Abstract

Flow cytometry is a well-established and powerful high- throughput fluorescence measurement tool that also allows for the sorting and enrichment of subpopulations of cells expressing unique fluorescence signatures. Owing to the reliance on intensity-only signals, flow cytometry sorters cannot easily discriminate between fluorophores that spectrally overlap. In this paper we demonstrate a new method of cell sorting using a fluorescence lifetime-dependent methodology. This approach, referred to herein as phase-filtered cell sorting (PFCS), permits sorting based on the average fluorescence lifetime of a fluorophore by separating fluorescence signals from species that emit differing average fluorescence lifetimes. Using lifetime-dependent hardware, cells and microspheres labeled with fluorophores were sorted with purities up to 90%. PFCS is a practical approach for separating populations of cells that are stained with spectrally overlapping fluorophores or that have interfering autofluorescence signals.

© 2013 OSA

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    [CrossRef] [PubMed]
  2. W. A. Bonner, H. R. Hulett, R. G. Sweet, and L. A. Herzenberg, “Fluorescence activated cell sorting,” Rev. Sci. Instrum.43(3), 404–409 (1972).
    [CrossRef] [PubMed]
  3. J. A. Steinkamp, M. J. Fulwyler, J. R. Coulter, R. D. Hiebert, J. L. Horney, and P. F. Mullancy, “A new multiparameter separator for microscopic particles and biological cells,” Rev. Sci. Instrum.44(9), 1301–1310 (1973).
    [CrossRef] [PubMed]
  4. J. H. Jett and R. G. Alexander, “Droplet sorting of large particles,” Cytometry6(5), 484–486 (1985).
    [CrossRef] [PubMed]
  5. M. F. Bartholdi, J. D. Parson, K. A. Albright, and L. S. Cram, “System for flow sorting chromosomes on the basis of pulse shape,” Cytometry11(1), 165–172 (1990).
    [CrossRef] [PubMed]
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  8. C. Deka, L. A. Sklar, and J. A. Steinkamp, “Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique,” Cytometry17(1), 94–101 (1994).
    [CrossRef] [PubMed]
  9. J. A. Steinkamp, “Phase-sensitive detection methods for resolving fluorescence emission signals and directly quantifying lifetime,” Methods in cell biology 42, 627–640 (1994).
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  21. D. P. Heller and C. L. Greenstock, “Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye,” Biophys. Chem.50(3), 305–312 (1994).
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    [CrossRef]
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  24. T. C. George, D. A. Basiji, B. E. Hall, D. H. Lynch, W. E. Ortyn, D. J. Perry, M. J. Seo, C. A. Zimmerman, and P. J. Morrissey, “Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer,” Cytometry A59A(2), 237–245 (2004).
    [CrossRef] [PubMed]
  25. A. Mittag, D. Lenz, A. O. H. Gerstner, U. Sack, M. Steinbrecher, M. Koksch, A. Raffael, J. Bocsi, and A. Tárnok, “Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets,” Cytometry A65A(2), 103–115 (2005).
    [CrossRef] [PubMed]
  26. S. C. De Rosa, L. A. Herzenberg, L. A. Herzenberg, and M. Roederer, “11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity,” Nat. Med.7(2), 245–248 (2001).
    [CrossRef] [PubMed]
  27. N. Baumgarth and M. Roederer, “A practical approach to multicolor flow cytometry for immunophenotyping,” J. Immunol. Methods243(1-2), 77–97 (2000).
    [CrossRef] [PubMed]
  28. S. C. De Rosa, J. M. Brenchley, and M. Roederer, “Beyond six colors: a new era in flow cytometry,” Nat. Med.9(1), 112–117 (2003).
    [CrossRef] [PubMed]
  29. M. Roederer, S. De Rosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg, and L. Herzenberg, “8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity,” Cytometry29(4), 328–339 (1997).
    [CrossRef] [PubMed]
  30. C. C. Stewart and S. J. Stewart, “Four color compensation,” Cytometry38(4), 161–175 (1999).
    [CrossRef] [PubMed]
  31. C. B. Bagwell and E. G. Adams, “Fluorescence Spectral Overlap Compensation for Any Number of Flow Cytometry Parameters,” Ann. N. Y. Acad. Sci.677(1 Clinical Flow), 167–184 (1993).
    [CrossRef] [PubMed]
  32. M. Bigos, N. Baumgarth, G. C. Jager, O. C. Herman, T. Nozaki, R. T. Stovel, D. R. Parks, and L. A. Herzenberg, “Nine color eleven parameter immunophenotyping using three laser flow cytometry,” Cytometry36(1), 36–45 (1999).
    [CrossRef] [PubMed]

2010 (1)

J. P. Houston, M. A. Naivar, and J. P. Freyer, “Digital analysis and sorting of fluorescence lifetime by flow cytometry,” Cytometry A.77, 861-872 (2010).

2007 (1)

M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A. 71, 915–924 (2007).

2006 (1)

D. Baskić, S. Popović, P. Ristić, and N. N. Arsenijević, “Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide,” Cell Biol. Int.30(11), 924–932 (2006).
[CrossRef] [PubMed]

2005 (1)

A. Mittag, D. Lenz, A. O. H. Gerstner, U. Sack, M. Steinbrecher, M. Koksch, A. Raffael, J. Bocsi, and A. Tárnok, “Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets,” Cytometry A65A(2), 103–115 (2005).
[CrossRef] [PubMed]

2004 (1)

T. C. George, D. A. Basiji, B. E. Hall, D. H. Lynch, W. E. Ortyn, D. J. Perry, M. J. Seo, C. A. Zimmerman, and P. J. Morrissey, “Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer,” Cytometry A59A(2), 237–245 (2004).
[CrossRef] [PubMed]

2003 (1)

S. C. De Rosa, J. M. Brenchley, and M. Roederer, “Beyond six colors: a new era in flow cytometry,” Nat. Med.9(1), 112–117 (2003).
[CrossRef] [PubMed]

2001 (1)

