Abstract

A Michelson interferometer has been adapted as an excitation source for fluorescence spectroscopy. A moving fringe pattern was generated by linear displacement of the movable mirror of the Michelson interferometer coupled to a xenon-arc lamp. This spectrally modulated source was monitored by a reference photomultiplier and used for exciting a Rhodamine B solution. The fluorescence emission at >645 nm was detected by a second photomultiplier. The two interferograms were acquired by a dual-channel digital oscilloscope, and their discrete Fourier transforms and corresponding power spectra were generated in a computer. The power spectrum of the emission signal represented the excitation spectrum, as was confirmed by comparison with the absorption spectrum of Rhodamine B. This optical arrangement is well suited for acquiring fluorescence excitation spectra in the optical microscopy of biological specimens.

© 1998 Optical Society of America

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References

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  1. G. Weber, “Enumeration of components in complex systems by fluorescence spectrophotometry,” Nature (London) 190, 27–29 (1961).
    [CrossRef]
  2. A. G. Marshall, F. R. Verdun, Fourier Transforms in NMR, Optical and Mass Spectrometry (Elsevier, Amsterdam, 1990).
  3. J. G. Hirschberg, E. Kohen, “Instrumentation design for study of metabolic control in living cells,” in Analytical Use of Fluorescent Probes in Oncology,ASI Series, E. Kohen, J. G. Hirschberg, eds., Vol. A286 of NATO ASI Series (Plenum, New York, 1996), pp. 293–298.
  4. J. G. Hirschberg, W. I. Fried, L. Hazelton, A. Wouters, “Multiplex Fabry-Perot interferometer,” Appl. Opt. 10, 1979–1980 (1971).
    [CrossRef] [PubMed]
  5. P. Fellgett, “Spectomètre interférential multiplex pour measures infra-rouges sur les étoiles,” J. Phys. Rad. 19, 237–240 (1958).
    [CrossRef]
  6. J. Connes, “Réchèrches sur la spectroscopie par transformation de Fourier,” Rev. Opt. Theor. Instrum. 40, 75–79 (1961).
  7. R. Paul, A. Steiner, R. Gemperlein, “Spectral sensitivity of Calliphora erythrocephala and other insect species studied with Fourier interferometric stimulation (FIS),” J. Comp. Physiol. A 158, 669–680 (1986).
    [CrossRef]
  8. A. Steiner, R. Paul, R. Gemperlein, “Retinal receptor types in Aglaais urticae and Pieris brassicae (Lepidoptera), revealed by the analysis of the electroretinogram obtained with Fourier interferometric stimulation (FIS),” J. Comp. Physiol. A 160, 247–258 (1987).
    [CrossRef]
  9. P. Jacquinot, “The luminosity of spectrometers with prisms, gratings, or Fabry-Perot étalons,” J. Opt. Soc. Am. 44, 761–765 (1954).
    [CrossRef]
  10. R. F. Chen, “Practical aspects of the calibration and use of the Aminco–Bowman spectrophotofluorometer,” Anal. Biochem. 20, 339–357 (1967).
    [CrossRef] [PubMed]
  11. Our research and R. Gemperlein, Zoological Institute, Faculty of Biology, Ludwig Maximilian University of München, Theresienstadt 39, D-80333 Mn̈chen, Germany (personal communication, 1997).

1987 (1)

A. Steiner, R. Paul, R. Gemperlein, “Retinal receptor types in Aglaais urticae and Pieris brassicae (Lepidoptera), revealed by the analysis of the electroretinogram obtained with Fourier interferometric stimulation (FIS),” J. Comp. Physiol. A 160, 247–258 (1987).
[CrossRef]

1986 (1)

R. Paul, A. Steiner, R. Gemperlein, “Spectral sensitivity of Calliphora erythrocephala and other insect species studied with Fourier interferometric stimulation (FIS),” J. Comp. Physiol. A 158, 669–680 (1986).
[CrossRef]

1971 (1)

1967 (1)

R. F. Chen, “Practical aspects of the calibration and use of the Aminco–Bowman spectrophotofluorometer,” Anal. Biochem. 20, 339–357 (1967).
[CrossRef] [PubMed]

1961 (2)

G. Weber, “Enumeration of components in complex systems by fluorescence spectrophotometry,” Nature (London) 190, 27–29 (1961).
[CrossRef]

J. Connes, “Réchèrches sur la spectroscopie par transformation de Fourier,” Rev. Opt. Theor. Instrum. 40, 75–79 (1961).

1958 (1)

P. Fellgett, “Spectomètre interférential multiplex pour measures infra-rouges sur les étoiles,” J. Phys. Rad. 19, 237–240 (1958).
[CrossRef]

1954 (1)

Chen, R. F.

