ANALYTICAL
BIOCHEMISTRY
Sensitive
90,
488-500 (1978)
Detection of Small Shifts in Fluorescence Spectra
L. D. BURTNICK Dvpartmrnt
AND I. Z. STEINBERG
of Chrmicnl Physics. Weizmctnn Institute of Sckncr. Rehovot. lsruel Received May 8, 1978
Instrumentation is described for the sensitive detection of small shifts in the emission peaks of fluorescence spectra. The method is based on the modulation with time of the wavelength of the detected light at the emission maximum of the starting spectrum. Modulation ofthe wavelength is achieved by a novel use of a photoelastic light modulator and a specific arrangement of polarizing filters placed at the exit slit of the emission monochromator. A shit? in the initial emission spectrum results in an alternating current in the detected light at double the fundamental frequency of the photoelastic modulator. which is detected with a phasesensitive amplifier. The instrument is sensitive to shifts of 0.2 to 0.5 nm in the wavelength of maximal emission. verified with solutions of Sdimethylaminonaphthalene-1-sulfonyl-oL-glutamic acid and reduced /3-nicotinamide adenine dinucleotide by following fluorescence shifts resulting from alterations in solvent polarity. Also, the fluorescence of quinine (at micromolar levels) is detectable in the presence of a tenfold higher emission intensity of 9-aminoacridine, although the emission maxima of the two compounds are separated by less than 10 nm. The results of these experiments suggest applications of the technique to problems of biological interest which require sensitive detection of minute changes in fluorescence spectra, changes which are due to a shift in the emission spectrum of the chromophore studied or to the occurrence of a new emitting species which has a slightly shifted fluorescence spectrum.
In in vitro biological systems, both intrinsic and extrinsic fluorescent groups have been used extensively to report on the environment in which they are situated and on changes in that environment which occur upon performance of a physiologically relevant function (1). Often, however, information transmitted by such fluorescent probes is difficult to retrieve due to spectral interference from a second fluorophore emitting at a similar wavelength or because shifts in the maximum wavelength of emission are small and not readily detectable in a precise manner. O’Haver and Parks (2) have described a technique for the resolution of overlapping fluorescence emission or excitation spectra. They modulated the wavelength of light transmitted by one monochromator of a commercial spectrofluorometer, scanned the second monochromator, and detected the resultant ac photodetector signal with a lock-in amplifier. 0003-2697/78/0902-0488$02.00/O Copyright All rights
6 1978 by Academic Press, Inc. of reproduction in any form reserved.
488
DETECTION
OF
FLUORESCENCE
SHIFTS
489
To obtain wavelength modulation, O’Haver C? al. replaced the grating drive mechanisms of a commercial spectrofluorometer with limitedrotation torque motors and applied appropriate ramp and ac waveforms to the motors (3). Such a mechanical system is not a satisfactory one in terms of the stress and premature wear that the monochromator gratings must endure, even at relatively low modulation frequencies (15 Hz). The possible introduction of vibrational noise due to mechanical oscillations is also a danger. In this paper a method to obtain wavelength modulation is described which employs a photoelastic light modulator in combination with a special arrangement of polarizing filters (“modulation unit”). The problems of moving mechanical components are eliminated. An additional advantage is gained in that the light modulator power supply frequency is 50 kHz, far removed from any component of electrical line noise. This high frequency should also permit kinetic measurements to be performed down to the submillisecond time range. Experiments are described which test the resolving capabilities of the instrument and which may serve as models for application to biochemical problems. PRINCIPLES
The principles of a technique for the resolution of closely overlapping spectra are outlined in Fig. 1, representing the emission spectra of two fluorescent compounds, A and B. The total emission intensity from a solution containing both A and B is monitored alternately at two wavelengths, A, and AZ, chosen to be symmetric about the emission maximum of A. At X, and h,, the contributions of the emission of A to the total fluorescence, IA, and ZA2, respectively, will be equal. But the contribution of the emission of B at A,, Za,. will not equal that at A,, ZB2,and an alternating component will appear in the total intensity. This ac signal, which is due only to compound B, can be measured using a lock-in amplifier. In practice, we detect the total fluorescence intensity not at two distinct wavelengths, but over two adjacent wavelength intervals which are symmetric about the emission maximum of A. Thus by allowing more light to reach the photodetector, noise levels are suppressed. The differences between Fig. 1 and the analogous figure presented by O’Haver and Parks (2) result from the different methods employed to modulate the wavelength of light emerging from a monochromator. To modulate the wavelength of the light reaching the photodetector, we take advantage of the dispersion in the wavelength of the emergent light across the exit slit of the emission monochromator. A modulation unit, comprising a photoelastic light modulator (Morvue M-FS3, HINDS International,
490
BURTNICK
AND STEINBERG
A
B
1I
t
h
x2
Wavelength FIG. I. Generation of an ac signal upon modulation of the wavelength of light emitted by a sample containing two compounds, A and B, which possess slightly different emission spectra. (For explanation. see text.)