S. C. De Rosa, L. A. Herzenberg, L. A. Herzenberg, and M. Roederer, “11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity,” Nat. Med.7(2), 245–248 (2001).
[CrossRef] [PubMed]

2000 (1)

N. Baumgarth and M. Roederer, “A practical approach to multicolor flow cytometry for immunophenotyping,” J. Immunol. Methods243(1-2), 77–97 (2000).
[CrossRef] [PubMed]

1999 (4)

C. C. Stewart and S. J. Stewart, “Four color compensation,” Cytometry38(4), 161–175 (1999).
[CrossRef] [PubMed]

J. A. Steinkamp and J. F. Keij, “Fluorescence intensity and lifetime measurement of free and particle-bound fluorophore in a sample stream by phase-sensitive flow cytometry,” Journal Name: Rev. Sci. Instrum.70(12), 4682–4688 (1999).
[CrossRef]

M. Bigos, N. Baumgarth, G. C. Jager, O. C. Herman, T. Nozaki, R. T. Stovel, D. R. Parks, and L. A. Herzenberg, “Nine color eleven parameter immunophenotyping using three laser flow cytometry,” Cytometry36(1), 36–45 (1999).
[CrossRef] [PubMed]

J. A. Steinkamp, B. E. Lehnert, and N. M. Lehnert, “Discrimination of damaged/dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry,” J. Immunol. Methods226(1-2), 59–70 (1999).
[CrossRef] [PubMed]

1997 (1)

M. Roederer, S. De Rosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg, and L. Herzenberg, “8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity,” Cytometry29(4), 328–339 (1997).
[CrossRef] [PubMed]

1994 (3)

D. P. Heller and C. L. Greenstock, “Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye,” Biophys. Chem.50(3), 305–312 (1994).
[CrossRef] [PubMed]

C. Deka, L. A. Sklar, and J. A. Steinkamp, “Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique,” Cytometry17(1), 94–101 (1994).
[CrossRef] [PubMed]

J. A. Steinkamp, “Phase-sensitive detection methods for resolving fluorescence emission signals and directly quantifying lifetime,” Methods in cell biology 42, 627–640 (1994).

1993 (3)

J. A. Steinkamp and H. A. Crissman, “Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase-sensitive flow cytometry,” Cytometry14(2), 210–216 (1993).
[CrossRef] [PubMed]

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, and R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry14(2), 123–135 (1993).
[CrossRef] [PubMed]

C. B. Bagwell and E. G. Adams, “Fluorescence Spectral Overlap Compensation for Any Number of Flow Cytometry Parameters,” Ann. N. Y. Acad. Sci.677(1 Clinical Flow), 167–184 (1993).
[CrossRef] [PubMed]

1990 (1)

M. F. Bartholdi, J. D. Parson, K. A. Albright, and L. S. Cram, “System for flow sorting chromosomes on the basis of pulse shape,” Cytometry11(1), 165–172 (1990).
[CrossRef] [PubMed]

1986 (1)

J. K. Larsen, B. Munch-Petersen, J. Christiansen, and K. Jørgensen, “Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde,” Cytometry7(1), 54–63 (1986).
[CrossRef] [PubMed]

1985 (2)

K. H. Jones and J. A. Senft, “An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide,” The journal of histochemistry and cytochemistry: official journal of the Histochemistry Society 33, 77–79 (1985).

J. H. Jett and R. G. Alexander, “Droplet sorting of large particles,” Cytometry6(5), 484–486 (1985).
[CrossRef] [PubMed]

1981 (1)

R. M. Böhmer and J. Ellwart, “Cell cycle analysis by combining the 5-bromodeoxyuridine/33258 Hoechst technique with DNA-specific ethidium bromide staining,” Cytometry2(1), 31–34 (1981).
[CrossRef] [PubMed]

1975 (1)

A. Krishan, “Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining,” J. Cell Biol.66(1), 188–193 (1975).
[CrossRef] [PubMed]

1973 (1)

J. A. Steinkamp, M. J. Fulwyler, J. R. Coulter, R. D. Hiebert, J. L. Horney, and P. F. Mullancy, “A new multiparameter separator for microscopic particles and biological cells,” Rev. Sci. Instrum.44(9), 1301–1310 (1973).
[CrossRef] [PubMed]

1972 (1)

W. A. Bonner, H. R. Hulett, R. G. Sweet, and L. A. Herzenberg, “Fluorescence activated cell sorting,” Rev. Sci. Instrum.43(3), 404–409 (1972).
[CrossRef] [PubMed]

1970 (1)

M. Edidin, “A rapid, quantitative fluorescence assay for cell damage by cytotoxic antibodies,” J. Immunol.104(5), 1303–1306 (1970).
[PubMed]

1965 (1)

M. J. Fulwyler, “Electronic separation of biological cells by volume,” Science150(3698), 910–911 (1965).
[CrossRef] [PubMed]

Adams, E. G.

C. B. Bagwell and E. G. Adams, “Fluorescence Spectral Overlap Compensation for Any Number of Flow Cytometry Parameters,” Ann. N. Y. Acad. Sci.677(1 Clinical Flow), 167–184 (1993).
[CrossRef] [PubMed]

Albright, K. A.

M. F. Bartholdi, J. D. Parson, K. A. Albright, and L. S. Cram, “System for flow sorting chromosomes on the basis of pulse shape,” Cytometry11(1), 165–172 (1990).
[CrossRef] [PubMed]

Alexander, R. G.

J. H. Jett and R. G. Alexander, “Droplet sorting of large particles,” Cytometry6(5), 484–486 (1985).
[CrossRef] [PubMed]

Anderson, M.

M. Roederer, S. De Rosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg, and L. Herzenberg, “8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity,” Cytometry29(4), 328–339 (1997).
[CrossRef] [PubMed]

Arsenijevic, N. N.

D. Baskić, S. Popović, P. Ristić, and N. N. Arsenijević, “Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide,” Cell Biol. Int.30(11), 924–932 (2006).
[CrossRef] [PubMed]

Bagwell, C. B.

