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Pentaferometer: a solid Sagnac interferometer

Joseph G. Hirschberg and Elli Kohen

A solid two-beam Sagnac-type interferometer is described that is especially adapted for use with amicroscope for Fourier excitation and emission fluorescence spectroscopy. Its advantages are its com-pactness and stability, and because it is an integral optical element, the need for eliminating vibrationand for shielding from air currents is greatly reduced. © 1999 Optical Society of America

OCIS codes: 120.2650, 120.3180, 170.2520, 170.4090, 300.6280, 300.6300.

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In fluorescence spectroscopy there are two distinctspectra: excitation and emission. Of the two,emission spectra have been more often utilized forthe study of biological materials, such as living cells.There are, however, good reasons for measuring ex-citation as well as emission fluorescence spectra.Among these reasons is that the shape of the excita-tion spectrum is dependent on more highly excitedenergy levels of the fluorescent molecule than that ofthe emission spectrum. Therefore the excitationspectrum is more sensitive to the environment of thefluorochrome.

As has been pointed out,1 in the case for which it isnecessary to distinguish between the spectra of sev-eral fluorophores, knowledge of the excitation as wellas the emission spectrum may provide crucial addi-tional information that is unavailable when the emis-sion spectrum alone is measured.

For excitation spectra, the spectral selection ele-ment is placed in the light path before the fluorescingobject instead of after it. When possible damage oralteration of the object to be studied ~e.g., a living cell!sets a limit to the intensity of the exciting radiationthat can be used, there is an important advantage inusing the excitation spectrum. The light through-put of a spectral selection element ~e.g., a prism, adiffraction grating, or an interferometer! is generallysignificantly less than 100%. Therefore, for thesame fluorescence yield, significantly less illumina-

The authors are with the University of Miami, Coral Gables,Florida, 33124. J. G. Hirschberg ~HIRSCHBERG@phyvax.ir.miami.edu! is with the Department of Physics. E. Kohen~KOHEN@umiami.ir.miami.edu! is with the Department of Biol-gy.Received 1 June 1998; revised manuscript received 31 August

998.0003-6935y99y010136-03$15.00y0© 1999 Optical Society of America

136 APPLIED OPTICS y Vol. 38, No. 1 y 1 January 1999

tion will fall on the object to be studied if the spectralselection element’s light losses occur in the excitationoptical path before the object rather than in the emis-sion path.

It is advantageous to measure both the excitationand the emission spectra by a simultaneous methodin which the whole spectrum is measured at the sametime; this is because a change in the fluorescenceyield during the measurement does not alter theshape of the spectrum. Since the whole spectrum isbeing investigated at once, the measurement is fast,the so-called Fellgett Advantage.2

In a simultaneous method for excitation spectros-copy, the sample is continuously excited by radiationfrom the whole excitation wavelength band. In thiscase the exciting wavelengths must be coded.

The necessary coding can be accomplished by atwo-beam interferometer3–5 such as the Sagnac. ASagnac-type interferometer, giving a series of linearinterference fringes, provides the Fourier transformof the spectrum as the fringes are swept by a slit.This can be accomplished by rotating the interferom-eter as a linear function of the time. Interferom-eters can also provide the Jacquinot Advantage6; theyhave a greater optical throughput ~etendue! than ei-her the prism or the diffraction grating.

There are, however, complications attached to thenterferometer method that have heretofore served toiscourage its widespread application. Perhaps theost important of these is that interferometers ordi-arily are delicate and necessitate isolation from vi-ration and air currents. A further circumstance ishat two-beam interferometers provide only the Fou-ier transform, making it necessary to perform a cal-ulation to recover the spectrum. The secondifficulty has been practically eliminated by theresent availability of inexpensive and powerful mi-rocomputers, which provide speed that was unimag-nable only a few years ago.

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However, the vibration and the air-current prob-lem remain. To address this, we describe here asolid compact Sagnac-type interferometer, the Pen-taferometer. Such a unit is inherently far more sta-ble than a classical Sagnac interferometer becausethe beam splitter, spacers, and mirrors form an inte-gral piece. In addition, because the interfering op-tical paths are entirely through glass, air currents donot affect the interference fringes. It is also muchsmaller than the usual interferometers, which makesit more convenient to use when both emission andexcitation spectra are being simultaneously mea-sured.