Inc., Portland, Oreg.) and two polarizing filters, P, and PZ, alternately permits light from each half of the exit slit to reach the photodetector, providing modulation over a wavelength interval which depends on the slit width. The modulation unit is depicted in Fig. 2. Light emerging from the emission monochromator first passes through polarizer P,, which consists of a pair of adjacent polarizers (Polaroid HN 32) butted against each other along a straight edge such that the two axes of polarization each form a 45” angle with this edge and, therefore, are mutually orthogonal (Fig. 2B). Polarizer P, is attached to the modulator with the joining edge bisecting the circular entrance window to the modulator and parallel to the long axis of the fused silica plate in the modulator. Polarizer PZ (Polacoat 105 UVWRMR) is placed behind the modulator, with respect to the direction of travel of the light, with its axis of polarization oriented at 45” to the long axis of the modulator plate and. therefore, parallel to the polarization axis of one-half of P1 and perpendicular to that of the second half. The modulation unit is positioned at the emission monochromator exit slit with the joining edge of P, centered over the slit and parallel to its long axis. If one looks through the modulator with the attached polarizers, when the silica plate is not oscillating, one-half of P, appears dark and the second half light. The operation of a photoelastic light modulator, which contains a flat slab of fused silica that can be rendered birefringent by application
DETECTION
OF FLUORESCENCE
SHIFTS
491
FSP
FIG. 2. The modulation unit. (A) A representation of the relative orientations of the monochromator exit slit, S; polarizers, P, and PZ; and the fused silica plate in the photoelastic light modulator, FSP. D indicates the direction of light traveling through the modulation unit. Direction of polarization of the polarizers, . Oscillating slow and fast axes in the fused silica plate, . (B) A view from the position of the photomultiplier tube of the monochromator slit. S (bordered by .). polarizer P, (bordered by - --) and polarizer P2 (bordered by -). The axes of polarization of P, and of the two halves of P, are indicated. as is the centering of P, over the monochromator slit.
of an external stress, is described by Kemp (4). Placing a pair of crossed polarizers, one before and one after the modulator, each at 45” to the axis of induced birefringence, produces a time-dependent intensity modulation device. The intensity of the transmitted light, Ia’, is given as
BURTNICK
492
AND STElNBERG
Z,!(r) = ZaO[sin( $- sin M)r, where Z,” is the intensity of the linearly polarized light incident on the oscillating silica plate, &, is the amplitude of phase retardation in radians (i.e., the magnitude of the phase retardation at maximum stress in each cycle), and w is the fundamental frequency of the modulator plate. Clearly, Eq. [l] also describes the fate of light passing through the half of P, for which the polarization axis is perpendicular to that of P,. By a similar derivation, the fate of light passing through the second half of PI, for which the polarization axis is parallel to that of P,, is described by Z,‘(t) = Zbo[cos ($
sin M)r,
where Zbo and lb’ are, respectively, analogous to I,” and Zb’ in Eq. [l]. The total light intensity at the photodetector, I, is the sum ofl,’ and I,,‘, which upon rearrangement gives Z(f)=(zao:zbo
)+(
zbo~zaO)cos(~Osinwf).
[3]
Z(t) consists of a constant and an oscillating component. The oscillating component is monitored with a phase-sensitive amplifier and is a measure of the intensity difference passing through the two halves of the exit slit of the monochromator bisected by the joining edge of P,. Approximation of the oscillating component, Z,(t), of Z(t) to the first two terms of the Taylor expansion of the cosine term shows that the ac signal at the photodetector will possess a frequency double that of the fundamental frequency of the fused silica plate in the modulator:
Evidently, I, is a measure of the difference between the intensities of light passing through each half of polarizer PI and is related to the difference between the intensities of the two adjacent wavelength intervals of light sampled at the bisected monochromator exit slit. Calculations show that the ac signal at frequency 2w will be maximal for & = m (i.e., h/2 retardation).2 1 Equation [l] is equivalent to the first equation of Ref. (4), except that the factor of onehalf in the innermost parentheses in that article is missing, apparently due to a misprint. z In an actual experiment, the ac signal is maximized empirically by altering the voltage applied to the modulator. A voltage giving I& = r is a convenient, near-maximal starting point.
DETECTION P2
MOD
P,
OF FLUORESCENCE L
Fp S
L
SHIFTS L
F,
M,,
493 L
LAMP
0tu00IO 0ql cl PS
FIG. 3. A block diagram of the instrument. M, and M, are, respectively, the excitation and emission monochromators. F, and F, are filters. L represents a lens and S, the sample holder. P, and P, are polarizers. MOD is the photoelastic light modulator and MPS is its power supply, which also feeds a reference signal, REF, into the lock-in amplifier. PM is the photomultiplier. PS represents a power supply and MET, a digital multimeter.