C. B. Bagwell and E. G. Adams, “Fluorescence Spectral Overlap Compensation for Any Number of Flow Cytometry Parameters,” Ann. N. Y. Acad. Sci.677(1 Clinical Flow), 167–184 (1993).
[CrossRef] [PubMed]

Bartholdi, M. F.

M. F. Bartholdi, J. D. Parson, K. A. Albright, and L. S. Cram, “System for flow sorting chromosomes on the basis of pulse shape,” Cytometry11(1), 165–172 (1990).
[CrossRef] [PubMed]

Basiji, D. A.

T. C. George, D. A. Basiji, B. E. Hall, D. H. Lynch, W. E. Ortyn, D. J. Perry, M. J. Seo, C. A. Zimmerman, and P. J. Morrissey, “Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer,” Cytometry A59A(2), 237–245 (2004).
[CrossRef] [PubMed]

Baskic, D.

D. Baskić, S. Popović, P. Ristić, and N. N. Arsenijević, “Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide,” Cell Biol. Int.30(11), 924–932 (2006).
[CrossRef] [PubMed]

Baumgarth, N.

N. Baumgarth and M. Roederer, “A practical approach to multicolor flow cytometry for immunophenotyping,” J. Immunol. Methods243(1-2), 77–97 (2000).
[CrossRef] [PubMed]

M. Bigos, N. Baumgarth, G. C. Jager, O. C. Herman, T. Nozaki, R. T. Stovel, D. R. Parks, and L. A. Herzenberg, “Nine color eleven parameter immunophenotyping using three laser flow cytometry,” Cytometry36(1), 36–45 (1999).
[CrossRef] [PubMed]

Berndt, K.

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, and R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry14(2), 123–135 (1993).
[CrossRef] [PubMed]

Bigos, M.

M. Bigos, N. Baumgarth, G. C. Jager, O. C. Herman, T. Nozaki, R. T. Stovel, D. R. Parks, and L. A. Herzenberg, “Nine color eleven parameter immunophenotyping using three laser flow cytometry,” Cytometry36(1), 36–45 (1999).
[CrossRef] [PubMed]

M. Roederer, S. De Rosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg, and L. Herzenberg, “8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity,” Cytometry29(4), 328–339 (1997).
[CrossRef] [PubMed]

Bocsi, J.

A. Mittag, D. Lenz, A. O. H. Gerstner, U. Sack, M. Steinbrecher, M. Koksch, A. Raffael, J. Bocsi, and A. Tárnok, “Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets,” Cytometry A65A(2), 103–115 (2005).
[CrossRef] [PubMed]

Böhmer, R. M.

R. M. Böhmer and J. Ellwart, “Cell cycle analysis by combining the 5-bromodeoxyuridine/33258 Hoechst technique with DNA-specific ethidium bromide staining,” Cytometry2(1), 31–34 (1981).
[CrossRef] [PubMed]

Bonner, W. A.

W. A. Bonner, H. R. Hulett, R. G. Sweet, and L. A. Herzenberg, “Fluorescence activated cell sorting,” Rev. Sci. Instrum.43(3), 404–409 (1972).
[CrossRef] [PubMed]

Brenchley, J. M.

S. C. De Rosa, J. M. Brenchley, and M. Roederer, “Beyond six colors: a new era in flow cytometry,” Nat. Med.9(1), 112–117 (2003).
[CrossRef] [PubMed]

Christiansen, J.

J. K. Larsen, B. Munch-Petersen, J. Christiansen, and K. Jørgensen, “Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde,” Cytometry7(1), 54–63 (1986).
[CrossRef] [PubMed]

Coulter, J. R.

J. A. Steinkamp, M. J. Fulwyler, J. R. Coulter, R. D. Hiebert, J. L. Horney, and P. F. Mullancy, “A new multiparameter separator for microscopic particles and biological cells,” Rev. Sci. Instrum.44(9), 1301–1310 (1973).
[CrossRef] [PubMed]

Cram, L. S.

M. F. Bartholdi, J. D. Parson, K. A. Albright, and L. S. Cram, “System for flow sorting chromosomes on the basis of pulse shape,” Cytometry11(1), 165–172 (1990).
[CrossRef] [PubMed]

Crissman, H. A.

J. A. Steinkamp and H. A. Crissman, “Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase-sensitive flow cytometry,” Cytometry14(2), 210–216 (1993).
[CrossRef] [PubMed]

De Rosa, S.

M. Roederer, S. De Rosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg, and L. Herzenberg, “8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity,” Cytometry29(4), 328–339 (1997).
[CrossRef] [PubMed]

De Rosa, S. C.

S. C. De Rosa, J. M. Brenchley, and M. Roederer, “Beyond six colors: a new era in flow cytometry,” Nat. Med.9(1), 112–117 (2003).
[CrossRef] [PubMed]

S. C. De Rosa, L. A. Herzenberg, L. A. Herzenberg, and M. Roederer, “11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity,” Nat. Med.7(2), 245–248 (2001).
[CrossRef] [PubMed]

Deka, C.

C. Deka, L. A. Sklar, and J. A. Steinkamp, “Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique,” Cytometry17(1), 94–101 (1994).
[CrossRef] [PubMed]

Edidin, M.

M. Edidin, “A rapid, quantitative fluorescence assay for cell damage by cytotoxic antibodies,” J. Immunol.104(5), 1303–1306 (1970).
[PubMed]

Edwards, B. S.

M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A. 71, 915–924 (2007).

Ellwart, J.

R. M. Böhmer and J. Ellwart, “Cell cycle analysis by combining the 5-bromodeoxyuridine/33258 Hoechst technique with DNA-specific ethidium bromide staining,” Cytometry2(1), 31–34 (1981).
[CrossRef] [PubMed]

Freyer, J. P.

J. P. Houston, M. A. Naivar, and J. P. Freyer, “Digital analysis and sorting of fluorescence lifetime by flow cytometry,” Cytometry A.77, 861-872 (2010).

M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A. 71, 915–924 (2007).

Fulwyler, M. J.

