Abstract

A novel fluorescence spectrometer and method for the simultaneous detection of multiple-fluorophore species in a no-moving-parts, instantaneous manner is described. In the reported embodiment of the instrument, a tapered Fabry–Perot filter is used to spatially encode the fluorescence spectrum from a multiple-dye-containing test sample. Using a pseudoinverse reconstruction algorithm, we spectrally decode the particle concentration for each dye specie in the test sample. Experimental results are reported along with a theoretical treatment of the method.

© 2005 Optical Society of America

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References

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  1. T. B. Hirschfeld, M. J. Block, “Fluorescent immunoassay using optical fibers and antibodies immobilized on surfaces,” presented at the Federation of Analytical Chemistry and Spectroscopy Societies Eleventh Annual Meeting, Philadelphia, Pa., 16 September 1984.
  2. M. J. Block, T. B. Hirschfeld, “Apparatus including optical fiber for fluorescence immunoassay,” U.S. patent4,582,809 (15April1986).
  3. E. W. Saaski, C. C. Jung, “Assay methods and apparatus,” U.S. patent6,136,611 (24October2000).
  4. C. C. Jung, E. W. Saaski, D. A. McCrae, B. M. Lingerfelt, G. P. Anderson, “RAPTOR: a fluoroimmunoassay-based fiber optic sensor for detection of biological threats,” IEEE Sensors J. 3, 352–360 (2003).
    [CrossRef]
  5. M. Gouzman, N. Lifshitz, S. Luryi, O. Semyonov, D. Gavrilov, V. Kuzminskiy, “Excitation-emission fluorimeter based on linear interference filters,” Appl. Opt. 43, 3066–3072 (2004).
    [CrossRef] [PubMed]
  6. J. A. Wahl, J. S. Van Delden, S. Tiwari, “Tapered Fabry–Perot filters,” IEEE Photon. Technol. Lett. 16, 1873–1875 (2004).
    [CrossRef]
  7. J. D. Gaskill, Linear Systems, Fourier Transforms, and Optics (Wiley, 1978).
  8. H. H. Barrett, K. J. Myers, Foundations of Image Science (Wiley, 2003).
  9. Y. Garini, A. Gil, I. Bar-Am, D. Cabib, N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35, 214–226 (1999).
    [CrossRef] [PubMed]
  10. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic, 1999).
    [CrossRef]

2004 (2)

2003 (1)

C. C. Jung, E. W. Saaski, D. A. McCrae, B. M. Lingerfelt, G. P. Anderson, “RAPTOR: a fluoroimmunoassay-based fiber optic sensor for detection of biological threats,” IEEE Sensors J. 3, 352–360 (2003).
[CrossRef]

1999 (1)

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35, 214–226 (1999).
[CrossRef] [PubMed]

Anderson, G. P.

C. C. Jung, E. W. Saaski, D. A. McCrae, B. M. Lingerfelt, G. P. Anderson, “RAPTOR: a fluoroimmunoassay-based fiber optic sensor for detection of biological threats,” IEEE Sensors J. 3, 352–360 (2003).
[CrossRef]

Bar-Am, I.

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35, 214–226 (1999).
[CrossRef] [PubMed]

Barrett, H. H.

H. H. Barrett, K. J. Myers, Foundations of Image Science (Wiley, 2003).

Block, M. J.

T. B. Hirschfeld, M. J. Block, “Fluorescent immunoassay using optical fibers and antibodies immobilized on surfaces,” presented at the Federation of Analytical Chemistry and Spectroscopy Societies Eleventh Annual Meeting, Philadelphia, Pa., 16 September 1984.

M. J. Block, T. B. Hirschfeld, “Apparatus including optical fiber for fluorescence immunoassay,” U.S. patent4,582,809 (15April1986).

Cabib, D.

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35, 214–226 (1999).
[CrossRef] [PubMed]

Garini, Y.

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35, 214–226 (1999).
[CrossRef] [PubMed]

Gaskill, J. D.

J. D. Gaskill, Linear Systems, Fourier Transforms, and Optics (Wiley, 1978).

Gavrilov, D.

Gil, A.

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35, 214–226 (1999).
[CrossRef] [PubMed]

Gouzman, M.

Hirschfeld, T. B.

T. B. Hirschfeld, M. J. Block, “Fluorescent immunoassay using optical fibers and antibodies immobilized on surfaces,” presented at the Federation of Analytical Chemistry and Spectroscopy Societies Eleventh Annual Meeting, Philadelphia, Pa., 16 September 1984.

M. J. Block, T. B. Hirschfeld, “Apparatus including optical fiber for fluorescence immunoassay,” U.S. patent4,582,809 (15April1986).

Jung, C. C.