R. F. Chen, “Practical aspects of the calibration and use of the Aminco–Bowman spectrophotofluorometer,” Anal. Biochem. 20, 339–357 (1967).
[CrossRef] [PubMed]

Connes, J.

J. Connes, “Réchèrches sur la spectroscopie par transformation de Fourier,” Rev. Opt. Theor. Instrum. 40, 75–79 (1961).

Fellgett, P.

P. Fellgett, “Spectomètre interférential multiplex pour measures infra-rouges sur les étoiles,” J. Phys. Rad. 19, 237–240 (1958).
[CrossRef]

Fried, W. I.

Gemperlein, R.

A. Steiner, R. Paul, R. Gemperlein, “Retinal receptor types in Aglaais urticae and Pieris brassicae (Lepidoptera), revealed by the analysis of the electroretinogram obtained with Fourier interferometric stimulation (FIS),” J. Comp. Physiol. A 160, 247–258 (1987).
[CrossRef]

R. Paul, A. Steiner, R. Gemperlein, “Spectral sensitivity of Calliphora erythrocephala and other insect species studied with Fourier interferometric stimulation (FIS),” J. Comp. Physiol. A 158, 669–680 (1986).
[CrossRef]

Our research and R. Gemperlein, Zoological Institute, Faculty of Biology, Ludwig Maximilian University of München, Theresienstadt 39, D-80333 Mn̈chen, Germany (personal communication, 1997).

Hazelton, L.

Hirschberg, J. G.

J. G. Hirschberg, W. I. Fried, L. Hazelton, A. Wouters, “Multiplex Fabry-Perot interferometer,” Appl. Opt. 10, 1979–1980 (1971).
[CrossRef] [PubMed]

J. G. Hirschberg, E. Kohen, “Instrumentation design for study of metabolic control in living cells,” in Analytical Use of Fluorescent Probes in Oncology,ASI Series, E. Kohen, J. G. Hirschberg, eds., Vol. A286 of NATO ASI Series (Plenum, New York, 1996), pp. 293–298.

Jacquinot, P.

Kohen, E.

J. G. Hirschberg, E. Kohen, “Instrumentation design for study of metabolic control in living cells,” in Analytical Use of Fluorescent Probes in Oncology,ASI Series, E. Kohen, J. G. Hirschberg, eds., Vol. A286 of NATO ASI Series (Plenum, New York, 1996), pp. 293–298.

Marshall, A. G.

A. G. Marshall, F. R. Verdun, Fourier Transforms in NMR, Optical and Mass Spectrometry (Elsevier, Amsterdam, 1990).

Paul, R.

A. Steiner, R. Paul, R. Gemperlein, “Retinal receptor types in Aglaais urticae and Pieris brassicae (Lepidoptera), revealed by the analysis of the electroretinogram obtained with Fourier interferometric stimulation (FIS),” J. Comp. Physiol. A 160, 247–258 (1987).
[CrossRef]

R. Paul, A. Steiner, R. Gemperlein, “Spectral sensitivity of Calliphora erythrocephala and other insect species studied with Fourier interferometric stimulation (FIS),” J. Comp. Physiol. A 158, 669–680 (1986).
[CrossRef]

Steiner, A.

A. Steiner, R. Paul, R. Gemperlein, “Retinal receptor types in Aglaais urticae and Pieris brassicae (Lepidoptera), revealed by the analysis of the electroretinogram obtained with Fourier interferometric stimulation (FIS),” J. Comp. Physiol. A 160, 247–258 (1987).
[CrossRef]

R. Paul, A. Steiner, R. Gemperlein, “Spectral sensitivity of Calliphora erythrocephala and other insect species studied with Fourier interferometric stimulation (FIS),” J. Comp. Physiol. A 158, 669–680 (1986).
[CrossRef]

Verdun, F. R.