J. A. Steinkamp, M. J. Fulwyler, J. R. Coulter, R. D. Hiebert, J. L. Horney, and P. F. Mullancy, “A new multiparameter separator for microscopic particles and biological cells,” Rev. Sci. Instrum.44(9), 1301–1310 (1973).
[CrossRef] [PubMed]

M. J. Fulwyler, “Electronic separation of biological cells by volume,” Science150(3698), 910–911 (1965).
[CrossRef] [PubMed]

George, T. C.

T. C. George, D. A. Basiji, B. E. Hall, D. H. Lynch, W. E. Ortyn, D. J. Perry, M. J. Seo, C. A. Zimmerman, and P. J. Morrissey, “Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer,” Cytometry A59A(2), 237–245 (2004).
[CrossRef] [PubMed]

Gerstein, R.

M. Roederer, S. De Rosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg, and L. Herzenberg, “8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity,” Cytometry29(4), 328–339 (1997).
[CrossRef] [PubMed]

Gerstner, A. O. H.

A. Mittag, D. Lenz, A. O. H. Gerstner, U. Sack, M. Steinbrecher, M. Koksch, A. Raffael, J. Bocsi, and A. Tárnok, “Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets,” Cytometry A65A(2), 103–115 (2005).
[CrossRef] [PubMed]

Graves, S. W.

M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A. 71, 915–924 (2007).

Greenstock, C. L.

D. P. Heller and C. L. Greenstock, “Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye,” Biophys. Chem.50(3), 305–312 (1994).
[CrossRef] [PubMed]

Habbersett, R. C.

M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A. 71, 915–924 (2007).

Hall, B. E.

T. C. George, D. A. Basiji, B. E. Hall, D. H. Lynch, W. E. Ortyn, D. J. Perry, M. J. Seo, C. A. Zimmerman, and P. J. Morrissey, “Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer,” Cytometry A59A(2), 237–245 (2004).
[CrossRef] [PubMed]

Heller, D. P.

D. P. Heller and C. L. Greenstock, “Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye,” Biophys. Chem.50(3), 305–312 (1994).
[CrossRef] [PubMed]

Herman, O. C.

M. Bigos, N. Baumgarth, G. C. Jager, O. C. Herman, T. Nozaki, R. T. Stovel, D. R. Parks, and L. A. Herzenberg, “Nine color eleven parameter immunophenotyping using three laser flow cytometry,” Cytometry36(1), 36–45 (1999).
[CrossRef] [PubMed]

Herzenberg, L.

M. Roederer, S. De Rosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg, and L. Herzenberg, “8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity,” Cytometry29(4), 328–339 (1997).
[CrossRef] [PubMed]

M. Roederer, S. De Rosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg, and L. Herzenberg, “8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity,” Cytometry29(4), 328–339 (1997).
[CrossRef] [PubMed]

Herzenberg, L. A.

S. C. De Rosa, L. A. Herzenberg, L. A. Herzenberg, and M. Roederer, “11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity,” Nat. Med.7(2), 245–248 (2001).
[CrossRef] [PubMed]

S. C. De Rosa, L. A. Herzenberg, L. A. Herzenberg, and M. Roederer, “11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity,” Nat. Med.7(2), 245–248 (2001).
[CrossRef] [PubMed]

M. Bigos, N. Baumgarth, G. C. Jager, O. C. Herman, T. Nozaki, R. T. Stovel, D. R. Parks, and L. A. Herzenberg, “Nine color eleven parameter immunophenotyping using three laser flow cytometry,” Cytometry36(1), 36–45 (1999).
[CrossRef] [PubMed]

W. A. Bonner, H. R. Hulett, R. G. Sweet, and L. A. Herzenberg, “Fluorescence activated cell sorting,” Rev. Sci. Instrum.43(3), 404–409 (1972).
[CrossRef] [PubMed]

Hiebert, R. D.

J. A. Steinkamp, M. J. Fulwyler, J. R. Coulter, R. D. Hiebert, J. L. Horney, and P. F. Mullancy, “A new multiparameter separator for microscopic particles and biological cells,” Rev. Sci. Instrum.44(9), 1301–1310 (1973).
[CrossRef] [PubMed]

Hoffman, R. A.

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, and R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry14(2), 123–135 (1993).
[CrossRef] [PubMed]

Horney, J. L.

J. A. Steinkamp, M. J. Fulwyler, J. R. Coulter, R. D. Hiebert, J. L. Horney, and P. F. Mullancy, “A new multiparameter separator for microscopic particles and biological cells,” Rev. Sci. Instrum.44(9), 1301–1310 (1973).
[CrossRef] [PubMed]

Houston, J. P.

J. P. Houston, M. A. Naivar, and J. P. Freyer, “Digital analysis and sorting of fluorescence lifetime by flow cytometry,” Cytometry A.77, 861-872 (2010).

Hulett, H. R.

W. A. Bonner, H. R. Hulett, R. G. Sweet, and L. A. Herzenberg, “Fluorescence activated cell sorting,” Rev. Sci. Instrum.43(3), 404–409 (1972).
[CrossRef] [PubMed]

Jager, G. C.

M. Bigos, N. Baumgarth, G. C. Jager, O. C. Herman, T. Nozaki, R. T. Stovel, D. R. Parks, and L. A. Herzenberg, “Nine color eleven parameter immunophenotyping using three laser flow cytometry,” Cytometry36(1), 36–45 (1999).
[CrossRef] [PubMed]

Jett, J. H.

M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A. 71, 915–924 (2007).

J. H. Jett and R. G. Alexander, “Droplet sorting of large particles,” Cytometry6(5), 484–486 (1985).
[CrossRef] [PubMed]

Jones, K. H.

K. H. Jones and J. A. Senft, “An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide,” The journal of histochemistry and cytochemistry: official journal of the Histochemistry Society 33, 77–79 (1985).

Jørgensen, K.

J. K. Larsen, B. Munch-Petersen, J. Christiansen, and K. Jørgensen, “Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde,” Cytometry7(1), 54–63 (1986).
[CrossRef] [PubMed]

Keij, J. F.

