Abstract

The aim of this study is to combine multiple excitation wavelengths in order to improve accuracy of fluorescence characterization of labeled cells. The experimental demonstration is realized with a hematology analyzer based on flow cytometry and a CW laser source emitting two visible wavelengths. A given optical encoding associated to each wavelength allows fluorescence identification coming from specific fluorochromes and avoiding the use of noisy compensation method.

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References

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  1. H. M. Shapiro, Practical Flow Cytometry, 2nd ed. (A. R. Liss, Inc., 1988).
  2. F. E. Craig and K. A. Foon, “Flow cytometric immunophenotyping for hematologic neoplasms,” Blood 111(8), 3941–3967 (2008).
    [CrossRef] [PubMed]
  3. J. L. Faucher, C. Lacronique-Gazaille, E. Frébet, F. Trimoreau, M. Donnard, D. Bordessoule, F. Lacombe, and J. Feuillard, ““6 markers/5 colors” extended white blood cell differential by flow cytometry,” Cytometry A 71A(11), 934–944 (2007).
    [CrossRef] [PubMed]
  4. C. Arnoulet, M. C. Béné, F. Durrieu, J. Feuillard, C. Fossat, B. Husson, H. Jouault, M. Maynadié, and F. Lacombe, “Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL,” Cytometry B Clin. Cytom. 78(1), 4–10 (2010).
    [PubMed]
  5. C. C. Stewart and S. J. Stewart, “Four color compensation,” Cytometry B 38(4), 161–175 (1999).
    [CrossRef]
  6. M. Roederer, “Spectral compensation for flow cytometry: visualization artefacts, limitations and caveats,” Cytometry A 45(3), 194–205 (2001).
    [CrossRef]
  7. P. Nérin and D. Lefevre, “Device and method for multiparametric analysis of microscopic elements,” U.S. patent 7,777,869 (14 April 2006).
  8. R. Kapoor and M. Casstevens, “Evaluation of multicomponent mixtures using modulated light beam,” U.S. 2007/0096039 A1 (14 April 2006).
  9. G. Durack and J. Hatcher, “System and method for the measurement of multiple fluorescence emissions in a flow cytometry system,” U.S. patent 2008/0213915 A1 (23 April 2007).
  10. K. Luong, P. Lundquist, R. Dalal, J. Lyle, and S. Turner, “Methods and systems for analysis of fluorescent reactions with modulated excitation,” CA patent 2711560 A1 (9 January 2009).
  11. D. Jameson, E. Gratton, and R. Hall, “The measurement and analysis of heterogenous emissions by multifrequency phase and modulation fluorometry,” Appl. Spectrosc. Rev. 20(1), 55–106 (1984).
    [CrossRef]
  12. G. Durack and J. P. Robinson, Emerging Tools for Single-Cell Analysis: Advances in Optical Measurement Technologies, ed. (Wiley-Liss., 2000).
  13. N. Åslund and K. Carlsson, “Confocal scanning microfluorometry of dual-labelled specimens using two excitation wavelengths and lock-in detection technique,” Micron 24(6), 603–609 (1993).
    [CrossRef]
  14. S. W. Lin, C. H. Chang, D. Y. Wu, and C. H. Lin, “Digitally synchronized LCD projector for multi-color fluorescence excitation in parallel capillary electrophoresis detection,” Biosens. Bioelectron. 26(2), 717–722 (2010).
    [CrossRef] [PubMed]
  15. A. D. Donnenberg and V. S. Donnenberg, “Configuration of initial control parameters in photodetectors for multi-color flow cytometry,” U.S. patent 2010/0256943 A1 (1 April 2010).
  16. M. Dinkelmann and C. Rogers, “Expanding fluorescence detection options with the accuri C6 flow cytometer system,” Nat. Meth. 7, ••• (2010).
  17. K. Mossberg and M. Ericsson, “Detection of doubly stained fluorescent specimens using confocal microscopy,” J. Microsc. 158(Pt 2), 215–224 (1990).
    [CrossRef] [PubMed]
  18. X. Wang and I. Kurtz, “H+/base transport in principal cells characterized by confocal fluorescence imaging,” Am. J. Physiol. 259(2 Pt 1), C365–C373 (1990).
    [PubMed]
  19. F. De Bisshop, “Electronic gating for particle/cell counting and sizing, DSP-operated,” IEEE Trans. Instrum. Meas. 58(9), 3159–3166 (2009).
    [CrossRef]
  20. A. Kudlinski, G. Bouwmans, O. Vanvincq, Y. Quiquempois, A. Le Rouge, L. Bigot, G. Mélin, and A. Mussot, “White-light cw-pumped supercontinuum generation in highly GeO(2)-doped-core photonic crystal fibers,” Opt. Lett. 34(23), 3631–3633 (2009).
    [CrossRef] [PubMed]
  21. B. A. Cumberland, J. C. Travers, S. V. Popov, and J. R. Taylor, “Toward visible cw-pumped supercontinua,” Opt. Lett. 33(18), 2122–2124 (2008).
    [CrossRef] [PubMed]
  22. J. C. Travers, R. E. Kennedy, S. V. Popov, J. R. Taylor, H. Sabert, and B. Mangan, “Extended continuous-wave supercontinuum generation in a low-water-loss holey fiber,” Opt. Lett. 30(15), 1938–1940 (2005).
    [CrossRef] [PubMed]

