Abstract

Fluorescence-based single particle counting devices have become very powerful tools for human health-related applications such as the detection of blood-borne pathogens. Instead of passing the sample fluid through a thin tube or microfluidic chip, as it is commonly practiced in flow cytometers and sorter devices, single particle counters scan the fluid volume by rotation and translation of the sample container. Hence, single particle counters are not limited by the fluid flow friction and can scan a large volume in a short timeframe while maintaining high sensitivity. A single particle can be detected in a milliliter of the fluid sample within minutes, and diagnostics are being developed using this principle. Until now, signal detection with particle counters has been based on signal intensity and signal separation into multiple wavelength bands coupled with multiple detectors, which limits the number of species that can be resolved. In this paper, we applied fluorescence lifetime detection to single particle counting to increase specificity and enable multiplexing with a single detector. We demonstrate how this principle can be used for diagnostic assays based on fluorescence quenching.

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

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References

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  1. D. K. Kang, M. M. Ali, K. Zhang, S. S. Huang, E. Peterson, M. A. Digman, E. Gratton, and W. Zhao, “Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection,” Nat. Commun. 5(1), 5427 (2014).
    [Crossref] [PubMed]
  2. J. P. Skinner, K. M. Swift, Q. Ruan, S. Perfetto, E. Gratton, and S. Y. Tetin, “Simplified confocal microscope for counting particles at low concentrations,” Rev. Sci. Instrum. 84(7), 074301 (2013).
    [Crossref] [PubMed]
  3. I. Altamore, L. Lanzano, and E. Gratton, “Dual channel detection of ultra low concentration of bacteria in real time by scanning FCS,” Meas. Sci. Technol. 24(6), 65702 (2013).
    [Crossref] [PubMed]
  4. M. Bouzin and E. Gratton, “Multi-slit detection,” (patent pending).
  5. S. P. Perfetto, P. K. Chattopadhyay, and M. Roederer, “Seventeen-colour flow cytometry: unravelling the immune system,” Nat. Rev. Immunol. 4(8), 648–655 (2004).
    [Crossref] [PubMed]
  6. R. A. Colyer, C. Lee, and E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
    [Crossref] [PubMed]
  7. E. Terpetschnig and D. M. Jameson, “Fluorescence Lifetime,” (ISS Inc., 2005).
  8. ISS, “Lifetime Data of Selected Fluorophores,” (ISS Inc., 2018).
  9. G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
    [Crossref] [PubMed]
  10. S. Ranjit, L. Malacrida, D. M. Jameson, and E. Gratton, “Fit-free analysis of fluorescence lifetime imaging data using the phasor approach,” Nat. Protoc. 13(9), 1979–2004 (2018).
    [Crossref] [PubMed]
  11. B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
    [Crossref] [PubMed]

2018 (1)

S. Ranjit, L. Malacrida, D. M. Jameson, and E. Gratton, “Fit-free analysis of fluorescence lifetime imaging data using the phasor approach,” Nat. Protoc. 13(9), 1979–2004 (2018).
[Crossref] [PubMed]

2014 (1)

D. K. Kang, M. M. Ali, K. Zhang, S. S. Huang, E. Peterson, M. A. Digman, E. Gratton, and W. Zhao, “Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection,” Nat. Commun. 5(1), 5427 (2014).
[Crossref] [PubMed]

2013 (2)

J. P. Skinner, K. M. Swift, Q. Ruan, S. Perfetto, E. Gratton, and S. Y. Tetin, “Simplified confocal microscope for counting particles at low concentrations,” Rev. Sci. Instrum. 84(7), 074301 (2013).
[Crossref] [PubMed]

I. Altamore, L. Lanzano, and E. Gratton, “Dual channel detection of ultra low concentration of bacteria in real time by scanning FCS,” Meas. Sci. Technol. 24(6), 65702 (2013).
[Crossref] [PubMed]

2011 (1)

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

2008 (1)

R. A. Colyer, C. Lee, and E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
[Crossref] [PubMed]

2005 (1)

B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
[Crossref] [PubMed]

2004 (1)

S. P. Perfetto, P. K. Chattopadhyay, and M. Roederer, “Seventeen-colour flow cytometry: unravelling the immune system,” Nat. Rev. Immunol. 4(8), 648–655 (2004).
[Crossref] [PubMed]

Ali, M. M.