INSTRUMENTATION
A block diagram of the instrument is presented in Fig. 3. As depicted, we detect the fluorescence emission in the same direction as the exciting beam. Such a geometry is not required, but happens to be convenient for us, as the same optical bench, with minor modifications, is employed in our laboratory for the detection of circular polarization of luminescence (5). This alignment necessitates the use of filters F, and Fz to eliminate excitation light from reaching the photomultiplier as scattered light (5). A high-pressure mercury arc lamp (Osram HBO 2OOW/2) is employed in an Oriel lamp housing and powered by an Oriel supply. A Bausch and Lomb high intensity monochromator (Model 5) with a uv-visible grating is used in the excitation beam. The emission monochromator is a JarrellAsh 25cm double monochromator (Model 82-410) with a resolution of 1.6 nm per l-mm slit width. The photodetector consists of an EM1 type 9558QB photomultiplier tube. The dc signal from the photomultiplier, read directly with a Fluke Model 8000A digital multimeter, provides a measure of the intensity of the emitted light. The ac signal is processed by an Ithaca Dynatrac 291A lock-in amplifier. The reference signal for the phase-sensitive amplifier at double the modulator frequency is obtained from the power supply of the modulator. A section of the instrument comprising the photoelastic light modulator and the polarizers P1 and P2 (Fig. 2) is responsible for wavelength-modulation of the light reaching the photomultiplier. This modulation unit is mounted on an adjustable stand between the emission monochromator and the photomultiplier. The light emerging from the monochromator is partially linearly polarized along the verticle axis of the exit slit, as determined by placing a polarizing filter between the slit and the photomultiplier,
494
BURTNICK
AND
STEINBERG
rotating the polarizer, and monitoring the output dc signal; the joining edge of polarizer P, must therefore be centered along the slit axis to obtain equal transmission from both halves of the slit. The light emerging from the excitation monochromator is partially linearly polarized in the vertical direction. Therefore, vertical polarization may be introduced into the emission of the sample. But the joining edge of polarizer P, is also aligned vertically, so any vertical polarization of the fluorescence of the sample is not going to affect the ac measurements, whether the excitation is head-on (Fig. 3) or at 90” to the direction of the detection of the fluorescence. In cases where for any reason the polarization of the emission is in a direction other than the vertical or horizontal, a depolarizer [as described in Ref. (5)] can be inserted between the sample and the emission monochromator to eliminate artifactual ac signals. PERFORMANCE
1. Dansyl-rx-Glutamic
Acid
Fluorescent probes which bind to macromolecules, whether covalently or noncovalently, provide information concerning the environment of the attachment site. The emission properties of such conjugate systems often are sensitive to conformational changes which occur in the macromolecule during a physiological event, such as the binding of a specific ligand, and provide mechanistic information about that event. Many common fluorescent probes involve the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl or Dns)3 group as the reporter fluorophore. Chen (6) has studied the emission properties of several dansyl-amino acids in solvents of different polarity and in protein-containing solutions. Similarly, we employed dansyl-DL-glutamic acid (Sigma Chemical Co.) dissolved in methanol (dielectric constant, E = 32.6) as a model system for a protein-bound dansyl group. To represent changes in the environment of the dansyl group, n-propanol (E = 20.1) was introduced into the solvent. A 1 IrIM stock solution of dansyl-glutamate was prepared in 50% ethanol-50% 10 mM Tris-HCl, pH 7.5, and diluted 50-fold with either methanol or n-propanol to obtain 10e5 M starting solutions. The two starting solutions were mixed in varying proportions to achieve samples of constant dansyl-glutamate concentration but different solvent polarities. The samples were excited at 365 nm. Filter F, was a No. 5840 Corning glass filter transmitting light between 320 and 390 nm. Filter Fz was a Schott KV cutoff filter transmitting only light above 460 nm. The maximal dc reading, representing total fluorescence intensity, for dansyl-glutamate in methanol 5.dimethylaminonaphthalene-I-sulfonyl: :’ Abbreviations used: dansyl-, constant of a medium relative to that of a vacuum.
E. dielectric
DETECTION 16
OF
FLUORESCENCE
SHIFTS
495
r
E
FIG. 4. The ac signal resulting from solutions containing dansyl-r>L-glutamic acid dissolved in various proportions of methanol and n-propanol. A change of one unit in E represents a wavelength shift of approximately 0.5 nm. Fluctuations in the ac voltage were maximally 20.2 V. as indicated by the size of the data circles. See text for experimental details.
was obtained at 520 nm. and for dansyl-glutamate in rz-propanol at 514 nm. The ac signal was measured with the emission monochromator set at 520 nm and the voltage on the modulator power supply adjusted so as to achieve & = r at 520 nm. The monochromator slits were fully open (12 mm) to allow a maximum of light into the monochromator and to have a relatively large dispersion of wavelengths emerging from it. Fused silica spectrophotometer cells (0.5 cm: Hellma QS) were used in the light beam. The lock-in amplifier sensitivity was set to 0.3 mV and a time constant of 12.5 s was employed. Photomultiplier voltage was set to 940 V. The ac output was zeroed against lop5 M dansyl-glutamate in methanol by moving the joining edge of the split polarizer (PJ across the monochromator slit until the digital readout was 0.00 V, ensuring that the joining edge was centered over the slit. The results of measurements on a series of samples are presented in Fig. 4. The ac signal responded linearly to the decrement in dielectric constant of the solvent. Changes of one unit in E produced significant changes in the ac output under the experimental conditions described. Such changes reflect shifts in the emission maximum of the order of 0.5 nm. 2. Reduced PNicotinamide
Adrnine Dinucleotide
Reduced /3-nicotinamide adenine dinucleotide (NADH) is a cofactor in many enzyme-catalyzed reactions. It is fluorescent in aqueous solution and its emission maximum shifts on binding to enzymes from about 470 to 445 nm (7,8). In 50% ethanol we observed maxima1 fluorescence of NADH (Sigma Chemical Co.) at 442 nm. close to that for protein-bound NADH. The NADH emission maximum could be shifted 6 nm to 448 nm. by
496
BURTNICK 81 ,
I
I
% FIG. 5. The ac signal ethanol concentration from shift was 6 nm. Fluctuations size of the data circles. See
AND STEINBERG I
I
Ethanol
from solutions of NADH obtained upon decreasing the 50 to 40% in a water-ethanol solvent. The total wavelength in the ac voltage were maximally kO.2 V, as indicated by the text for experimental details.