C. C. Jung, E. W. Saaski, D. A. McCrae, B. M. Lingerfelt, G. P. Anderson, “RAPTOR: a fluoroimmunoassay-based fiber optic sensor for detection of biological threats,” IEEE Sensors J. 3, 352–360 (2003).
[CrossRef]

E. W. Saaski, C. C. Jung, “Assay methods and apparatus,” U.S. patent6,136,611 (24October2000).

Katzir, N.

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35, 214–226 (1999).
[CrossRef] [PubMed]

Kuzminskiy, V.

Lakowicz, J. R.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic, 1999).
[CrossRef]

Lifshitz, N.

Lingerfelt, B. M.

C. C. Jung, E. W. Saaski, D. A. McCrae, B. M. Lingerfelt, G. P. Anderson, “RAPTOR: a fluoroimmunoassay-based fiber optic sensor for detection of biological threats,” IEEE Sensors J. 3, 352–360 (2003).
[CrossRef]

Luryi, S.

McCrae, D. A.

C. C. Jung, E. W. Saaski, D. A. McCrae, B. M. Lingerfelt, G. P. Anderson, “RAPTOR: a fluoroimmunoassay-based fiber optic sensor for detection of biological threats,” IEEE Sensors J. 3, 352–360 (2003).
[CrossRef]

Myers, K. J.

H. H. Barrett, K. J. Myers, Foundations of Image Science (Wiley, 2003).

Saaski, E. W.

C. C. Jung, E. W. Saaski, D. A. McCrae, B. M. Lingerfelt, G. P. Anderson, “RAPTOR: a fluoroimmunoassay-based fiber optic sensor for detection of biological threats,” IEEE Sensors J. 3, 352–360 (2003).
[CrossRef]

E. W. Saaski, C. C. Jung, “Assay methods and apparatus,” U.S. patent6,136,611 (24October2000).

Semyonov, O.

Tiwari, S.

J. A. Wahl, J. S. Van Delden, S. Tiwari, “Tapered Fabry–Perot filters,” IEEE Photon. Technol. Lett. 16, 1873–1875 (2004).
[CrossRef]

Van Delden, J. S.

J. A. Wahl, J. S. Van Delden, S. Tiwari, “Tapered Fabry–Perot filters,” IEEE Photon. Technol. Lett. 16, 1873–1875 (2004).
[CrossRef]

Wahl, J. A.

J. A. Wahl, J. S. Van Delden, S. Tiwari, “Tapered Fabry–Perot filters,” IEEE Photon. Technol. Lett. 16, 1873–1875 (2004).
[CrossRef]

Appl. Opt. (1)

Cytometry (1)

Y. Garini, A. Gil, I. Bar-Am, D. Cabib, N. Katzir, “Signal to noise analysis of multiple color fluorescence imaging microscopy,” Cytometry 35, 214–226 (1999).
[CrossRef] [PubMed]

IEEE Photon. Technol. Lett. (1)

J. A. Wahl, J. S. Van Delden, S. Tiwari, “Tapered Fabry–Perot filters,” IEEE Photon. Technol. Lett. 16, 1873–1875 (2004).
[CrossRef]

IEEE Sensors J. (1)

C. C. Jung, E. W. Saaski, D. A. McCrae, B. M. Lingerfelt, G. P. Anderson, “RAPTOR: a fluoroimmunoassay-based fiber optic sensor for detection of biological threats,” IEEE Sensors J. 3, 352–360 (2003).
[CrossRef]

Other (6)

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic, 1999).
[CrossRef]

J. D. Gaskill, Linear Systems, Fourier Transforms, and Optics (Wiley, 1978).

H. H. Barrett, K. J. Myers, Foundations of Image Science (Wiley, 2003).

T. B. Hirschfeld, M. J. Block, “Fluorescent immunoassay using optical fibers and antibodies immobilized on surfaces,” presented at the Federation of Analytical Chemistry and Spectroscopy Societies Eleventh Annual Meeting, Philadelphia, Pa., 16 September 1984.

M. J. Block, T. B. Hirschfeld, “Apparatus including optical fiber for fluorescence immunoassay,” U.S. patent4,582,809 (15April1986).

E. W. Saaski, C. C. Jung, “Assay methods and apparatus,” U.S. patent6,136,611 (24October2000).

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Figures (8)

Fig. 1
Fig. 1

Schematic of the TFP fluorescence spectrometer.

Fig. 2
Fig. 2

Photo of the TFP fluorescence spectrometer on a 2 ft (0.61 m) × 3 ft (0.91 m) optical breadboard.

Fig. 3
Fig. 3

Measured spectra for six calibration concentrations of SR 101 in pure ethanol for 25 ms exposures and 100 averaged frames.

Fig. 4
Fig. 4

Maximum counts minus the background for each of the six SR 101 calibration concentrations. The solid line is a LS linear fit.