D. K. Kang, M. M. Ali, K. Zhang, S. S. Huang, E. Peterson, M. A. Digman, E. Gratton, and W. Zhao, “Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection,” Nat. Commun. 5(1), 5427 (2014).
[Crossref] [PubMed]

Altamore, I.

I. Altamore, L. Lanzano, and E. Gratton, “Dual channel detection of ultra low concentration of bacteria in real time by scanning FCS,” Meas. Sci. Technol. 24(6), 65702 (2013).
[Crossref] [PubMed]

Bates, M.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

Bräuchle, C.

B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
[Crossref] [PubMed]

Chattopadhyay, P. K.

S. P. Perfetto, P. K. Chattopadhyay, and M. Roederer, “Seventeen-colour flow cytometry: unravelling the immune system,” Nat. Rev. Immunol. 4(8), 648–655 (2004).
[Crossref] [PubMed]

Chen, K. H.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

Colyer, R. A.

R. A. Colyer, C. Lee, and E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
[Crossref] [PubMed]

Dempsey, G. T.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

Digman, M. A.

D. K. Kang, M. M. Ali, K. Zhang, S. S. Huang, E. Peterson, M. A. Digman, E. Gratton, and W. Zhao, “Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection,” Nat. Commun. 5(1), 5427 (2014).
[Crossref] [PubMed]

Gratton, E.

S. Ranjit, L. Malacrida, D. M. Jameson, and E. Gratton, “Fit-free analysis of fluorescence lifetime imaging data using the phasor approach,” Nat. Protoc. 13(9), 1979–2004 (2018).
[Crossref] [PubMed]

D. K. Kang, M. M. Ali, K. Zhang, S. S. Huang, E. Peterson, M. A. Digman, E. Gratton, and W. Zhao, “Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection,” Nat. Commun. 5(1), 5427 (2014).
[Crossref] [PubMed]

I. Altamore, L. Lanzano, and E. Gratton, “Dual channel detection of ultra low concentration of bacteria in real time by scanning FCS,” Meas. Sci. Technol. 24(6), 65702 (2013).
[Crossref] [PubMed]

J. P. Skinner, K. M. Swift, Q. Ruan, S. Perfetto, E. Gratton, and S. Y. Tetin, “Simplified confocal microscope for counting particles at low concentrations,” Rev. Sci. Instrum. 84(7), 074301 (2013).
[Crossref] [PubMed]

R. A. Colyer, C. Lee, and E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
[Crossref] [PubMed]

Huang, S. S.

D. K. Kang, M. M. Ali, K. Zhang, S. S. Huang, E. Peterson, M. A. Digman, E. Gratton, and W. Zhao, “Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection,” Nat. Commun. 5(1), 5427 (2014).
[Crossref] [PubMed]

Jameson, D. M.

S. Ranjit, L. Malacrida, D. M. Jameson, and E. Gratton, “Fit-free analysis of fluorescence lifetime imaging data using the phasor approach,” Nat. Protoc. 13(9), 1979–2004 (2018).
[Crossref] [PubMed]

Kang, D. K.

D. K. Kang, M. M. Ali, K. Zhang, S. S. Huang, E. Peterson, M. A. Digman, E. Gratton, and W. Zhao, “Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection,” Nat. Commun. 5(1), 5427 (2014).
[Crossref] [PubMed]

Lamb, D. C.

B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
[Crossref] [PubMed]

Lanzano, L.

I. Altamore, L. Lanzano, and E. Gratton, “Dual channel detection of ultra low concentration of bacteria in real time by scanning FCS,” Meas. Sci. Technol. 24(6), 65702 (2013).
[Crossref] [PubMed]

Lee, C.

R. A. Colyer, C. Lee, and E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
[Crossref] [PubMed]

Malacrida, L.

S. Ranjit, L. Malacrida, D. M. Jameson, and E. Gratton, “Fit-free analysis of fluorescence lifetime imaging data using the phasor approach,” Nat. Protoc. 13(9), 1979–2004 (2018).
[Crossref] [PubMed]

Müller, B. K.

B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
[Crossref] [PubMed]

Perfetto, S.