reducing the ethanol concentration to 40%. To follow the shift, we prepared 10 /..LM NADH solutions in 2 mM Tris-HCl, pH 7.5, containing various ethanol concentrations from 40% to 50%. The samples, in 1.O-cm fused silica cells (Hellma QS), were excited at 365 nm through a 5840 Corning glass filter (F,). The filter for the emitted light (F,) consisted of 2 M aqueous NaNO, of a l.O-cm optical path, transmitting light only above 400 nm. The ac signal was zeroed using 10 PM NADH in 50% ethanol with the emission monochromator set to 442 nm and the modulator power supply voltage adjusted to give &, = 7r at 442 nm. The monochromator slits were fully open (12 mm). The lock-in amplifier sensitivity was set to 1 .O mV and the time constant to 12.5 s. The ac response to the decreasing ethanol concentration was linear (Fig. 5). Shifts of 0.2 nm in the emission maximum could be detected. 3. Quinine
and 9-Aminoacridine
Information concerning the environment of a fluorescent probe may be lost due to interference from a second fluorescent species in solution whose emission properties are similar to those of the first. This situation may represent two populations of the same fluorophore, for example a bound form and a form free in solution, or two bound forms in different environments, perhaps resulting from a conformational change experienced by a portion of the molecules. To test the resolving ability of the selective modulation technique, two blue fluorescing compounds, quinine and 9aminoacridine, were mixed in various proportions and studied.
DETECTION
OF FLUORESCENCE
SHIFTS
497
-12-lO- -8 r 0 2 -6:: -4-
CQuininel
(pM)
FIG. 6. The ac signal from solutions containing a fixed concentration of 9-aminoacridine and increasing amounts of quinine. The contribution of 9-aminoacridine to the total fluorescence intensity was 5 times that of the lowest (nonzero) quinine concentration used. Fluctuations in the ac voltage were maximally 20.1 V, as indicated by the size of the data circles. See text for experimental details.
The samples were excited at 365 nm. Filter F, was a Corning 5840 glass filter and F2 was a Schott KV cutoff filter transmitting only light above 390 nm. In 0.1 N HzS04, the emission maximum of quinine was obtained at 447 nm and that of 9-aminoacridine at 438 nm. Fused silica spectrophotometer cells (0.2 cm; Hellma QS) were used. The emission monochromator was set at 438 nm and the modulator power supply voltage adjusted so as to achieve +,, = rr at 438 nm. The monochromator slits were open as in the previous experiments. The lock-in amplifier sensitivity was set to 0.3 mV and a time constant of 4.0 s was chosen. The photomultiplier voltage employed was 820 V. A stock 3.75 x 10P5 M quinine solution was prepared in 0.1 N H,SO,. The concentration of the 9-aminoacridine stock solution was adjusted such that the dc signal at 438 nm was equal to that of the quinine stock solution (within a few percent). Samples were prepared by taking l.O-ml aliquots of the 9-aminoacridine stock, adding increasing amounts of the quinine solution and bringing the total sample volume to 3.0 ml with 0.1 N H,SO,. The samples contained a fixed 9-aminoacridine concentration and increasing levels of quinine. The ac output was set to zero, as described previously, against a solution containing 1.0 ml of the 9-aminoacridine stock and 2.0 ml of 0.1 N H,SO,. Figure 6 shows that as the quinine concentration increased, the ac signal became proportionally more negative, suggesting a red shift in the emission of the sample mixture. Emission from micromolar concentrations of
498
BLJRTNICK
AND
STEINBERG
quinine could be detected successfully in the presence of a fivefold higher emission from the 9-aminoacridine. A variation of the above experiment was also performed in which the concentrations of both quinine and 9-aminoacridine were changed. The principles of the selective modulation technique suggest that the concentration of the substance about whose emission maximum the wavelength is modulated should not be an important factor in measuring the ac signal due to the second fluorescing component. In practice it was found that the ac zero could be set most precisely against a concentration of the fluorophore approximately equal to the maximum concentration to be encountered in the experiment. Portions of a 2.2 x 10e5 M stock solution of quinine were mixed with portions of a stock 9-aminoacridine solution of the same fluorescent intensity at 438 nm such that the final sample volume was constant at 2.0 ml. The ac zero was set using the stock 9-aminoacridine solution with the emission monochromator set at 438 nm. All instrumental settings were as described for the previous experiment in this section, except that the photomultiplier voltage employed was 780 V. Again, the ac response was directly proportional to the quinine concentration. The emission from micromolar levels of quinine could be detected in the presence of more than tenfold higher light intensities due to 9-aminoacridine emission. DISCUSSION
