Abstract

By simple modification of the pattern of fluorescence excitation light in an epi-illumination inverted microscope, one can achieve conditions that produce total internal reflection fluorescence (TIRF) by evanescent wave excitation. Though traditionally requiring a collimated beam traversing through a special prism, TIRF also can be achieved by epi-illumination through the periphery of a 1.4 numerical aperture objective. An opaque disk of appropriate size is placed in the illumination path external to the microscope so as to cast a sharp, real-image shadow at the objective's back focal plane. This shadow allows a hollow cone of epi-illumination rays traveling at only super-critical angles to reach the glass/water interface at the sample plane. Three kinds of TIRF illumination patterns can be produced by variations of this scheme: (1) a small spot of illumination of 1.5 μm radius by use of a laser light source, (2) a large region of illumination by use of a laser-illuminated diffusing screen located upbeam from the opaque disk, and (3) a large region of illumination by use of a conventional mercury arc.

© 1989 Optical Society of America

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References

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  1. D. Axelrod, T. P. Burghardt, N. L. Thompson, “Total Internal Reflection Fluorescence,” Annu. Rev. Biophys. Bioeng. 13, 247–268 (1984).
    [CrossRef] [PubMed]
  2. E. H. Hellen, D. Axelrod, “Fluorescence Emission at Dielectric and Metal-Film Interfaces,” J. Opt. Soc. Am. B 4, 337–350 (1987).
    [CrossRef]
  3. D. Axelrod, R. M. Fulbright, E. H. Hellen, “Adsorption Kinetics on Biological Membranes: Measurement by Total Internal Reflection Fluorescence,” Applications of Fluorescence in the Biomedical Sciences, D. L. Taylor, A. J. Waggoner, R. F. Murphy, F. Lanni, R. R. Birge, Eds. (Liss, New York, 1986), pp. 461–476.

1987 (1)

1984 (1)

D. Axelrod, T. P. Burghardt, N. L. Thompson, “Total Internal Reflection Fluorescence,” Annu. Rev. Biophys. Bioeng. 13, 247–268 (1984).
[CrossRef] [PubMed]

Axelrod, D.

E. H. Hellen, D. Axelrod, “Fluorescence Emission at Dielectric and Metal-Film Interfaces,” J. Opt. Soc. Am. B 4, 337–350 (1987).
[CrossRef]

D. Axelrod, T. P. Burghardt, N. L. Thompson, “Total Internal Reflection Fluorescence,” Annu. Rev. Biophys. Bioeng. 13, 247–268 (1984).
[CrossRef] [PubMed]

D. Axelrod, R. M. Fulbright, E. H. Hellen, “Adsorption Kinetics on Biological Membranes: Measurement by Total Internal Reflection Fluorescence,” Applications of Fluorescence in the Biomedical Sciences, D. L. Taylor, A. J. Waggoner, R. F. Murphy, F. Lanni, R. R. Birge, Eds. (Liss, New York, 1986), pp. 461–476.

Burghardt, T. P.

D. Axelrod, T. P. Burghardt, N. L. Thompson, “Total Internal Reflection Fluorescence,” Annu. Rev. Biophys. Bioeng. 13, 247–268 (1984).
[CrossRef] [PubMed]

Fulbright, R. M.

D. Axelrod, R. M. Fulbright, E. H. Hellen, “Adsorption Kinetics on Biological Membranes: Measurement by Total Internal Reflection Fluorescence,” Applications of Fluorescence in the Biomedical Sciences, D. L. Taylor, A. J. Waggoner, R. F. Murphy, F. Lanni, R. R. Birge, Eds. (Liss, New York, 1986), pp. 461–476.

Hellen, E. H.

E. H. Hellen, D. Axelrod, “Fluorescence Emission at Dielectric and Metal-Film Interfaces,” J. Opt. Soc. Am. B 4, 337–350 (1987).
[CrossRef]

D. Axelrod, R. M. Fulbright, E. H. Hellen, “Adsorption Kinetics on Biological Membranes: Measurement by Total Internal Reflection Fluorescence,” Applications of Fluorescence in the Biomedical Sciences, D. L. Taylor, A. J. Waggoner, R. F. Murphy, F. Lanni, R. R. Birge, Eds. (Liss, New York, 1986), pp. 461–476.

Thompson, N. L.

D. Axelrod, T. P. Burghardt, N. L. Thompson, “Total Internal Reflection Fluorescence,” Annu. Rev. Biophys. Bioeng. 13, 247–268 (1984).
[CrossRef] [PubMed]

Annu. Rev. Biophys. Bioeng. (1)

D. Axelrod, T. P. Burghardt, N. L. Thompson, “Total Internal Reflection Fluorescence,” Annu. Rev. Biophys. Bioeng. 13, 247–268 (1984).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B (1)

Other (1)

D. Axelrod, R. M. Fulbright, E. H. Hellen, “Adsorption Kinetics on Biological Membranes: Measurement by Total Internal Reflection Fluorescence,” Applications of Fluorescence in the Biomedical Sciences, D. L. Taylor, A. J. Waggoner, R. F. Murphy, F. Lanni, R. R. Birge, Eds. (Liss, New York, 1986), pp. 461–476.

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Figures (4)

Fig. 1
Fig. 1

Schematic diagram of supercritical rays in the epi-illuminator of a fluorescence microscope.

Fig. 2
Fig. 2

Optical arrangements before the epi-illuminator for three configurations: (a) laser source focused with a single lens to a spot, (b) laser source focused to a spot with two lenses, (c) laser source converted to an extended source by a latex bead suspension for illuminating a large area (size of the illuminated region on the DSP is exaggerated for pictoral clarity), (d) mercury arc source, including the use of a conical lens to increase the power of supercritical light at the expense of subcritical light.

Fig. 3
Fig. 3

TIRF on fluorescence-labeled erythrocyte ghosts, laser-illuminated by the configuration depicted in Fig. 2(c). (a) TIRF, focused at coverslip surface, (b) epi-illumination, focused at coverslip surface, (c) TIRF focused at midplane of spherical ghosts, (d) epi-illumination focused at midplane of spherical ghosts. Exposure time = 12 s for both TIRF and epi-illumination, on Kodak TMAX P3200 film. For TIRF the total laser power at λ = 514.5 nm was about 0.4 W before the OL; for epi-illumination this power was reduced by at least a factor of 10. Space bar = 10 μm.

Fig. 4
Fig. 4

Same as Fig. 3, except illuminated by the mercury arc source with the configuration depicted in Fig. 2(d).

Equations (4)

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θ c = sin 1 ( n 1 / n 2 ) ,
A = n g sin θ m .
sin θ m > sin θ c .
A > n w .

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