2010 (3)

C. Arnoulet, M. C. Béné, F. Durrieu, J. Feuillard, C. Fossat, B. Husson, H. Jouault, M. Maynadié, and F. Lacombe, “Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL,” Cytometry B Clin. Cytom. 78(1), 4–10 (2010).
[PubMed]

S. W. Lin, C. H. Chang, D. Y. Wu, and C. H. Lin, “Digitally synchronized LCD projector for multi-color fluorescence excitation in parallel capillary electrophoresis detection,” Biosens. Bioelectron. 26(2), 717–722 (2010).
[CrossRef] [PubMed]

M. Dinkelmann and C. Rogers, “Expanding fluorescence detection options with the accuri C6 flow cytometer system,” Nat. Meth. 7, ••• (2010).

2009 (2)

2008 (2)

B. A. Cumberland, J. C. Travers, S. V. Popov, and J. R. Taylor, “Toward visible cw-pumped supercontinua,” Opt. Lett. 33(18), 2122–2124 (2008).
[CrossRef] [PubMed]

F. E. Craig and K. A. Foon, “Flow cytometric immunophenotyping for hematologic neoplasms,” Blood 111(8), 3941–3967 (2008).
[CrossRef] [PubMed]

2007 (1)

J. L. Faucher, C. Lacronique-Gazaille, E. Frébet, F. Trimoreau, M. Donnard, D. Bordessoule, F. Lacombe, and J. Feuillard, ““6 markers/5 colors” extended white blood cell differential by flow cytometry,” Cytometry A 71A(11), 934–944 (2007).
[CrossRef] [PubMed]

2005 (1)

2001 (1)

M. Roederer, “Spectral compensation for flow cytometry: visualization artefacts, limitations and caveats,” Cytometry A 45(3), 194–205 (2001).
[CrossRef]

1999 (1)

C. C. Stewart and S. J. Stewart, “Four color compensation,” Cytometry B 38(4), 161–175 (1999).
[CrossRef]

1993 (1)

N. Åslund and K. Carlsson, “Confocal scanning microfluorometry of dual-labelled specimens using two excitation wavelengths and lock-in detection technique,” Micron 24(6), 603–609 (1993).
[CrossRef]

1990 (2)

K. Mossberg and M. Ericsson, “Detection of doubly stained fluorescent specimens using confocal microscopy,” J. Microsc. 158(Pt 2), 215–224 (1990).
[CrossRef] [PubMed]

X. Wang and I. Kurtz, “H+/base transport in principal cells characterized by confocal fluorescence imaging,” Am. J. Physiol. 259(2 Pt 1), C365–C373 (1990).
[PubMed]

1984 (1)

D. Jameson, E. Gratton, and R. Hall, “The measurement and analysis of heterogenous emissions by multifrequency phase and modulation fluorometry,” Appl. Spectrosc. Rev. 20(1), 55–106 (1984).
[CrossRef]

Arnoulet, C.

C. Arnoulet, M. C. Béné, F. Durrieu, J. Feuillard, C. Fossat, B. Husson, H. Jouault, M. Maynadié, and F. Lacombe, “Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL,” Cytometry B Clin. Cytom. 78(1), 4–10 (2010).
[PubMed]

Åslund, N.