Fig. 5
Fig. 5

Measured spectrum for 100 nM SR 101 in pure ethanol for 25 ms integration and 100 averages.

Fig. 6
Fig. 6

Measured spectra of (a) YG FluoSpheres and (b) CR Fluospheres on a log-linear scale for 100 ms exposure and ten averaged frames.

Fig. 7
Fig. 7

Concentration versus gmax for (a) YG FluoSpheres and (b) CR FluoSpheres for 100 ms exposures and ten averaged frames with LS linear fits (solid lines).

Fig. 8
Fig. 8

Measured spectra of 1:1 (by volume) mixture of YG and CR beads along with the measured spectra of the constituent components. The exposure time was 100 ms and the spectra were averaged ten times.

Equations (17)

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Φ tot ( λ ) = 0 d λ Φ 0 ( λ ) m = 1 M η m ( λ ) { 1 - exp [ - m ( λ ) l eff c m ] } ,
T TFP ( x ,     y ,     λ ) = τ f π x lw 2 L [ x - x 0 ( λ ) ;     0.5 x lw ] × exp [ - π ( x x bw ) 2 ] rect ( x w x ) rect ( y w y ) ,
E d ( x ,     y ,     λ ) = π τ c τ f π r x lw ( 1 - 1 - NA 2 ) 4 A i m r y L ( x - m r x x 0 ( λ ) ;     0.5 m r x x lw ) × exp [ - π ( x m r x x bw ) 2 ] rect ( x m r x w x ) × rect ( y m r y w y ) 0 d λ Φ 0 ( λ ) × m = 1 M η m F m e ( λ ) { 1 - exp [ - m F m a ( λ ) l eff c m ] } ,
g i = π τ c τ f τ r x lw ( 1 - 1 - NA 2 ) 4 A i m r y 0 d λ 0 d λ × - d x d y R ( λ ) pix i ( x ,     y ) m = 1 M η m F m e ( λ ) Φ 0 ( λ ) { 1 - exp [ - m F m a ( λ ) l eff c m ] } L [ x - m r x x 0 ( λ ) ;     0.5 m r x x lw ] exp [ - π ( x m r x x bw ) 2 ] × rect ( x m r x w x ) rect ( y m r y w y ) ,
pix i ( x ,     y ) = rect ( x - x i p x ) rect ( y p y ) ,
- d x d y pix i ( x ,     y ) L [ x - m r x x 0 ( λ ) ;     0.5 m r x x lw ] × exp [ - π ( x m r x x bw ) 2 ] A p L ( x i - m r x x 0 ( λ ) ;     0.5 m r x x lw ) exp [ - π ( x i m r x x bw ) 2 ]
g i = π τ c τ f τ r η m x lw ( 1 - 1 - NA 2 ) A p 4 A i m r y × 0 d λ 0 d λ R ( λ ) m = 1 M F m e ( λ ) Φ 0 ( λ ) { 1 - exp [ - m F m a ( λ ) l eff c m ] } L [ x i - m r x x 0 ( λ ) ;     0.5 m r x x lw ] exp [ - π ( x i m r x x bw ) 2 ] .
g i π τ c τ f τ r η m l eff x lw ( 1 - 1 - NA 2 ) A p 4 A i m r y × exp [ - π ( x i m r x x bw ) 2 ] 0 d λ 0 d λ R ( λ ) Φ 0 ( λ ) L ( x i - m r x x 0 ( λ ) ;     0.5 m r x x lw ) m = 1 M F m e ( λ ) m F m a ( λ ) c m .
g i = m = 1 M h i m c m ,
h i m = π τ c τ f τ r η m m x lw ( 1 - 1 - NA 2 ) A p 4 m r y m c 2 A 0 1 / 2 × exp [ - π ( x i m r x x bw ) 2 ] 0 d λ F m a ( λ ) Φ ( λ ) × 0 d λ R ( λ ) F m e ( λ ) L ( x i - m r x x 0 ( λ ) ;     0.5 m r x x lw ) .
g max p = m = 1 M h max p m c m ,
G max = H max · C ,
[ g max 1 g max 2 g max M ] = [ h max 11 h max 12 h max 1 M h max 21 h max 22 h max 2 M h max M 1 h max M 2 h max M M ] [ c 1 c 2 c M ] .
C = H max - 1 · G max .
[ g max p ( 1 ) g max p ( 2 ) g max p ( N c ) ] = [ c 1 ( 1 ) c 2 ( 1 ) c M ( 1 ) c 1 ( 2 ) c 2 ( 2 ) c M ( 2 ) c 1 ( N c ) c 2 ( N c ) c M ( N c ) ] [ h maxLS p 1 h maxLS p 2 h maxLS p M ] .
H maxLS = C + · G ,
N m ( no . of particles ) = 0.001 A 0 l eff N A c m ,

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