J. A. Steinkamp and J. F. Keij, “Fluorescence intensity and lifetime measurement of free and particle-bound fluorophore in a sample stream by phase-sensitive flow cytometry,” Journal Name: Rev. Sci. Instrum.70(12), 4682–4688 (1999).
[CrossRef]

Koksch, M.

A. Mittag, D. Lenz, A. O. H. Gerstner, U. Sack, M. Steinbrecher, M. Koksch, A. Raffael, J. Bocsi, and A. Tárnok, “Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets,” Cytometry A65A(2), 103–115 (2005).
[CrossRef] [PubMed]

Krishan, A.

A. Krishan, “Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining,” J. Cell Biol.66(1), 188–193 (1975).
[CrossRef] [PubMed]

Ladasky, J. J.

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, and R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry14(2), 123–135 (1993).
[CrossRef] [PubMed]

Lakowicz, J. R.

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, and R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry14(2), 123–135 (1993).
[CrossRef] [PubMed]

J. R. Lakowicz, Time-resolved laser Spectroscopy in Biochemistry (1988).

Larsen, J. K.

J. K. Larsen, B. Munch-Petersen, J. Christiansen, and K. Jørgensen, “Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde,” Cytometry7(1), 54–63 (1986).
[CrossRef] [PubMed]

Lehnert, B. E.

J. A. Steinkamp, B. E. Lehnert, and N. M. Lehnert, “Discrimination of damaged/dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry,” J. Immunol. Methods226(1-2), 59–70 (1999).
[CrossRef] [PubMed]

Lehnert, N. M.

J. A. Steinkamp, B. E. Lehnert, and N. M. Lehnert, “Discrimination of damaged/dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry,” J. Immunol. Methods226(1-2), 59–70 (1999).
[CrossRef] [PubMed]

Lenz, D.

A. Mittag, D. Lenz, A. O. H. Gerstner, U. Sack, M. Steinbrecher, M. Koksch, A. Raffael, J. Bocsi, and A. Tárnok, “Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets,” Cytometry A65A(2), 103–115 (2005).
[CrossRef] [PubMed]

Lynch, D. H.

T. C. George, D. A. Basiji, B. E. Hall, D. H. Lynch, W. E. Ortyn, D. J. Perry, M. J. Seo, C. A. Zimmerman, and P. J. Morrissey, “Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer,” Cytometry A59A(2), 237–245 (2004).
[CrossRef] [PubMed]

Martin, J. C.

M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A. 71, 915–924 (2007).

Mittag, A.

A. Mittag, D. Lenz, A. O. H. Gerstner, U. Sack, M. Steinbrecher, M. Koksch, A. Raffael, J. Bocsi, and A. Tárnok, “Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets,” Cytometry A65A(2), 103–115 (2005).
[CrossRef] [PubMed]

Morrissey, P. J.

T. C. George, D. A. Basiji, B. E. Hall, D. H. Lynch, W. E. Ortyn, D. J. Perry, M. J. Seo, C. A. Zimmerman, and P. J. Morrissey, “Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer,” Cytometry A59A(2), 237–245 (2004).
[CrossRef] [PubMed]

Mullancy, P. F.

J. A. Steinkamp, M. J. Fulwyler, J. R. Coulter, R. D. Hiebert, J. L. Horney, and P. F. Mullancy, “A new multiparameter separator for microscopic particles and biological cells,” Rev. Sci. Instrum.44(9), 1301–1310 (1973).
[CrossRef] [PubMed]

Munch-Petersen, B.

J. K. Larsen, B. Munch-Petersen, J. Christiansen, and K. Jørgensen, “Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde,” Cytometry7(1), 54–63 (1986).
[CrossRef] [PubMed]

Naivar, M. A.

J. P. Houston, M. A. Naivar, and J. P. Freyer, “Digital analysis and sorting of fluorescence lifetime by flow cytometry,” Cytometry A.77, 861-872 (2010).

M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A. 71, 915–924 (2007).

Nolan, J. P.

M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A. 71, 915–924 (2007).

Nozaki, T.

M. Bigos, N. Baumgarth, G. C. Jager, O. C. Herman, T. Nozaki, R. T. Stovel, D. R. Parks, and L. A. Herzenberg, “Nine color eleven parameter immunophenotyping using three laser flow cytometry,” Cytometry36(1), 36–45 (1999).
[CrossRef] [PubMed]

M. Roederer, S. De Rosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg, and L. Herzenberg, “8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity,” Cytometry29(4), 328–339 (1997).
[CrossRef] [PubMed]

Ortyn, W. E.

T. C. George, D. A. Basiji, B. E. Hall, D. H. Lynch, W. E. Ortyn, D. J. Perry, M. J. Seo, C. A. Zimmerman, and P. J. Morrissey, “Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer,” Cytometry A59A(2), 237–245 (2004).
[CrossRef] [PubMed]

Parks, D.

M. Roederer, S. De Rosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg, and L. Herzenberg, “8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity,” Cytometry29(4), 328–339 (1997).
[CrossRef] [PubMed]

Parks, D. R.

M. Bigos, N. Baumgarth, G. C. Jager, O. C. Herman, T. Nozaki, R. T. Stovel, D. R. Parks, and L. A. Herzenberg, “Nine color eleven parameter immunophenotyping using three laser flow cytometry,” Cytometry36(1), 36–45 (1999).
[CrossRef] [PubMed]

Parson, J. D.

M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A. 71, 915–924 (2007).

M. F. Bartholdi, J. D. Parson, K. A. Albright, and L. S. Cram, “System for flow sorting chromosomes on the basis of pulse shape,” Cytometry11(1), 165–172 (1990).
[CrossRef] [PubMed]

Perry, D. J.

T. C. George, D. A. Basiji, B. E. Hall, D. H. Lynch, W. E. Ortyn, D. J. Perry, M. J. Seo, C. A. Zimmerman, and P. J. Morrissey, “Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer,” Cytometry A59A(2), 237–245 (2004).
[CrossRef] [PubMed]

Pinsky, B. G.

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, and R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry14(2), 123–135 (1993).
[CrossRef] [PubMed]

Popovic, S.