N. Åslund and K. Carlsson, “Confocal scanning microfluorometry of dual-labelled specimens using two excitation wavelengths and lock-in detection technique,” Micron 24(6), 603–609 (1993).
[CrossRef]

Béné, M. C.

C. Arnoulet, M. C. Béné, F. Durrieu, J. Feuillard, C. Fossat, B. Husson, H. Jouault, M. Maynadié, and F. Lacombe, “Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL,” Cytometry B Clin. Cytom. 78(1), 4–10 (2010).
[PubMed]

Bigot, L.

Bordessoule, D.

J. L. Faucher, C. Lacronique-Gazaille, E. Frébet, F. Trimoreau, M. Donnard, D. Bordessoule, F. Lacombe, and J. Feuillard, ““6 markers/5 colors” extended white blood cell differential by flow cytometry,” Cytometry A 71A(11), 934–944 (2007).
[CrossRef] [PubMed]

Bouwmans, G.

Carlsson, K.

N. Åslund and K. Carlsson, “Confocal scanning microfluorometry of dual-labelled specimens using two excitation wavelengths and lock-in detection technique,” Micron 24(6), 603–609 (1993).
[CrossRef]

Chang, C. H.

S. W. Lin, C. H. Chang, D. Y. Wu, and C. H. Lin, “Digitally synchronized LCD projector for multi-color fluorescence excitation in parallel capillary electrophoresis detection,” Biosens. Bioelectron. 26(2), 717–722 (2010).
[CrossRef] [PubMed]

Craig, F. E.

F. E. Craig and K. A. Foon, “Flow cytometric immunophenotyping for hematologic neoplasms,” Blood 111(8), 3941–3967 (2008).
[CrossRef] [PubMed]

Cumberland, B. A.

De Bisshop, F.

F. De Bisshop, “Electronic gating for particle/cell counting and sizing, DSP-operated,” IEEE Trans. Instrum. Meas. 58(9), 3159–3166 (2009).
[CrossRef]

Dinkelmann, M.

M. Dinkelmann and C. Rogers, “Expanding fluorescence detection options with the accuri C6 flow cytometer system,” Nat. Meth. 7, ••• (2010).

Donnard, M.

J. L. Faucher, C. Lacronique-Gazaille, E. Frébet, F. Trimoreau, M. Donnard, D. Bordessoule, F. Lacombe, and J. Feuillard, ““6 markers/5 colors” extended white blood cell differential by flow cytometry,” Cytometry A 71A(11), 934–944 (2007).
[CrossRef] [PubMed]

Durrieu, F.

C. Arnoulet, M. C. Béné, F. Durrieu, J. Feuillard, C. Fossat, B. Husson, H. Jouault, M. Maynadié, and F. Lacombe, “Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL,” Cytometry B Clin. Cytom. 78(1), 4–10 (2010).
[PubMed]

Ericsson, M.

K. Mossberg and M. Ericsson, “Detection of doubly stained fluorescent specimens using confocal microscopy,” J. Microsc. 158(Pt 2), 215–224 (1990).
[CrossRef] [PubMed]

Faucher, J. L.

J. L. Faucher, C. Lacronique-Gazaille, E. Frébet, F. Trimoreau, M. Donnard, D. Bordessoule, F. Lacombe, and J. Feuillard, ““6 markers/5 colors” extended white blood cell differential by flow cytometry,” Cytometry A 71A(11), 934–944 (2007).
[CrossRef] [PubMed]

Feuillard, J.

C. Arnoulet, M. C. Béné, F. Durrieu, J. Feuillard, C. Fossat, B. Husson, H. Jouault, M. Maynadié, and F. Lacombe, “Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL,” Cytometry B Clin. Cytom. 78(1), 4–10 (2010).
[PubMed]

J. L. Faucher, C. Lacronique-Gazaille, E. Frébet, F. Trimoreau, M. Donnard, D. Bordessoule, F. Lacombe, and J. Feuillard, ““6 markers/5 colors” extended white blood cell differential by flow cytometry,” Cytometry A 71A(11), 934–944 (2007).
[CrossRef] [PubMed]

Foon, K. A.

F. E. Craig and K. A. Foon, “Flow cytometric immunophenotyping for hematologic neoplasms,” Blood 111(8), 3941–3967 (2008).
[CrossRef] [PubMed]

Fossat, C.

