: photon economy and acquisition speed,” J. Opt. Soc. Am. A 24, 3261–3273 (2007).
[CrossRef]

H. C. Gerritsen, M. A. H. Assselbergs, A. V. Agronskaia, and W. G. Van Sark, “Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution,” J. Microsc. 206, 218–224 (2002).
[CrossRef] [PubMed]

J. M. Vroom, K. J. De Grauw, H. C. Gerritsen, D. J. Bradshaw, P. D. Marsh, G. K. Watson, J. J. Birmingham, and C. Allison, “Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy,” Appl. Environ. Microbiol. 65, 3502–3511 (1999).
[PubMed]

H. C. Gerritsen, A. Draaijer, D. J. van den Heuvel, and A. V. Agronskaia, “Fluorescence lifetime imaging in scanning microscopy,” in Handbook of Biological Confocal Microscopy, 3rd ed., J.B.Pawley, ed. (Springer, 2006).
[CrossRef]

Han, W. T.

Hartig, R.

Houpt, P.

R. Sanders, H. Gerritsen, A. Draaijer, P. Houpt, and Y. K. Levine, “Fluorescence lifetime imaging of free calcium in single cells,” Bioimaging 2, 131–138 (1994).
[CrossRef]

Jose, M.

Kaminski, C. F.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS One 3, 1–10 (2008).
[CrossRef]

Kim, D. U.

Kim, D. Y.

Kuner, T.

Levine, Y. K.

R. Sanders, H. Gerritsen, A. Draaijer, P. Houpt, and Y. K. Levine, “Fluorescence lifetime imaging of free calcium in single cells,” Bioimaging 2, 131–138 (1994).
[CrossRef]

Lew, V. L.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS One 3, 1–10 (2008).
[CrossRef]

Marsh, P. D.

J. M. Vroom, K. J. De Grauw, H. C. Gerritsen, D. J. Bradshaw, P. D. Marsh, G. K. Watson, J. J. Birmingham, and C. Allison, “Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy,” Appl. Environ. Microbiol. 65, 3502–3511 (1999).
[PubMed]

Mauritz, J. M. A.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS One 3, 1–10 (2008).
[CrossRef]

Merajver, S. D.

Merrick, K. A.

Moon, S.

Mycek, M.

Nair, D. K.

Neefjes, J.

J. Neefjes and N. P. Dantuma, “Fluorescence probes for proteolysis: tools for drug discovery,” Nat. Rev. Drug Discov. 3, 58–69 (2004).
[CrossRef] [PubMed]

Otto, C.

H.-J. van Manen, P. Verkuijlen, Paul Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94, L67–L69 (2008).
[CrossRef] [PubMed]

Reynolds, A. R.

P. J. Verveer, F. S. Wouters, A. R. Reynolds, and P. I. Bastiaens, “Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane,” Science 290, 1567–1570(2000).
[CrossRef] [PubMed]

Roos, D.

H.-J. van Manen, P. Verkuijlen, Paul Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94, L67–L69 (2008).
[CrossRef] [PubMed]

Sanders, R.

R. Sanders, H. Gerritsen, A. Draaijer, P. Houpt, and Y. K. Levine, “Fluorescence lifetime imaging of free calcium in single cells,” Bioimaging 2, 131–138 (1994).
[CrossRef]

Schlachter, S.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS One 3, 1–10 (2008).
[CrossRef]

Subramaniam, V.

H.-J. van Manen, P. Verkuijlen, Paul Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94, L67–L69 (2008).
[CrossRef] [PubMed]

Tiffert, T.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS One 3, 1–10 (2008).
[CrossRef]

van den Berg, T. K.

H.-J. van Manen, P. Verkuijlen, Paul Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94, L67–L69 (2008).
[CrossRef] [PubMed]

van den Heuvel, D. J.

H. C. Gerritsen, A. Draaijer, D. J. van den Heuvel, and A. V. Agronskaia, “Fluorescence lifetime imaging in scanning microscopy,” in Handbook of Biological Confocal Microscopy, 3rd ed., J.B.Pawley, ed. (Springer, 2006).
[CrossRef]

van Manen, H.-J.

H.-J. van Manen, P. Verkuijlen, Paul Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94, L67–L69 (2008).
[CrossRef] [PubMed]

Van Sark, W. G.

H. C. Gerritsen, M. A. H. Assselbergs, A. V. Agronskaia, and W. G. Van Sark, “Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution,” J. Microsc. 206, 218–224 (2002).
[CrossRef] [PubMed]

Verkuijlen, P.

H.-J. van Manen, P. Verkuijlen, Paul Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94, L67–L69 (2008).
[CrossRef] [PubMed]

Verveer, P. J.

P. J. Verveer, F. S. Wouters, A. R. Reynolds, and P. I. Bastiaens, “Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane,” Science 290, 1567–1570(2000).
[CrossRef] [PubMed]

Vroom, J. M.

J. M. Vroom, K. J. De Grauw, H. C. Gerritsen, D. J. Bradshaw, P. D. Marsh, G. K. Watson, J. J. Birmingham, and C. Allison, “Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy,” Appl. Environ. Microbiol. 65, 3502–3511 (1999).
[PubMed]

Watson, G. K.

