Abstract

The sensitivity of chemical or biological analyses using fluorescent tagged reagents is limited by concentration quenching of the fluorescence at high tag concentrations and by photochemical bleaching if high illumination intensities are used. This paper shows by theoretical analysis and experimental verification that concentration quenching lengthens bleaching lifetimes, and the integrated fluorescent emission obtained on complete bleaching is invariant with fluorescent quantum efficiency, absorption cross section, and the illumination’s intensity and duration. This can be used to produce extremely sensitive fluorescent tagging procedes. The procedure also improves photometric reproducibility, allows a fluorescent stain to act as a probe of several properties in its microenvironment, and allows sample background differentiation even when the free stain background is more fluorescent than the bound one.

© 1976 Optical Society of America

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References

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  1. M. Goldman, Fluorescent Antibody Methods (Academic, New York, 1968).
  2. I. Vierosanu, J. Chim Phys. 71, 61 (1974).
  3. S. Shu, J. Deng, S. Vetter, W. H. Bentner, Ann. N.Y. Acad. Sci. 180, 550 (1974).
  4. T. Hirschfeld, “Staining Antibodies,” Application for Letters Patent (1973).
  5. T. Hirschfeld, D. Eaton, “Antigen Detecting Reagents,” Applications for Letters Patent (1974).
  6. G. I. Kaufman, J. F. Nester, D. E. Wasserman, J. Histochem Cytochem. 19, 469 (1971).
    [CrossRef] [PubMed]
  7. G. G. Guilbault, Practical Fluorescence (Marcel Dekker, New York, 1973).
  8. R. F. Chen, G. V. Vurek, N. Alexander, Science 156, 949 (1967).
    [CrossRef] [PubMed]
  9. A. D. Britt, W. B. Moriz, IEEE J. Quantum Electron. QE-8, 913 (1972).
    [CrossRef]
  10. W. J. Dreyer, U.S. Patent3,853,987 (10December1974).
  11. W. A. Bonner, H. R. Hulett, R. G. Sweet, L. A. Herzenberg, Rev. Sci. Instrum. 43, 404 (1972).
    [CrossRef] [PubMed]
  12. L. Stryer, Science 162, 526 (1968).
    [CrossRef] [PubMed]
  13. S. Udenfriend, Fluorescence Assay in Biology and Medicine (Academic, New York, 1969), Vol. 2.
  14. J. B. Le Pecq, C. Paoletti, J. Mol. Biol. 27, 87 (1967).
    [CrossRef]
  15. V. W. Burns, Biophys. J. 14, 189 (1974).
    [CrossRef]
  16. T. Hirschfeld, W. Mueller, Histochem. J., to be published.
  17. T. Hirschfeld, Appl. Opt. 15, 2965 (1976).
    [CrossRef] [PubMed]

1976 (1)

1974 (4)

V. W. Burns, Biophys. J. 14, 189 (1974).
[CrossRef]

I. Vierosanu, J. Chim Phys. 71, 61 (1974).

S. Shu, J. Deng, S. Vetter, W. H. Bentner, Ann. N.Y. Acad. Sci. 180, 550 (1974).

T. Hirschfeld, D. Eaton, “Antigen Detecting Reagents,” Applications for Letters Patent (1974).

1973 (1)

T. Hirschfeld, “Staining Antibodies,” Application for Letters Patent (1973).

1972 (2)

A. D. Britt, W. B. Moriz, IEEE J. Quantum Electron. QE-8, 913 (1972).
[CrossRef]

W. A. Bonner, H. R. Hulett, R. G. Sweet, L. A. Herzenberg, Rev. Sci. Instrum. 43, 404 (1972).
[CrossRef] [PubMed]

1971 (1)

G. I. Kaufman, J. F. Nester, D. E. Wasserman, J. Histochem Cytochem. 19, 469 (1971).
[CrossRef] [PubMed]

1968 (1)

L. Stryer, Science 162, 526 (1968).
[CrossRef] [PubMed]

1967 (2)

J. B. Le Pecq, C. Paoletti, J. Mol. Biol. 27, 87 (1967).
[CrossRef]

R. F. Chen, G. V. Vurek, N. Alexander, Science 156, 949 (1967).
[CrossRef] [PubMed]

Alexander, N.