J. P. Skinner, K. M. Swift, Q. Ruan, S. Perfetto, E. Gratton, and S. Y. Tetin, “Simplified confocal microscope for counting particles at low concentrations,” Rev. Sci. Instrum. 84(7), 074301 (2013).
[Crossref] [PubMed]

Perfetto, S. P.

S. P. Perfetto, P. K. Chattopadhyay, and M. Roederer, “Seventeen-colour flow cytometry: unravelling the immune system,” Nat. Rev. Immunol. 4(8), 648–655 (2004).
[Crossref] [PubMed]

Peterson, E.

D. K. Kang, M. M. Ali, K. Zhang, S. S. Huang, E. Peterson, M. A. Digman, E. Gratton, and W. Zhao, “Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection,” Nat. Commun. 5(1), 5427 (2014).
[Crossref] [PubMed]

Ranjit, S.

S. Ranjit, L. Malacrida, D. M. Jameson, and E. Gratton, “Fit-free analysis of fluorescence lifetime imaging data using the phasor approach,” Nat. Protoc. 13(9), 1979–2004 (2018).
[Crossref] [PubMed]

Roederer, M.

S. P. Perfetto, P. K. Chattopadhyay, and M. Roederer, “Seventeen-colour flow cytometry: unravelling the immune system,” Nat. Rev. Immunol. 4(8), 648–655 (2004).
[Crossref] [PubMed]

Ruan, Q.

J. P. Skinner, K. M. Swift, Q. Ruan, S. Perfetto, E. Gratton, and S. Y. Tetin, “Simplified confocal microscope for counting particles at low concentrations,” Rev. Sci. Instrum. 84(7), 074301 (2013).
[Crossref] [PubMed]

Skinner, J. P.

J. P. Skinner, K. M. Swift, Q. Ruan, S. Perfetto, E. Gratton, and S. Y. Tetin, “Simplified confocal microscope for counting particles at low concentrations,” Rev. Sci. Instrum. 84(7), 074301 (2013).
[Crossref] [PubMed]

Swift, K. M.

J. P. Skinner, K. M. Swift, Q. Ruan, S. Perfetto, E. Gratton, and S. Y. Tetin, “Simplified confocal microscope for counting particles at low concentrations,” Rev. Sci. Instrum. 84(7), 074301 (2013).
[Crossref] [PubMed]

Tetin, S. Y.

J. P. Skinner, K. M. Swift, Q. Ruan, S. Perfetto, E. Gratton, and S. Y. Tetin, “Simplified confocal microscope for counting particles at low concentrations,” Rev. Sci. Instrum. 84(7), 074301 (2013).
[Crossref] [PubMed]

Vaughan, J. C.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

Zaychikov, E.

B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
[Crossref] [PubMed]

Zhang, K.

D. K. Kang, M. M. Ali, K. Zhang, S. S. Huang, E. Peterson, M. A. Digman, E. Gratton, and W. Zhao, “Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection,” Nat. Commun. 5(1), 5427 (2014).
[Crossref] [PubMed]

Zhao, W.

D. K. Kang, M. M. Ali, K. Zhang, S. S. Huang, E. Peterson, M. A. Digman, E. Gratton, and W. Zhao, “Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection,” Nat. Commun. 5(1), 5427 (2014).
[Crossref] [PubMed]

Zhuang, X.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

Biophys. J. (1)

B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
[Crossref] [PubMed]

Meas. Sci. Technol. (1)

I. Altamore, L. Lanzano, and E. Gratton, “Dual channel detection of ultra low concentration of bacteria in real time by scanning FCS,” Meas. Sci. Technol. 24(6), 65702 (2013).
[Crossref] [PubMed]

Microsc. Res. Tech. (1)

R. A. Colyer, C. Lee, and E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
[Crossref] [PubMed]

Nat. Commun. (1)

D. K. Kang, M. M. Ali, K. Zhang, S. S. Huang, E. Peterson, M. A. Digman, E. Gratton, and W. Zhao, “Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection,” Nat. Commun. 5(1), 5427 (2014).
[Crossref] [PubMed]

Nat. Methods (1)

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

Nat. Protoc. (1)

S. Ranjit, L. Malacrida, D. M. Jameson, and E. Gratton, “Fit-free analysis of fluorescence lifetime imaging data using the phasor approach,” Nat. Protoc. 13(9), 1979–2004 (2018).
[Crossref] [PubMed]