A. G. Marshall, F. R. Verdun, Fourier Transforms in NMR, Optical and Mass Spectrometry (Elsevier, Amsterdam, 1990).

Weber, G.

G. Weber, “Enumeration of components in complex systems by fluorescence spectrophotometry,” Nature (London) 190, 27–29 (1961).
[CrossRef]

Wouters, A.

Anal. Biochem. (1)

R. F. Chen, “Practical aspects of the calibration and use of the Aminco–Bowman spectrophotofluorometer,” Anal. Biochem. 20, 339–357 (1967).
[CrossRef] [PubMed]

Appl. Opt. (1)

J. Comp. Physiol. A (2)

R. Paul, A. Steiner, R. Gemperlein, “Spectral sensitivity of Calliphora erythrocephala and other insect species studied with Fourier interferometric stimulation (FIS),” J. Comp. Physiol. A 158, 669–680 (1986).
[CrossRef]

A. Steiner, R. Paul, R. Gemperlein, “Retinal receptor types in Aglaais urticae and Pieris brassicae (Lepidoptera), revealed by the analysis of the electroretinogram obtained with Fourier interferometric stimulation (FIS),” J. Comp. Physiol. A 160, 247–258 (1987).
[CrossRef]

J. Opt. Soc. Am. (1)

J. Phys. Rad. (1)

P. Fellgett, “Spectomètre interférential multiplex pour measures infra-rouges sur les étoiles,” J. Phys. Rad. 19, 237–240 (1958).
[CrossRef]

Nature (London) (1)

G. Weber, “Enumeration of components in complex systems by fluorescence spectrophotometry,” Nature (London) 190, 27–29 (1961).
[CrossRef]

Rev. Opt. Theor. Instrum. (1)

J. Connes, “Réchèrches sur la spectroscopie par transformation de Fourier,” Rev. Opt. Theor. Instrum. 40, 75–79 (1961).

Other (3)

A. G. Marshall, F. R. Verdun, Fourier Transforms in NMR, Optical and Mass Spectrometry (Elsevier, Amsterdam, 1990).

J. G. Hirschberg, E. Kohen, “Instrumentation design for study of metabolic control in living cells,” in Analytical Use of Fluorescent Probes in Oncology,ASI Series, E. Kohen, J. G. Hirschberg, eds., Vol. A286 of NATO ASI Series (Plenum, New York, 1996), pp. 293–298.

Our research and R. Gemperlein, Zoological Institute, Faculty of Biology, Ludwig Maximilian University of München, Theresienstadt 39, D-80333 Mn̈chen, Germany (personal communication, 1997).

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Figures (4)

Fig. 1
Fig. 1

Schematic of the optical system for fluorescence spectroscopy based on a Michelson interferometer for spectral modulation of excitation. See text for details.

Fig. 2
Fig. 2

Fluorescence and reference signals modulated by the interferometer in the time domain. Michelson interferograms for (a) the Rhodamine fluorescence and (b) the excitation light. The fluorescence (a) and reference (b) PM signals were collected in parallel, started by the same trigger. Data collection was for 1 s, with a sampling frequency of 8000 Hz.

Fig. 3
Fig. 3

Fourier transforms and power spectra. (a) Magnitude and (b) phase of discrete Fourier transforms and (c) calculated power spectra of the fluorescence signal (fl PM) and the reference signal (ref PM). The range of both the DFT and the PSP was 8000 Hz. The PSP of the reference PM signal [(c), thinner curve line] corresponds to the spectrum of the exciting light (in arbitrary units), whereas the power spectrum of the fluorescence signal [(c), thicker curve line] represents the uncorrected excitation spectrum of Rhodamine B.

Fig. 4
Fig. 4

Interferometric excitation spectrum of Rhodamine B compared with its absorption spectrum. Correspondence of the excitation spectrum of Rhodamine B (open circles) to its absorption spectrum (solid curve) obtained in a spectrophotometer. To be able to compare the two plots, we plotted the absorbance of Rhodamine in arbitrary units against wave number (lower abscissa and left ordinate) and plotted the power spectrum of the dye fluorescence in the frequency domain (upper abscissa and right ordinate).

Equations (2)

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m λ = 2 x ,
d m d t = 2 λ d x d t .

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