The use of a photoelastic light modulator allows the wavelength of light emerging from a monochromator to be modulated without the necessity of moving mechanical parts, making the technique a practical and reliable one. The sensitivity achievable in the detection of small shifts in emission spectra depends on various experimental factors. Over the modulation interval, the slope of the shifted emission spectrum affects the amplitude of the ac signal detected. A broad, flat maximum will yield a lower signal than a sharp, well-defined peak. Two limiting sources of noise exist in the detected ac signal. One is instrumental in nature, arising from the various electronic components or from lamp instability. The second is shot noise due to the quantum nature of light. At this time, we cannot distinguish which contribution is dominant, but their sum ultimately limits the sensitivity of the instrument. In our experiments, fluctuations in detected ac voltages were roughly + 100 to +-200 mV and depended somewhat on the chromophore studied and the solvent used. Increasing the time constant on the phase-sensitive amplifier may further improve the signal-to-noise ratio. In our investigations of the fluorescence of dansyl-glutamic acid and NADH, shifts in the emission maximum of half a nanometer were detected readily. The fluorescence maxima of dansyl-glutamic acid in
DETECTION
OF FLUORESCENCE
499
SHIFTS
methanol and n-propanol, respectively, closely resemble those of dansylcysteine linked covalently to the monomeric and polymerized forms of the muscle protein, actin (9). The concentration of label we employed, however, was one to two orders of magnitude lower than those reported in the above study on actin. Similarly, the emission maxima of NADA in 40 to 50% ethanol resemble those of protein-bound NADH (7,8). The concentrations of NADH we employed resembled those reported in the studies referenced. The sensitivity achieved in these experiments suggests that precise titrations of spectral shifts will be possible with the instrument described. Preliminary results from our laboratory confirm selective wavelength modulation to be useful in detecting shifts in the emission of dansylcysteine linked to a protein through a disulfide bridge. The use of uv polarizers to study emission from protein tyrosine and tryptophan residues is also being investigated. Our data also demonstrate that the emission from micromolar levels of quinine can be quantitated in the presence of a more than tenfold excess intensity of emission from 9-aminoacridine, which possesses similar emission properties. This was achieved by detecting shifts in the total fluorescence emission maximum in solutions containing both fluorophores. The emission maximum of neither component changed during these experiments; only changes in their concentration ratio led to the overall shift detected. Such experiments may be of interest in studies of ligand-macromolecule interactions where ligand binding causes a conformational change in the macromolecule. If the two populations of macromolecules (ligand-bound and ligand-free) emit at slightly different wavelengths, the appearance of the ligand-bound form could be followed as ligand concentration increases. The application of wavelength modulation to the resolution of spectral components or of spectral shifts is not limited to fluorescence emission. Such a method may be as readily applied to absorption spectrometry. The modulation unit also in this case would be inserted at the exit slit of the monochromator. The wavelength modulation technique may also be applied to the measurement of fluorescence excitation spectra, as was demonstrated by O’Haver and Parks (2) by their experimental approach to the problem. Furthermore, the high frequency of wavelength modulation obtained in our case (100 kHz) suggests the possibility of very fast detection of spectral shifts. Kinetics of fast processes occurring in milliseconds or less which are accompanied by spectral shifts may thus be monitored by this technique in conjunction with stop-flow methods. ACKNOWLEDGMENT L.D.B. wishes to thank the Medical in the form of a MRC Fellowship.
Research
Council
of Canada
for personal
support
500
BURTNICK
AND STEINBERG
REFERENCES 1. Chen, R. D., and Edelhoch, H. (eds.) (1975) Biochemical Fluorescence: Concepts, Vol. 2, Dekker, New York. 2. O’Haver, T. C., and Parks, W. M. ( 1974) And. Chem. 46, 1886- 1894. 3. O’Haver, T. C., Green, G. L., and Keppler, B. R. (1973)Chem. Instrum. 4, 197-201. 4. Kemp, J. C. (1969) J. Opt. Sot. Amer. 59, 950-954. 5. Steinberg, I. Z., and Gafni, A. (1972) Rev. Sci. Instrum. 43, 409-413. 6. Chen, R. F. (1967) Arch. Biochem. Biophys. 120, 609-620. 7. Yonetani, T., and Theorell, H. (1962) Arch. Biochem. Biophys. 99, 433-446. 8. Scott, T. G., Spencer, R. D., Leonard, N. J., and Weber, G. (1970)3. Amer. Chem. Sot. 92, 687-695. 9. Cheung, H. C., Cooke, R., and Smith, L. (1971)Arch. Biochem. Biophys. 142,333-339.