Nat. Rev. Immunol. (1)

S. P. Perfetto, P. K. Chattopadhyay, and M. Roederer, “Seventeen-colour flow cytometry: unravelling the immune system,” Nat. Rev. Immunol. 4(8), 648–655 (2004).
[Crossref] [PubMed]

Rev. Sci. Instrum. (1)

J. P. Skinner, K. M. Swift, Q. Ruan, S. Perfetto, E. Gratton, and S. Y. Tetin, “Simplified confocal microscope for counting particles at low concentrations,” Rev. Sci. Instrum. 84(7), 074301 (2013).
[Crossref] [PubMed]

Other (3)

M. Bouzin and E. Gratton, “Multi-slit detection,” (patent pending).

E. Terpetschnig and D. M. Jameson, “Fluorescence Lifetime,” (ISS Inc., 2005).

ISS, “Lifetime Data of Selected Fluorophores,” (ISS Inc., 2018).

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Figures (6)

Fig. 1
Fig. 1 Schematic of the particle counter setup as described in the text.
Fig. 2
Fig. 2 Lifetime measurement with a particle counting device. (A) Principle of heterodyning, a pulsed/modulated light source is used to illuminate the sample, the photons detected are placed in certain bins with respect to the excitation pulse period. (B) From the counts accumulated in each bin, a phase histogram is constructed showing the average lifetime response. (C) From the phase histogram the lifetime phase and modulation can be calculated with the uncertainty scaling with the inverse of the square root of the number of photons collected. For each particle detected (D), the phase histogram is calculated (E) and transformed to a position on the phasor plot (F).
Fig. 3
Fig. 3 (A) One second long portion of the fluorescence time trace of a solution of unlabeled lipid droplets mixed with FITC labeled droplets at a ratio of 1000 to 1. (B) Exemplary peak indentified with the correlation filter. (C) Cumulative phasor plot positions of all peaks identified overlaid with the position of the Coumarin 6 reference solution (2.5 ns). Each blue dot represents the average phasor coordinates of the photons detected for each hit.
Fig. 4
Fig. 4 (A) Comparison of peak intensities found for solutions of unlabeled lipid droplets mixed with lipid droplets containing FITC and Cy2, respectively. Each dot represents the total number of photons detected for a particle passing through the detection volume indentified by the correlation filter. (B) Comparison of peak lifetimes found for the data of the lipid droplet solutions presented in panel A. Clearly, the two populations can only be separated by lifetime, not intensity. Laser modulation frequency, 20 MHz.
Fig. 5
Fig. 5 (A) Comparison of peak intensities found for a solution of beads prepared in a clean environment with the same solution after adding shelf dust as contaminant. Each dot represents the photons counted while a particle was passing through the detection volume. (B) Comparison of peak lifetimes found for the data shown in panel A. Again, the two populations can only be separated by lifetime, not intensity. Laser modulation frequency, 20 MHz.
Fig. 6
Fig. 6 Lifetime analysis of Kras3.0 quenched droplets (negative) and Kras3.0 quenched droplets mixed with unquenched droplets (positive) data acquired with the particle counter. (A) Intensity distribution of the “hits” detected for the negative and positive sample, each dot represents the photons counted for a given particle passing through the detection volume. (B) Phasor plot distributions of lifetimes of the “hits” detected for the negative and positive samples. The dotted line indicates the quenching trajectory from fully unquenched to fully quenched with the limits indicated by circles on the phasor plot. (C) Distribution of the positions of the “hits” detected along the quenching trajectory. Arrows indicate the boundary between quenched and unquenched populations. (D) Plot of “hit” quenching trajectory positions as a function of “hit” intensity. The median values plus standard deviations of the intensity and fraction unquenched determined by lifetime of the negative sample are represented by dotted lines, dividing the plot area in quadrants 1-4. (E) Fluorescence microscopy image of the positive sample. (F) Corresponding FLIM image with the pixels color coded according to their phasor plot positions shown in panel G. (G) Phasor histogram of pixel lifetimes. Two cursors mark the positions of the quenched (cyan) and unquenched (magenta) dye. Pixels within the perimeter of those cursors are painted accordingly in panel F. Laser modulation frequency, 80 MHz. Scale bar, 100 µm.

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