C. Arnoulet, M. C. Béné, F. Durrieu, J. Feuillard, C. Fossat, B. Husson, H. Jouault, M. Maynadié, and F. Lacombe, “Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL,” Cytometry B Clin. Cytom. 78(1), 4–10 (2010).
[PubMed]

Frébet, E.

J. L. Faucher, C. Lacronique-Gazaille, E. Frébet, F. Trimoreau, M. Donnard, D. Bordessoule, F. Lacombe, and J. Feuillard, ““6 markers/5 colors” extended white blood cell differential by flow cytometry,” Cytometry A 71A(11), 934–944 (2007).
[CrossRef] [PubMed]

Gratton, E.

D. Jameson, E. Gratton, and R. Hall, “The measurement and analysis of heterogenous emissions by multifrequency phase and modulation fluorometry,” Appl. Spectrosc. Rev. 20(1), 55–106 (1984).
[CrossRef]

Hall, R.

D. Jameson, E. Gratton, and R. Hall, “The measurement and analysis of heterogenous emissions by multifrequency phase and modulation fluorometry,” Appl. Spectrosc. Rev. 20(1), 55–106 (1984).
[CrossRef]

Husson, B.

C. Arnoulet, M. C. Béné, F. Durrieu, J. Feuillard, C. Fossat, B. Husson, H. Jouault, M. Maynadié, and F. Lacombe, “Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL,” Cytometry B Clin. Cytom. 78(1), 4–10 (2010).
[PubMed]

Jameson, D.

D. Jameson, E. Gratton, and R. Hall, “The measurement and analysis of heterogenous emissions by multifrequency phase and modulation fluorometry,” Appl. Spectrosc. Rev. 20(1), 55–106 (1984).
[CrossRef]

Jouault, H.

C. Arnoulet, M. C. Béné, F. Durrieu, J. Feuillard, C. Fossat, B. Husson, H. Jouault, M. Maynadié, and F. Lacombe, “Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL,” Cytometry B Clin. Cytom. 78(1), 4–10 (2010).
[PubMed]

Kennedy, R. E.

Kudlinski, A.

Kurtz, I.

X. Wang and I. Kurtz, “H+/base transport in principal cells characterized by confocal fluorescence imaging,” Am. J. Physiol. 259(2 Pt 1), C365–C373 (1990).
[PubMed]

Lacombe, F.

C. Arnoulet, M. C. Béné, F. Durrieu, J. Feuillard, C. Fossat, B. Husson, H. Jouault, M. Maynadié, and F. Lacombe, “Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL,” Cytometry B Clin. Cytom. 78(1), 4–10 (2010).
[PubMed]

J. L. Faucher, C. Lacronique-Gazaille, E. Frébet, F. Trimoreau, M. Donnard, D. Bordessoule, F. Lacombe, and J. Feuillard, ““6 markers/5 colors” extended white blood cell differential by flow cytometry,” Cytometry A 71A(11), 934–944 (2007).
[CrossRef] [PubMed]

Lacronique-Gazaille, C.

J. L. Faucher, C. Lacronique-Gazaille, E. Frébet, F. Trimoreau, M. Donnard, D. Bordessoule, F. Lacombe, and J. Feuillard, ““6 markers/5 colors” extended white blood cell differential by flow cytometry,” Cytometry A 71A(11), 934–944 (2007).
[CrossRef] [PubMed]

Le Rouge, A.

Lin, C. H.

S. W. Lin, C. H. Chang, D. Y. Wu, and C. H. Lin, “Digitally synchronized LCD projector for multi-color fluorescence excitation in parallel capillary electrophoresis detection,” Biosens. Bioelectron. 26(2), 717–722 (2010).
[CrossRef] [PubMed]

Lin, S. W.

S. W. Lin, C. H. Chang, D. Y. Wu, and C. H. Lin, “Digitally synchronized LCD projector for multi-color fluorescence excitation in parallel capillary electrophoresis detection,” Biosens. Bioelectron. 26(2), 717–722 (2010).
[CrossRef] [PubMed]

Mangan, B.

Maynadié, M.

C. Arnoulet, M. C. Béné, F. Durrieu, J. Feuillard, C. Fossat, B. Husson, H. Jouault, M. Maynadié, and F. Lacombe, “Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL,” Cytometry B Clin. Cytom. 78(1), 4–10 (2010).
[PubMed]

Mélin, G.