BIOCHEMISTRY
Sensitive
90,
488-500 (1978)
Detection of Small Shifts in Fluorescence Spectra
L. D. BURTNICK Dvpartmrnt
AND I. Z. STEINBERG
of Chrmicnl Physics. Weizmctnn Institute of Sckncr. Rehovot. lsruel Received May 8, 1978
Instrumentation is described for the sensitive detection of small shifts in the emission peaks of fluorescence spectra. The method is based on the modulation with time of the wavelength of the detected light at the emission maximum of the starting spectrum. Modulation ofthe wavelength is achieved by a novel use of a photoelastic light modulator and a specific arrangement of polarizing filters placed at the exit slit of the emission monochromator. A shit? in the initial emission spectrum results in an alternating current in the detected light at double the fundamental frequency of the photoelastic modulator. which is detected with a phasesensitive amplifier. The instrument is sensitive to shifts of 0.2 to 0.5 nm in the wavelength of maximal emission. verified with solutions of Sdimethylaminonaphthalene-1-sulfonyl-oL-glutamic acid and reduced /3-nicotinamide adenine dinucleotide by following fluorescence shifts resulting from alterations in solvent polarity. Also, the fluorescence of quinine (at micromolar levels) is detectable in the presence of a tenfold higher emission intensity of 9-aminoacridine, although the emission maxima of the two compounds are separated by less than 10 nm. The results of these experiments suggest applications of the technique to problems of biological interest which require sensitive detection of minute changes in fluorescence spectra, changes which are due to a shift in the emission spectrum of the chromophore studied or to the occurrence of a new emitting species which has a slightly shifted fluorescence spectrum.
In in vitro biological systems, both intrinsic and extrinsic fluorescent groups have been used extensively to report on the environment in which they are situated and on changes in that environment which occur upon performance of a physiologically relevant function (1). Often, however, information transmitted by such fluorescent probes is difficult to retrieve due to spectral interference from a second fluorophore emitting at a similar wavelength or because shifts in the maximum wavelength of emission are small and not readily detectable in a precise manner. O’Haver and Parks (2) have described a technique for the resolution of overlapping fluorescence emission or excitation spectra. They modulated the wavelength of light transmitted by one monochromator of a commercial spectrofluorometer, scanned the second monochromator, and detected the resultant ac photodetector signal with a lock-in amplifier. 0003-2697/78/0902-0488$02.00/O Copyright All rights
6 1978 by Academic Press, Inc. of reproduction in any form reserved.
488
DETECTION
OF
FLUORESCENCE
SHIFTS
489
To obtain wavelength modulation, O’Haver C? al. replaced the grating drive mechanisms of a commercial spectrofluorometer with limitedrotation torque motors and applied appropriate ramp and ac waveforms to the motors (3). Such a mechanical system is not a satisfactory one in terms of the stress and premature wear that the monochromator gratings must endure, even at relatively low modulation frequencies (15 Hz). The possible introduction of vibrational noise due to mechanical oscillations is also a danger. In this paper a method to obtain wavelength modulation is described which employs a photoelastic light modulator in combination with a special arrangement of polarizing filters (“modulation unit”). The problems of moving mechanical components are eliminated. An additional advantage is gained in that the light modulator power supply frequency is 50 kHz, far removed from any component of electrical line noise. This high frequency should also permit kinetic measurements to be performed down to the submillisecond time range. Experiments are described which test the resolving capabilities of the instrument and which may serve as models for application to biochemical problems. PRINCIPLES
The principles of a technique for the resolution of closely overlapping spectra are outlined in Fig. 1, representing the emission spectra of two fluorescent compounds, A and B. The total emission intensity from a solution containing both A and B is monitored alternately at two wavelengths, A, and AZ, chosen to be symmetric about the emission maximum of A. At X, and h,, the contributions of the emission of A to the total fluorescence, IA, and ZA2, respectively, will be equal. But the contribution of the emission of B at A,, Za,. will not equal that at A,, ZB2,and an alternating component will appear in the total intensity. This ac signal, which is due only to compound B, can be measured using a lock-in amplifier. In practice, we detect the total fluorescence intensity not at two distinct wavelengths, but over two adjacent wavelength intervals which are symmetric about the emission maximum of A. Thus by allowing more light to reach the photodetector, noise levels are suppressed. The differences between Fig. 1 and the analogous figure presented by O’Haver and Parks (2) result from the different methods employed to modulate the wavelength of light emerging from a monochromator. To modulate the wavelength of the light reaching the photodetector, we take advantage of the dispersion in the wavelength of the emergent light across the exit slit of the emission monochromator. A modulation unit, comprising a photoelastic light modulator (Morvue M-FS3, HINDS International,
490
BURTNICK
AND STEINBERG
A
B
1I
t
h
x2
Wavelength FIG. I. Generation of an ac signal upon modulation of the wavelength of light emitted by a sample containing two compounds, A and B, which possess slightly different emission spectra. (For explanation. see text.)