R. F. Chen, G. V. Vurek, N. Alexander, Science 156, 949 (1967).
[CrossRef] [PubMed]

Bentner, W. H.

S. Shu, J. Deng, S. Vetter, W. H. Bentner, Ann. N.Y. Acad. Sci. 180, 550 (1974).

Bonner, W. A.

W. A. Bonner, H. R. Hulett, R. G. Sweet, L. A. Herzenberg, Rev. Sci. Instrum. 43, 404 (1972).
[CrossRef] [PubMed]

Britt, A. D.

A. D. Britt, W. B. Moriz, IEEE J. Quantum Electron. QE-8, 913 (1972).
[CrossRef]

Burns, V. W.

V. W. Burns, Biophys. J. 14, 189 (1974).
[CrossRef]

Chen, R. F.

R. F. Chen, G. V. Vurek, N. Alexander, Science 156, 949 (1967).
[CrossRef] [PubMed]

Deng, J.

S. Shu, J. Deng, S. Vetter, W. H. Bentner, Ann. N.Y. Acad. Sci. 180, 550 (1974).

Dreyer, W. J.

W. J. Dreyer, U.S. Patent3,853,987 (10December1974).

Eaton, D.

T. Hirschfeld, D. Eaton, “Antigen Detecting Reagents,” Applications for Letters Patent (1974).

Goldman, M.

M. Goldman, Fluorescent Antibody Methods (Academic, New York, 1968).

Guilbault, G. G.

G. G. Guilbault, Practical Fluorescence (Marcel Dekker, New York, 1973).

Herzenberg, L. A.

W. A. Bonner, H. R. Hulett, R. G. Sweet, L. A. Herzenberg, Rev. Sci. Instrum. 43, 404 (1972).
[CrossRef] [PubMed]

Hirschfeld, T.

T. Hirschfeld, Appl. Opt. 15, 2965 (1976).
[CrossRef] [PubMed]

T. Hirschfeld, D. Eaton, “Antigen Detecting Reagents,” Applications for Letters Patent (1974).

T. Hirschfeld, “Staining Antibodies,” Application for Letters Patent (1973).

T. Hirschfeld, W. Mueller, Histochem. J., to be published.

Hulett, H. R.

W. A. Bonner, H. R. Hulett, R. G. Sweet, L. A. Herzenberg, Rev. Sci. Instrum. 43, 404 (1972).
[CrossRef] [PubMed]

Kaufman, G. I.

G. I. Kaufman, J. F. Nester, D. E. Wasserman, J. Histochem Cytochem. 19, 469 (1971).
[CrossRef] [PubMed]

Le Pecq, J. B.

J. B. Le Pecq, C. Paoletti, J. Mol. Biol. 27, 87 (1967).
[CrossRef]

Moriz, W. B.

A. D. Britt, W. B. Moriz, IEEE J. Quantum Electron. QE-8, 913 (1972).
[CrossRef]

Mueller, W.

T. Hirschfeld, W. Mueller, Histochem. J., to be published.

Nester, J. F.

G. I. Kaufman, J. F. Nester, D. E. Wasserman, J. Histochem Cytochem. 19, 469 (1971).
[CrossRef] [PubMed]

Paoletti, C.

J. B. Le Pecq, C. Paoletti, J. Mol. Biol. 27, 87 (1967).
[CrossRef]

Shu, S.

S. Shu, J. Deng, S. Vetter, W. H. Bentner, Ann. N.Y. Acad. Sci. 180, 550 (1974).

Stryer, L.

L. Stryer, Science 162, 526 (1968).
[CrossRef] [PubMed]

Sweet, R. G.

W. A. Bonner, H. R. Hulett, R. G. Sweet, L. A. Herzenberg, Rev. Sci. Instrum. 43, 404 (1972).
[CrossRef] [PubMed]

Udenfriend, S.

S. Udenfriend, Fluorescence Assay in Biology and Medicine (Academic, New York, 1969), Vol. 2.

Vetter, S.

S. Shu, J. Deng, S. Vetter, W. H. Bentner, Ann. N.Y. Acad. Sci. 180, 550 (1974).

Vierosanu, I.