J. M. Vroom, K. J. De Grauw, H. C. Gerritsen, D. J. Bradshaw, P. D. Marsh, G. K. Watson, J. J. Birmingham, and C. Allison, “Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy,” Appl. Environ. Microbiol. 65, 3502–3511 (1999).
[PubMed]

Wittendorp, Paul

H.-J. van Manen, P. Verkuijlen, Paul Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94, L67–L69 (2008).
[CrossRef] [PubMed]

Won, Y. J.

Wouters, F. S.

A. Esposito, H. C. Gerritsen, and F. S. Wouters, “Optimizing frequency-domain fluorescence lifetime sensing for high-throughput applications: photon economy and acquisition speed,” J. Opt. Soc. Am. A 24, 3261–3273 (2007).
[CrossRef]

P. J. Verveer, F. S. Wouters, A. R. Reynolds, and P. I. Bastiaens, “Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane,” Science 290, 1567–1570(2000).
[CrossRef] [PubMed]

Wu, M.

Yang, W.

Zhong, W.

Zuschratter, W.

Zverev, A. I.

A. I. Zverev, Handbook of Filter Synthesis (Wiley & Sons, 2005).

Appl. Environ. Microbiol. (1)

J. M. Vroom, K. J. De Grauw, H. C. Gerritsen, D. J. Bradshaw, P. D. Marsh, G. K. Watson, J. J. Birmingham, and C. Allison, “Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy,” Appl. Environ. Microbiol. 65, 3502–3511 (1999).
[PubMed]

Bioimaging (1)

R. Sanders, H. Gerritsen, A. Draaijer, P. Houpt, and Y. K. Levine, “Fluorescence lifetime imaging of free calcium in single cells,” Bioimaging 2, 131–138 (1994).
[CrossRef]

Biophys. J. (1)

H.-J. van Manen, P. Verkuijlen, Paul Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94, L67–L69 (2008).
[CrossRef] [PubMed]

J. Microsc. (1)

H. C. Gerritsen, M. A. H. Assselbergs, A. V. Agronskaia, and W. G. Van Sark, “Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution,” J. Microsc. 206, 218–224 (2002).
[CrossRef] [PubMed]

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

Nat. Rev. Drug Discov. (1)

J. Neefjes and N. P. Dantuma, “Fluorescence probes for proteolysis: tools for drug discovery,” Nat. Rev. Drug Discov. 3, 58–69 (2004).
[CrossRef] [PubMed]

Opt. Express (4)

PLoS One (1)

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS One 3, 1–10 (2008).
[CrossRef]

Science (1)

P. J. Verveer, F. S. Wouters, A. R. Reynolds, and P. I. Bastiaens, “Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane,” Science 290, 1567–1570(2000).
[CrossRef] [PubMed]

Other (3)

A. I. Zverev, Handbook of Filter Synthesis (Wiley & Sons, 2005).

ISS, Inc., “Lifetime data of selected fluorophores,” http://www.iss.com/resources/fluorophores.html.

H. C. Gerritsen, A. Draaijer, D. J. van den Heuvel, and A. V. Agronskaia, “Fluorescence lifetime imaging in scanning microscopy,” in Handbook of Biological Confocal Microscopy, 3rd ed., J.B.Pawley, ed. (Springer, 2006).
[CrossRef]

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Figures (9)

Fig. 1
Fig. 1

Experimental setup for the AMD method.

Fig. 2
Fig. 2

Lifetime and its standard deviation as a function of the number of iterations for the proper center position of a window function used in the AMD method.

Fig. 3
Fig. 3

(a) Simulated IRF (dotted curve) and fluorescence (solid curves) signals whose lifetimes are 3, 5, 7, and 10 ns . (b) Simulation results of extracted lifetimes of various fluorophores by the AMD method versus their ideal lifetimes.

Fig. 4
Fig. 4

Minimum integration window sizes for an IRF with a FWHM of about 30 ns that make lifetime error to be less than 0.1% (blue squares), 1% (black triangles), and 3% (red circles) of its original value.

Fig. 5
Fig. 5

Average lifetime and standard deviation according to different window sizes for (a) Cy5 and (b) Alexa Fluor 633.

Fig. 6
Fig. 6

Figure of merit for lifetimes of 1, 3.2, 5, and 8 ns as estimated by Monte Carlo simulation.

Fig. 7
Fig. 7

Total standard error (blue triangles), standard error by shot noise (black squares), and standard error by system noise (red circles) for lifetimes of 1, 3.2, 5, and 8 ns .

Fig. 8
Fig. 8

IRF of the GLPF in the (a) time domain and (b) frequency domain.

Fig. 9
Fig. 9

(a) Optimum window size that satisfies a lifetime error rate of less than 1% with respect to the 50, 100, 200, and 500 MHz highest cutoff frequency of the GLPF. (b) Figures of merit for the cases shown in (a)

Equations (3)

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

τ = T e T e 0 ( t · i e ( t ) d t i e ( t ) d t ) ( t · i irf ( t ) d t i irf ( t ) d t ) ,
F = σ τ N τ ,
Δ τ tot = Δ τ shot 2 + Δ τ sys 2 .

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