Inc., Portland, Oreg.) and two polarizing filters, P, and PZ, alternately permits light from each half of the exit slit to reach the photodetector, providing modulation over a wavelength interval which depends on the slit width. The modulation unit is depicted in Fig. 2. Light emerging from the emission monochromator first passes through polarizer P,, which consists of a pair of adjacent polarizers (Polaroid HN 32) butted against each other along a straight edge such that the two axes of polarization each form a 45” angle with this edge and, therefore, are mutually orthogonal (Fig. 2B). Polarizer P, is attached to the modulator with the joining edge bisecting the circular entrance window to the modulator and parallel to the long axis of the fused silica plate in the modulator. Polarizer PZ (Polacoat 105 UVWRMR) is placed behind the modulator, with respect to the direction of travel of the light, with its axis of polarization oriented at 45” to the long axis of the modulator plate and. therefore, parallel to the polarization axis of one-half of P1 and perpendicular to that of the second half. The modulation unit is positioned at the emission monochromator exit slit with the joining edge of P, centered over the slit and parallel to its long axis. If one looks through the modulator with the attached polarizers, when the silica plate is not oscillating, one-half of P, appears dark and the second half light. The operation of a photoelastic light modulator, which contains a flat slab of fused silica that can be rendered birefringent by application
DETECTION
OF FLUORESCENCE
SHIFTS
491
FSP
FIG. 2. The modulation unit. (A) A representation of the relative orientations of the monochromator exit slit, S; polarizers, P, and PZ; and the fused silica plate in the photoelastic light modulator, FSP. D indicates the direction of light traveling through the modulation unit. Direction of polarization of the polarizers, . Oscillating slow and fast axes in the fused silica plate, . (B) A view from the position of the photomultiplier tube of the monochromator slit. S (bordered by .). polarizer P, (bordered by - --) and polarizer P2 (bordered by -). The axes of polarization of P, and of the two halves of P, are indicated. as is the centering of P, over the monochromator slit.
of an external stress, is described by Kemp (4). Placing a pair of crossed polarizers, one before and one after the modulator, each at 45” to the axis of induced birefringence, produces a time-dependent intensity modulation device. The intensity of the transmitted light, Ia’, is given as
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492
AND STElNBERG
Z,!(r) = ZaO[sin( $- sin M)r, where Z,” is the intensity of the linearly polarized light incident on the oscillating silica plate, &, is the amplitude of phase retardation in radians (i.e., the magnitude of the phase retardation at maximum stress in each cycle), and w is the fundamental frequency of the modulator plate. Clearly, Eq. [l] also describes the fate of light passing through the half of P, for which the polarization axis is perpendicular to that of P,. By a similar derivation, the fate of light passing through the second half of PI, for which the polarization axis is parallel to that of P,, is described by Z,‘(t) = Zbo[cos ($
sin M)r,
where Zbo and lb’ are, respectively, analogous to I,” and Zb’ in Eq. [l]. The total light intensity at the photodetector, I, is the sum ofl,’ and I,,‘, which upon rearrangement gives Z(f)=(zao:zbo
)+(
zbo~zaO)cos(~Osinwf).
[3]
Z(t) consists of a constant and an oscillating component. The oscillating component is monitored with a phase-sensitive amplifier and is a measure of the intensity difference passing through the two halves of the exit slit of the monochromator bisected by the joining edge of P,. Approximation of the oscillating component, Z,(t), of Z(t) to the first two terms of the Taylor expansion of the cosine term shows that the ac signal at the photodetector will possess a frequency double that of the fundamental frequency of the fused silica plate in the modulator:
Evidently, I, is a measure of the difference between the intensities of light passing through each half of polarizer PI and is related to the difference between the intensities of the two adjacent wavelength intervals of light sampled at the bisected monochromator exit slit. Calculations show that the ac signal at frequency 2w will be maximal for & = m (i.e., h/2 retardation).2 1 Equation [l] is equivalent to the first equation of Ref. (4), except that the factor of onehalf in the innermost parentheses in that article is missing, apparently due to a misprint. z In an actual experiment, the ac signal is maximized empirically by altering the voltage applied to the modulator. A voltage giving I& = r is a convenient, near-maximal starting point.
DETECTION P2
MOD
P,
OF FLUORESCENCE L
Fp S
L
SHIFTS L
F,
M,,
493 L
LAMP
0tu00IO 0ql cl PS
FIG. 3. A block diagram of the instrument. M, and M, are, respectively, the excitation and emission monochromators. F, and F, are filters. L represents a lens and S, the sample holder. P, and P, are polarizers. MOD is the photoelastic light modulator and MPS is its power supply, which also feeds a reference signal, REF, into the lock-in amplifier. PM is the photomultiplier. PS represents a power supply and MET, a digital multimeter.