D. Baskić, S. Popović, P. Ristić, and N. N. Arsenijević, “Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide,” Cell Biol. Int.30(11), 924–932 (2006).
[CrossRef] [PubMed]

Raffael, A.

A. Mittag, D. Lenz, A. O. H. Gerstner, U. Sack, M. Steinbrecher, M. Koksch, A. Raffael, J. Bocsi, and A. Tárnok, “Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets,” Cytometry A65A(2), 103–115 (2005).
[CrossRef] [PubMed]

Ristic, P.

D. Baskić, S. Popović, P. Ristić, and N. N. Arsenijević, “Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide,” Cell Biol. Int.30(11), 924–932 (2006).
[CrossRef] [PubMed]

Roederer, M.

S. C. De Rosa, J. M. Brenchley, and M. Roederer, “Beyond six colors: a new era in flow cytometry,” Nat. Med.9(1), 112–117 (2003).
[CrossRef] [PubMed]

S. C. De Rosa, L. A. Herzenberg, L. A. Herzenberg, and M. Roederer, “11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity,” Nat. Med.7(2), 245–248 (2001).
[CrossRef] [PubMed]

N. Baumgarth and M. Roederer, “A practical approach to multicolor flow cytometry for immunophenotyping,” J. Immunol. Methods243(1-2), 77–97 (2000).
[CrossRef] [PubMed]

M. Roederer, S. De Rosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg, and L. Herzenberg, “8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity,” Cytometry29(4), 328–339 (1997).
[CrossRef] [PubMed]

Sack, U.

A. Mittag, D. Lenz, A. O. H. Gerstner, U. Sack, M. Steinbrecher, M. Koksch, A. Raffael, J. Bocsi, and A. Tárnok, “Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets,” Cytometry A65A(2), 103–115 (2005).
[CrossRef] [PubMed]

Senft, J. A.

K. H. Jones and J. A. Senft, “An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide,” The journal of histochemistry and cytochemistry: official journal of the Histochemistry Society 33, 77–79 (1985).

Seo, M. J.

T. C. George, D. A. Basiji, B. E. Hall, D. H. Lynch, W. E. Ortyn, D. J. Perry, M. J. Seo, C. A. Zimmerman, and P. J. Morrissey, “Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer,” Cytometry A59A(2), 237–245 (2004).
[CrossRef] [PubMed]

Sklar, L.

M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A. 71, 915–924 (2007).

Sklar, L. A.

C. Deka, L. A. Sklar, and J. A. Steinkamp, “Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique,” Cytometry17(1), 94–101 (1994).
[CrossRef] [PubMed]

Steinbrecher, M.

A. Mittag, D. Lenz, A. O. H. Gerstner, U. Sack, M. Steinbrecher, M. Koksch, A. Raffael, J. Bocsi, and A. Tárnok, “Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets,” Cytometry A65A(2), 103–115 (2005).
[CrossRef] [PubMed]

Steinkamp, J. A.

J. A. Steinkamp, B. E. Lehnert, and N. M. Lehnert, “Discrimination of damaged/dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry,” J. Immunol. Methods226(1-2), 59–70 (1999).
[CrossRef] [PubMed]

J. A. Steinkamp and J. F. Keij, “Fluorescence intensity and lifetime measurement of free and particle-bound fluorophore in a sample stream by phase-sensitive flow cytometry,” Journal Name: Rev. Sci. Instrum.70(12), 4682–4688 (1999).
[CrossRef]

C. Deka, L. A. Sklar, and J. A. Steinkamp, “Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique,” Cytometry17(1), 94–101 (1994).
[CrossRef] [PubMed]

J. A. Steinkamp, “Phase-sensitive detection methods for resolving fluorescence emission signals and directly quantifying lifetime,” Methods in cell biology 42, 627–640 (1994).

J. A. Steinkamp and H. A. Crissman, “Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase-sensitive flow cytometry,” Cytometry14(2), 210–216 (1993).
[CrossRef] [PubMed]

J. A. Steinkamp, M. J. Fulwyler, J. R. Coulter, R. D. Hiebert, J. L. Horney, and P. F. Mullancy, “A new multiparameter separator for microscopic particles and biological cells,” Rev. Sci. Instrum.44(9), 1301–1310 (1973).
[CrossRef] [PubMed]

Stewart, C. C.

C. C. Stewart and S. J. Stewart, “Four color compensation,” Cytometry38(4), 161–175 (1999).
[CrossRef] [PubMed]

Stewart, S. J.

C. C. Stewart and S. J. Stewart, “Four color compensation,” Cytometry38(4), 161–175 (1999).
[CrossRef] [PubMed]

Stovel, R.

M. Roederer, S. De Rosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg, and L. Herzenberg, “8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity,” Cytometry29(4), 328–339 (1997).
[CrossRef] [PubMed]

Stovel, R. T.

M. Bigos, N. Baumgarth, G. C. Jager, O. C. Herman, T. Nozaki, R. T. Stovel, D. R. Parks, and L. A. Herzenberg, “Nine color eleven parameter immunophenotyping using three laser flow cytometry,” Cytometry36(1), 36–45 (1999).
[CrossRef] [PubMed]

Sweet, R. G.

W. A. Bonner, H. R. Hulett, R. G. Sweet, and L. A. Herzenberg, “Fluorescence activated cell sorting,” Rev. Sci. Instrum.43(3), 404–409 (1972).
[CrossRef] [PubMed]

Tárnok, A.

A. Mittag, D. Lenz, A. O. H. Gerstner, U. Sack, M. Steinbrecher, M. Koksch, A. Raffael, J. Bocsi, and A. Tárnok, “Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets,” Cytometry A65A(2), 103–115 (2005).
[CrossRef] [PubMed]

Wilder, M. E.

M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A. 71, 915–924 (2007).

Zimmerman, C. A.

T. C. George, D. A. Basiji, B. E. Hall, D. H. Lynch, W. E. Ortyn, D. J. Perry, M. J. Seo, C. A. Zimmerman, and P. J. Morrissey, “Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer,” Cytometry A59A(2), 237–245 (2004).
[CrossRef] [PubMed]

An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide (1)

K. H. Jones and J. A. Senft, “An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide,” The journal of histochemistry and cytochemistry: official journal of the Histochemistry Society 33, 77–79 (1985).