Mossberg, K.

K. Mossberg and M. Ericsson, “Detection of doubly stained fluorescent specimens using confocal microscopy,” J. Microsc. 158(Pt 2), 215–224 (1990).
[CrossRef] [PubMed]

Mussot, A.

Popov, S. V.

Quiquempois, Y.

Roederer, M.

M. Roederer, “Spectral compensation for flow cytometry: visualization artefacts, limitations and caveats,” Cytometry A 45(3), 194–205 (2001).
[CrossRef]

Rogers, C.

M. Dinkelmann and C. Rogers, “Expanding fluorescence detection options with the accuri C6 flow cytometer system,” Nat. Meth. 7, ••• (2010).

Sabert, H.

Stewart, C. C.

C. C. Stewart and S. J. Stewart, “Four color compensation,” Cytometry B 38(4), 161–175 (1999).
[CrossRef]

Stewart, S. J.

C. C. Stewart and S. J. Stewart, “Four color compensation,” Cytometry B 38(4), 161–175 (1999).
[CrossRef]

Taylor, J. R.

Travers, J. C.

Trimoreau, F.

J. L. Faucher, C. Lacronique-Gazaille, E. Frébet, F. Trimoreau, M. Donnard, D. Bordessoule, F. Lacombe, and J. Feuillard, ““6 markers/5 colors” extended white blood cell differential by flow cytometry,” Cytometry A 71A(11), 934–944 (2007).
[CrossRef] [PubMed]

Vanvincq, O.

Wang, X.

X. Wang and I. Kurtz, “H+/base transport in principal cells characterized by confocal fluorescence imaging,” Am. J. Physiol. 259(2 Pt 1), C365–C373 (1990).
[PubMed]

Wu, D. Y.

S. W. Lin, C. H. Chang, D. Y. Wu, and C. H. Lin, “Digitally synchronized LCD projector for multi-color fluorescence excitation in parallel capillary electrophoresis detection,” Biosens. Bioelectron. 26(2), 717–722 (2010).
[CrossRef] [PubMed]

Am. J. Physiol. (1)

X. Wang and I. Kurtz, “H+/base transport in principal cells characterized by confocal fluorescence imaging,” Am. J. Physiol. 259(2 Pt 1), C365–C373 (1990).
[PubMed]

Appl. Spectrosc. Rev. (1)

D. Jameson, E. Gratton, and R. Hall, “The measurement and analysis of heterogenous emissions by multifrequency phase and modulation fluorometry,” Appl. Spectrosc. Rev. 20(1), 55–106 (1984).
[CrossRef]

Biosens. Bioelectron. (1)

S. W. Lin, C. H. Chang, D. Y. Wu, and C. H. Lin, “Digitally synchronized LCD projector for multi-color fluorescence excitation in parallel capillary electrophoresis detection,” Biosens. Bioelectron. 26(2), 717–722 (2010).
[CrossRef] [PubMed]

Blood (1)

F. E. Craig and K. A. Foon, “Flow cytometric immunophenotyping for hematologic neoplasms,” Blood 111(8), 3941–3967 (2008).
[CrossRef] [PubMed]

Cytometry A (2)

J. L. Faucher, C. Lacronique-Gazaille, E. Frébet, F. Trimoreau, M. Donnard, D. Bordessoule, F. Lacombe, and J. Feuillard, ““6 markers/5 colors” extended white blood cell differential by flow cytometry,” Cytometry A 71A(11), 934–944 (2007).
[CrossRef] [PubMed]

M. Roederer, “Spectral compensation for flow cytometry: visualization artefacts, limitations and caveats,” Cytometry A 45(3), 194–205 (2001).
[CrossRef]

Cytometry B (1)

C. C. Stewart and S. J. Stewart, “Four color compensation,” Cytometry B 38(4), 161–175 (1999).
[CrossRef]

Cytometry B Clin. Cytom. (1)

C. Arnoulet, M. C. Béné, F. Durrieu, J. Feuillard, C. Fossat, B. Husson, H. Jouault, M. Maynadié, and F. Lacombe, “Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL,” Cytometry B Clin. Cytom. 78(1), 4–10 (2010).
[PubMed]

IEEE Trans. Instrum. Meas. (1)