The Pentaferometer is shown in Fig. 1. We pre-pared two identical quadrilateral glass prisms by slic-ing an existing penta prism in half. The two longestfaces of each prism were ground flat and polished.One of the prisms was then provided with a 50%reflecting coating over approximately half of its longface to serve as a beam splitter. The opposite sidesof each prism were aluminized to form mirrors, asshown. Each of the mirrors was provided with aprotective outer coating. The two prisms were fi-nally cemented together to form an integral unit.

The angular spacing of the resulting interferencefringes depends on the relative offset of the twoprisms as they are slid back and forth along their longsides before the cement becomes hard. A positionwas chosen to yield fringes with a convenient sepa-ration angle ~approximately 0.8°!.

The fringes in white and in quasi-monochromaticlight ~through an interference filter! are shown in Fig.. With white light, approximately eight coloredringes on either side of the zero-order black fringere visible; with the filter, many more could be seen.The application for which the interferometer was

esigned is for the study of fluorescence with a tissue-ulture microscope, shown in Fig. 3. Two Pen-aferometers are to be used, one in the excitation pathnd one in the emission path.The fluorescence excitation light is first coded ac-

Fig. 1. Interferometer for which the long sides of the two prismsare provided with a beam-splitting coating over approximately halfof their length and the opposite sides aluminized as shown. Theyare then cemented together with an offset that determines theangular separation of the fringes.

cording to wavelength by being passed through thefirst Pentaferometer, which is rotated, sweeping theinterference fringes over a slit, and then focused bythe microscope objective on the object. The result-ing fluorescent light is sent to a second rotating Pen-taferometer and slit, finally falling on a CCD detector~Fig. 3!.

The two interferometers are rotated at signifi-cantly different speeds, so as to distinguish betweenthe excitation and the emission spectra. The exci-

Fig. 2. ~a! Resulting fringes with white light and ~b! the fringeswith quasi-monochromatic light.

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tation and the emission spectra are then calculatedby the computer. The process of obtaining the spec-tra is explained in References 2–5.

Fig. 3. Proposed application with tissue-culture microscope.Light from a broad source falls on the excitation Pentaferometer at~a!. The Pentaferometer is rotated at constant speed, sweepingthe resulting interference fringes by a slit on which they are fo-cused by a fused-silica lens. A fused-silica clear beam splitterdiverts approximately 6% of the exciting light into the excitationphotodetector, providing a reference spectrum. The rest of theexciting light falls on the specimen, which is on the stage of themicroscope. The resulting fluorescence is passed to the emissionPentaferometer at ~b!. It is also rotated, but at a different speed,so that the two signals, representing the excitation and the emis-sion spectra, can be separated by the computer.

38 APPLIED OPTICS y Vol. 38, No. 1 y 1 January 1999

This research was partially supported by the U.S.National Science Foundation Instrumentation grantBIR9521478 and by the Max Planck Institute for Bio-physical Chemistry, Gottingen, Germany. The au-thors especially thank George C. Alexandrakis,Chairman of the University of Miami Department ofPhysics, and Thomas M. Jovin, Chairman of the De-partment of Molecular Biology of the Max PlanckInstitute for Biophysical Chemistry, Gottingen, Ger-many, for their encouragement and support. Theyalso thank Wolfgang Sauermann and his optical shopstaff at the Max Planck Institute for constructing theprototype.

References1. G. Weber, “Enumeration of components in complex systems by

fluorescence spectrophotometry,” Nature ~London! 190, 27–29~1961!.

2. P. Fellgett, “Spectrometre interferentiel multiplex pour mea-sures infrarouges sur les etoiles,” J. Phys. Radium 19, 237–240~1958!.

3. R. Gemperlein, “A new method for the study of spectral char-acteristics in visual systems,” Doc. Opthamol. Proc. Ser. 33,265–267 ~1980!.

4. A. Steiner, R. Paul, and R. Gemperlein, “Retinal receptor typesin Aglaais urticae and Pieris brassicae ~Lepidoptera!, revealedby the analysis of the electroretinogram obtained with Fourierinterferometric stimulation ~FIS!,” J. Comp. Physiol. A 160,247–258 ~1986!.

5. J. G. Hirschberg, G. Vereb, C. K. Meyer, A. K. Kirsch, E.Kohen, and T. M. Jovin, “Interferometric measurement offluorescence excitation spectra,” Appl. Opt. 37, 1953–1957~1998!.

6. P. Jacquinot, “The luminosity of spectrometers with prisms,gratings or Fabry–Perot etalons,” J. Opt. Soc. Am. 44, 761–765~1954!.