Ann. N. Y. Acad. Sci. (1)

C. B. Bagwell and E. G. Adams, “Fluorescence Spectral Overlap Compensation for Any Number of Flow Cytometry Parameters,” Ann. N. Y. Acad. Sci.677(1 Clinical Flow), 167–184 (1993).
[CrossRef] [PubMed]

Biophys. Chem. (1)

D. P. Heller and C. L. Greenstock, “Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye,” Biophys. Chem.50(3), 305–312 (1994).
[CrossRef] [PubMed]

Cell Biol. Int. (1)

D. Baskić, S. Popović, P. Ristić, and N. N. Arsenijević, “Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide,” Cell Biol. Int.30(11), 924–932 (2006).
[CrossRef] [PubMed]

Cytometry (10)

R. M. Böhmer and J. Ellwart, “Cell cycle analysis by combining the 5-bromodeoxyuridine/33258 Hoechst technique with DNA-specific ethidium bromide staining,” Cytometry2(1), 31–34 (1981).
[CrossRef] [PubMed]

J. K. Larsen, B. Munch-Petersen, J. Christiansen, and K. Jørgensen, “Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde,” Cytometry7(1), 54–63 (1986).
[CrossRef] [PubMed]

J. H. Jett and R. G. Alexander, “Droplet sorting of large particles,” Cytometry6(5), 484–486 (1985).
[CrossRef] [PubMed]

M. F. Bartholdi, J. D. Parson, K. A. Albright, and L. S. Cram, “System for flow sorting chromosomes on the basis of pulse shape,” Cytometry11(1), 165–172 (1990).
[CrossRef] [PubMed]

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, and R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry14(2), 123–135 (1993).
[CrossRef] [PubMed]

C. Deka, L. A. Sklar, and J. A. Steinkamp, “Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique,” Cytometry17(1), 94–101 (1994).
[CrossRef] [PubMed]

J. A. Steinkamp and H. A. Crissman, “Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase-sensitive flow cytometry,” Cytometry14(2), 210–216 (1993).
[CrossRef] [PubMed]

M. Roederer, S. De Rosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg, and L. Herzenberg, “8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity,” Cytometry29(4), 328–339 (1997).
[CrossRef] [PubMed]

C. C. Stewart and S. J. Stewart, “Four color compensation,” Cytometry38(4), 161–175 (1999).
[CrossRef] [PubMed]

M. Bigos, N. Baumgarth, G. C. Jager, O. C. Herman, T. Nozaki, R. T. Stovel, D. R. Parks, and L. A. Herzenberg, “Nine color eleven parameter immunophenotyping using three laser flow cytometry,” Cytometry36(1), 36–45 (1999).
[CrossRef] [PubMed]

Cytometry A (2)

T. C. George, D. A. Basiji, B. E. Hall, D. H. Lynch, W. E. Ortyn, D. J. Perry, M. J. Seo, C. A. Zimmerman, and P. J. Morrissey, “Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer,” Cytometry A59A(2), 237–245 (2004).
[CrossRef] [PubMed]

A. Mittag, D. Lenz, A. O. H. Gerstner, U. Sack, M. Steinbrecher, M. Koksch, A. Raffael, J. Bocsi, and A. Tárnok, “Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets,” Cytometry A65A(2), 103–115 (2005).
[CrossRef] [PubMed]

Digital analysis and sorting of fluorescence lifetime by flow cytometry (1)

J. P. Houston, M. A. Naivar, and J. P. Freyer, “Digital analysis and sorting of fluorescence lifetime by flow cytometry,” Cytometry A.77, 861-872 (2010).

J. Cell Biol. (1)

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[PubMed]

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J. A. Steinkamp, B. E. Lehnert, and N. M. Lehnert, “Discrimination of damaged/dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry,” J. Immunol. Methods226(1-2), 59–70 (1999).
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[CrossRef] [PubMed]

Journal Name: Rev. Sci. Instrum. (1)

J. A. Steinkamp and J. F. Keij, “Fluorescence intensity and lifetime measurement of free and particle-bound fluorophore in a sample stream by phase-sensitive flow cytometry,” Journal Name: Rev. Sci. Instrum.70(12), 4682–4688 (1999).
[CrossRef]

Nat. Med. (2)

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[CrossRef] [PubMed]

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[CrossRef] [PubMed]

Open, reconfigurable cytometric acquisition system: ORCAS (1)

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P. Jenkins, M. A. Naivar, and J. P. Houston, 1040 South Horseshoe Drive Las Cruces, NM 88003 are revising a mansucript to be called “Multi-frequency flow cytometry.”

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Figures (9)

Fig. 1
Fig. 1

Illustration of signal processing electronics and different stages required for phase-filtered cell sorting. A modulated Gaussian signal (solid black line) is input into stage 1; see also Eq. (7). After band-pass filtering this signal, stage 2 represents the “cleaned” and total fluorescence signal. The total signal is split in half and each half is mixed with separate phase-delayed reference signals (shown as a single sinusoidal input as solid black line) represented by Eq. (8) and (9). At stage 3, the output of each mixed signal represented by Eqs. (10) and (11) are then low-pass filtered. The final phase-sensitive fluorescence signals at stage 4 are delivered to a flow cytometry data acquisition system for routine analysis or sorting. The outputs after stage 4 are signals from Dye #1 (blue line) and Dye #2 (red line). The different stages are also simulated and exemplified in Fig. 3.

Fig. 2
Fig. 2

Simplified diagram of the PFCS system. Fluorescently labeled particles or cells rapidly transit a modulated laser beam during hydrodynamic focusing. The fluorescence and side scattered light are focused onto photomultiplier tube detectors. Signals from the PMTs are collected and routed to the PFCS hardware. A box represents all PFCS hardware, which are depicted in detail by Fig. 1. After phase-filtering, the signals are connected to the cytometry data system and normal sorting is accomplished. Sorting is based upon the formation of droplets and the selective charging of each droplet based on the phase-sensitive parameter. Once charged the droplets are attracted to either positively or negatively charged plates to be collected into separate vials. All sorting gates are set on the cytometry computer after the phase filtering is performed.