I. Vierosanu, J. Chim Phys. 71, 61 (1974).

Vurek, G. V.

R. F. Chen, G. V. Vurek, N. Alexander, Science 156, 949 (1967).
[CrossRef] [PubMed]

Wasserman, D. E.

G. I. Kaufman, J. F. Nester, D. E. Wasserman, J. Histochem Cytochem. 19, 469 (1971).
[CrossRef] [PubMed]

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

S. Shu, J. Deng, S. Vetter, W. H. Bentner, Ann. N.Y. Acad. Sci. 180, 550 (1974).

Appl. Opt. (1)

Application for Letters Patent (1)

T. Hirschfeld, “Staining Antibodies,” Application for Letters Patent (1973).

Applications for Letters Patent (1)

T. Hirschfeld, D. Eaton, “Antigen Detecting Reagents,” Applications for Letters Patent (1974).

Biophys. J. (1)

V. W. Burns, Biophys. J. 14, 189 (1974).
[CrossRef]

IEEE J. Quantum Electron. (1)

A. D. Britt, W. B. Moriz, IEEE J. Quantum Electron. QE-8, 913 (1972).
[CrossRef]

J. Chim Phys. (1)

I. Vierosanu, J. Chim Phys. 71, 61 (1974).

J. Histochem Cytochem. (1)

G. I. Kaufman, J. F. Nester, D. E. Wasserman, J. Histochem Cytochem. 19, 469 (1971).
[CrossRef] [PubMed]

J. Mol. Biol. (1)

J. B. Le Pecq, C. Paoletti, J. Mol. Biol. 27, 87 (1967).
[CrossRef]

Rev. Sci. Instrum. (1)

W. A. Bonner, H. R. Hulett, R. G. Sweet, L. A. Herzenberg, Rev. Sci. Instrum. 43, 404 (1972).
[CrossRef] [PubMed]

Science (2)

L. Stryer, Science 162, 526 (1968).
[CrossRef] [PubMed]

R. F. Chen, G. V. Vurek, N. Alexander, Science 156, 949 (1967).
[CrossRef] [PubMed]

Other (5)

S. Udenfriend, Fluorescence Assay in Biology and Medicine (Academic, New York, 1969), Vol. 2.

T. Hirschfeld, W. Mueller, Histochem. J., to be published.

G. G. Guilbault, Practical Fluorescence (Marcel Dekker, New York, 1973).

M. Goldman, Fluorescent Antibody Methods (Academic, New York, 1968).

W. J. Dreyer, U.S. Patent3,853,987 (10December1974).

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Figures (3)

Fig. 1
Fig. 1

Quantum efficiency and effective number of dye molecules in antibody labeled with fluoresceine loaded polyethyleneimine.

Fig. 2
Fig. 2

100-ppm fluoresceine bleaching curve at 1.2 × 104-W/cm2 illumination.

Fig. 3
Fig. 3

3330-ppm fluoresceine bleaching curve at 1.2 × 104-W/cm2 illumination.

Tables (1)

Tables Icon

Table I Experimental Verification of QF Independent Fluorimetry

Equations (14)

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S ( t ) = n Q F τ E 0 t g ( t ) d t ,
τ E = ( h c ) / I λ C ) ,
d g * ( t ) = k R g * ( t ) d t ,
d g ( t ) = [ ( τ L ) / ( τ E ) ] k R g ( t ) d t .
g ( t ) = exp ( - t / τ B ) ,
τ B = ( τ E τ R ) / ( τ L ) .
S ( t ) = n [ ( Q F τ B ) / ( τ E ) ] [ 1 - exp ( - t / τ B ) ] .
Q F = τ L / τ F ,
S ( t ) = n [ ( τ R ) / ( τ F ) ] .
t τ B = ( τ R h c ) / ( I λ C τ F Q F ) .
[ d S ( t ) d t ] t 0 = n Q F τ E .
1 τ B = [ d S ( t ) / d t ] t 0 S ( t τ B ) = τ F τ E Q F τ R ,
t d = τ B ln S L
R F = [ ( Q S ) / ( Q B ) ] S L ( 1 - Q B / Q S ) ,

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