F. De Bisshop, “Electronic gating for particle/cell counting and sizing, DSP-operated,” IEEE Trans. Instrum. Meas. 58(9), 3159–3166 (2009).
[CrossRef]

J. Microsc. (1)

K. Mossberg and M. Ericsson, “Detection of doubly stained fluorescent specimens using confocal microscopy,” J. Microsc. 158(Pt 2), 215–224 (1990).
[CrossRef] [PubMed]

Micron (1)

N. Åslund and K. Carlsson, “Confocal scanning microfluorometry of dual-labelled specimens using two excitation wavelengths and lock-in detection technique,” Micron 24(6), 603–609 (1993).
[CrossRef]

Nat. Meth. (1)

M. Dinkelmann and C. Rogers, “Expanding fluorescence detection options with the accuri C6 flow cytometer system,” Nat. Meth. 7, ••• (2010).

Opt. Lett. (3)

Other (7)

A. D. Donnenberg and V. S. Donnenberg, “Configuration of initial control parameters in photodetectors for multi-color flow cytometry,” U.S. patent 2010/0256943 A1 (1 April 2010).

G. Durack and J. P. Robinson, Emerging Tools for Single-Cell Analysis: Advances in Optical Measurement Technologies, ed. (Wiley-Liss., 2000).

H. M. Shapiro, Practical Flow Cytometry, 2nd ed. (A. R. Liss, Inc., 1988).

P. Nérin and D. Lefevre, “Device and method for multiparametric analysis of microscopic elements,” U.S. patent 7,777,869 (14 April 2006).

R. Kapoor and M. Casstevens, “Evaluation of multicomponent mixtures using modulated light beam,” U.S. 2007/0096039 A1 (14 April 2006).

G. Durack and J. Hatcher, “System and method for the measurement of multiple fluorescence emissions in a flow cytometry system,” U.S. patent 2008/0213915 A1 (23 April 2007).

K. Luong, P. Lundquist, R. Dalal, J. Lyle, and S. Turner, “Methods and systems for analysis of fluorescent reactions with modulated excitation,” CA patent 2711560 A1 (9 January 2009).

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Figures (6)

Fig. 1
Fig. 1

Optical system of the hematology analyzer. The particle flow circulates out of the plane, in our direction.

Fig. 2
Fig. 2

Absorption and emission spectra of green fluorescent microspheres. Absorption and emission spectra of red fluorescent microspheres. Excitation wavelengths of laser (λ = 491 nm and λ = 532 nm). Bandpass filters for two fluorescent detections (rectangles).

Fig. 3
Fig. 3

Optical modulation efficiency of the tunable acousto-optic filter versus frequency for laser beams at 491 nm and 532 nm.

Fig. 4
Fig. 4

(a) Incident light beams at 491 nm and 532 nm modulated with frequencies w 1(200 kHz) et w 2(300 kHz) respectively. (b) Fluorescence signal at 510 nm from channel 1. (c) Fluorescence signal at 610 nm from channel 2 (experimental data).

Fig. 5
Fig. 5

Optical signals of fluorescent microspheres extracted from experimental data recorded on channel 2 (S2). (a) Useful optical signal which is red microsphere fluorescent (FluoRED) and (b) useless optical signal.

Fig. 6
Fig. 6

Crosstalk between sm1 and sm2 signals with respect to the frequency detuning of the two frequency modulations.(a) w1 = 200kHz, w2 = 300kHz; (b) w1 = 200kHz, w2 = 250kHz.

Equations (6)

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

I ( t ) = s m 1 ( w 1 ) + s m 2 ( w 2 ) .
I ( t ) = I 10 ( t ) 2 [ 1 + M 1 cos ( w 1 t ) ] + I 20 ( t ) 2 [ 1 + M 2 cos ( w 2 t ) ] .
S m ( t ) = exp [ t t 0 ] ² [ ( k G + k ' R ) s m 1 ( w 1 ) + k R s m 2 ( w 2 ) ]
S m ( t ) = exp [ t t 0 ] ² [ ( k G + k ' R ) I 10 ( t ) 2 [ 1 + M 1 cos ( w 1 t ) ] + k R I 20 ( t ) 2 [ 1 + M 2 cos ( w 2 t ) ] ]
k G = 0 , 55. ρ G k R = 0 , 44. ρ R k ' R = 1. ρ R
F l u o R E D = S 2 ε . S 1

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