Fig. 3
Fig. 3

Simulation results at each stage during phase-resolved detection (see also Fig. 1 for each stage) where (a) is the total fluorescence signal from a mixture of two spectrally overlapping fluorophores (Eq. (7)) when the modulation frequency is 1 MHz. After band-pass filtering of this signal (b), the fluorescence signal is mixed with a 1-MHz phase-shifted reference signal (c). Simulation of low-pass filtering is a 3-stage process. First, a Fast Fourier Transform of (c) results in (d), where the 1-MHz high frequency from the FFT output is revealed. Then low pass filtering of (d) results in (e), where the 1-MHz peak is eliminated. Finally, an inverse FFT of (e) is accomplished to collect the final phase-sensitive signal (f).

Fig. 4
Fig. 4

Cytometric histograms displaying the number of fluorescence microspheres or cells having a measured fluorescence lifetime. Measurements were made with a modified FACSVantage SE digital lifetime cytometer. (a) Fluorescence lifetime histogram of Flow-Check fluorospheres, which is an average of 7 ns. The standard deviation of the fluorescence lifetime measured in this population is 1.302 ns. (b) Fluorescence lifetime histogram of EB stained cells. The mean fluorescence lifetime is 19.28 ns with 1.733 standard deviation (STD). (c) Fluorescence lifetime histogram of PI stained cells. The mean lifetime is 16.07 ns with 1.490 STD

Fig. 5
Fig. 5

Histograms and dot plots representing the number of Flow-Check fluorospheres having a specific side-scatter intensity (SSC), fluorescence intensity (FL1-H), or phase-sensitive intensity (PSD) signal before and after the phase-filtering process. Panels (a) and (b) present typical scatter and fluorescence histograms to illustrate the presence and detectability of the fluorospheres on the PFCS system. Panels (c) and (d) are the PSD fluorescence intensity counts measured by the same cytometer after phase filtering to maximize the fluorosphere signal (i.e. cos( ϕ 1 ϕ REF )=1 ). The dot plot shows the signal correlated to side scatter (SSC). Panels (e) and (f) are the PSD fluorescence intensity counts measured by the same cytometer after phase filtering to eliminate the fluorosphere signal (i.e. cos( ϕ 1 ϕ REF )=0 ).

Fig. 6
Fig. 6

Dot plots of phase-resolved detection prior to sorting. The parameters plotted are PSD No.1 vs. PSD No.2 (see Fig. 1). In panels (a) and (b) Flow-Check fluorospheres and YG microspheres are measured separately with the PFCS system. Panel (a): PSD No.1 is used to null YG fluorescence so that only fluorescence from Flow-Check fluorospheres is detectable; this demonstrates that the Flow-Check fluorosphere signals remain. Panel (b): PSD No.2 is adjusted to null Flow-Check fluorospheres so only signals from YG microspheres are detectable. Panel (c) is a dot plot of the phase-resolved detection of a mixed sample. Dots in gate 1 are Flow-Check fluorospheres and dots in gate 2 represent individual YG microspheres when the respective PSD channels are nulled to result in only one type of fluorescence signal per channel.

Fig. 7
Fig. 7

Dot plots demonstrating separation of mixed fluorescence microspheres using PFCS. A BD Accuri flow cytometer was used to obtain the three plots where panel (a) is pre-sort Flow-Check fluorosphere and YG microsphere mixture, panel (b) and (c) display the number of separated YG and Flow-Check fluorospheres when PSFC was implemented. YG microspheres were purified to 90.64% and Flow-Check fluorospheres were purified to 91.70%.

Fig. 8
Fig. 8

Dot plot of two correlated parameters, PSD No.1 vs PSD No.2 (see Fig. 1), for a mixture of EB and PI stained cells. The events show counts in the respective PSD channels where channel No.1 contains only signals from the EB-labeled cells and channel No.2 contains fluorescence signals from only PI-labeled cells. The plot was obtained after adjusting the respective PSD channels to null the signal from either the PI- or EB-stained samples. Therefore dots in Gate 01 are EB stained cells, which are approximately 29.91% of the total population, and dots in Gate 02 are approximately 14.91% of the total population.

Fig. 9
Fig. 9

Dot plots showing populations of sorted EB- and PI-labeled cells using an analog fluorescence lifetime flow cytometer. Panel (a) displays example results of a population of cells with only ethidium bromide as the primary fluorescent label. Likewise, panel (b) displays the number of cells sorted that only contain PI as the primary fluorescence label.

Tables (2)

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Table 1 Simulation parameters

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Table 2 Modulation frequencies for EB and PI

Equations (11)

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V(t)=A[1+msin(ωt-φ)] e a 2 (t t 0 ) 2
V(t)=A e a 2 (t t 0 ) 2 +Amsin(ωt-ϕ) e a 2 (t t 0 ) 2
V(t)= 1 2 mAcos(ϕ ϕ REF ) e a 2 (t t 0 ) 2
ϕ=arctan(ωτ)
V(t)= 1 2 m 1 A 1 cos( ϕ 1 ϕ RFE ) e a 2 (t t 0 ) 2
V(t)= 1 2 m 2 A 2 cos( ϕ 2 ϕ RFE ) e a 2 (t t 0 ) 2
V(t)= 1 2 m 1 A 1 cos( φ 1 φ RFE ) e a 2 (t t 0 ) 2 + 1 2 m 2 A 2 cos( ϕ 2 ϕ RFE ) e a 2 (t t 0 ) 2
ϕ REF1 = π 2 + ϕ 2
ϕ REF2 = π 2 + ϕ 1
V (t) 1 = 1 2 m 1 A 1 sin( ϕ 1 ϕ 2 ) e a 2 (t t 0 ) 2
V (t) 2 = 1 2 m 2 A 2 sin( ϕ 1 ϕ 2 ) e a 2 (t t 0 ) 2

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