INSTRUMENTATION
A block diagram of the instrument is presented in Fig. 3. As depicted, we detect the fluorescence emission in the same direction as the exciting beam. Such a geometry is not required, but happens to be convenient for us, as the same optical bench, with minor modifications, is employed in our laboratory for the detection of circular polarization of luminescence (5). This alignment necessitates the use of filters F, and Fz to eliminate excitation light from reaching the photomultiplier as scattered light (5). A high-pressure mercury arc lamp (Osram HBO 2OOW/2) is employed in an Oriel lamp housing and powered by an Oriel supply. A Bausch and Lomb high intensity monochromator (Model 5) with a uv-visible grating is used in the excitation beam. The emission monochromator is a JarrellAsh 25cm double monochromator (Model 82-410) with a resolution of 1.6 nm per l-mm slit width. The photodetector consists of an EM1 type 9558QB photomultiplier tube. The dc signal from the photomultiplier, read directly with a Fluke Model 8000A digital multimeter, provides a measure of the intensity of the emitted light. The ac signal is processed by an Ithaca Dynatrac 291A lock-in amplifier. The reference signal for the phase-sensitive amplifier at double the modulator frequency is obtained from the power supply of the modulator. A section of the instrument comprising the photoelastic light modulator and the polarizers P1 and P2 (Fig. 2) is responsible for wavelength-modulation of the light reaching the photomultiplier. This modulation unit is mounted on an adjustable stand between the emission monochromator and the photomultiplier. The light emerging from the monochromator is partially linearly polarized along the verticle axis of the exit slit, as determined by placing a polarizing filter between the slit and the photomultiplier,
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rotating the polarizer, and monitoring the output dc signal; the joining edge of polarizer P, must therefore be centered along the slit axis to obtain equal transmission from both halves of the slit. The light emerging from the excitation monochromator is partially linearly polarized in the vertical direction. Therefore, vertical polarization may be introduced into the emission of the sample. But the joining edge of polarizer P, is also aligned vertically, so any vertical polarization of the fluorescence of the sample is not going to affect the ac measurements, whether the excitation is head-on (Fig. 3) or at 90” to the direction of the detection of the fluorescence. In cases where for any reason the polarization of the emission is in a direction other than the vertical or horizontal, a depolarizer [as described in Ref. (5)] can be inserted between the sample and the emission monochromator to eliminate artifactual ac signals. PERFORMANCE
1. Dansyl-rx-Glutamic
Acid
Fluorescent probes which bind to macromolecules, whether covalently or noncovalently, provide information concerning the environment of the attachment site. The emission properties of such conjugate systems often are sensitive to conformational changes which occur in the macromolecule during a physiological event, such as the binding of a specific ligand, and provide mechanistic information about that event. Many common fluorescent probes involve the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl or Dns)3 group as the reporter fluorophore. Chen (6) has studied the emission properties of several dansyl-amino acids in solvents of different polarity and in protein-containing solutions. Similarly, we employed dansyl-DL-glutamic acid (Sigma Chemical Co.) dissolved in methanol (dielectric constant, E = 32.6) as a model system for a protein-bound dansyl group. To represent changes in the environment of the dansyl group, n-propanol (E = 20.1) was introduced into the solvent. A 1 IrIM stock solution of dansyl-glutamate was prepared in 50% ethanol-50% 10 mM Tris-HCl, pH 7.5, and diluted 50-fold with either methanol or n-propanol to obtain 10e5 M starting solutions. The two starting solutions were mixed in varying proportions to achieve samples of constant dansyl-glutamate concentration but different solvent polarities. The samples were excited at 365 nm. Filter F, was a No. 5840 Corning glass filter transmitting light between 320 and 390 nm. Filter Fz was a Schott KV cutoff filter transmitting only light above 460 nm. The maximal dc reading, representing total fluorescence intensity, for dansyl-glutamate in methanol 5.dimethylaminonaphthalene-I-sulfonyl: :’ Abbreviations used: dansyl-, constant of a medium relative to that of a vacuum.
E. dielectric
DETECTION 16
OF
FLUORESCENCE
SHIFTS
495
r
E
FIG. 4. The ac signal resulting from solutions containing dansyl-r>L-glutamic acid dissolved in various proportions of methanol and n-propanol. A change of one unit in E represents a wavelength shift of approximately 0.5 nm. Fluctuations in the ac voltage were maximally 20.2 V. as indicated by the size of the data circles. See text for experimental details.
was obtained at 520 nm. and for dansyl-glutamate in rz-propanol at 514 nm. The ac signal was measured with the emission monochromator set at 520 nm and the voltage on the modulator power supply adjusted so as to achieve & = r at 520 nm. The monochromator slits were fully open (12 mm) to allow a maximum of light into the monochromator and to have a relatively large dispersion of wavelengths emerging from it. Fused silica spectrophotometer cells (0.5 cm: Hellma QS) were used in the light beam. The lock-in amplifier sensitivity was set to 0.3 mV and a time constant of 12.5 s was employed. Photomultiplier voltage was set to 940 V. The ac output was zeroed against lop5 M dansyl-glutamate in methanol by moving the joining edge of the split polarizer (PJ across the monochromator slit until the digital readout was 0.00 V, ensuring that the joining edge was centered over the slit. The results of measurements on a series of samples are presented in Fig. 4. The ac signal responded linearly to the decrement in dielectric constant of the solvent. Changes of one unit in E produced significant changes in the ac output under the experimental conditions described. Such changes reflect shifts in the emission maximum of the order of 0.5 nm. 2. Reduced PNicotinamide
Adrnine Dinucleotide
Reduced /3-nicotinamide adenine dinucleotide (NADH) is a cofactor in many enzyme-catalyzed reactions. It is fluorescent in aqueous solution and its emission maximum shifts on binding to enzymes from about 470 to 445 nm (7,8). In 50% ethanol we observed maxima1 fluorescence of NADH (Sigma Chemical Co.) at 442 nm. close to that for protein-bound NADH. The NADH emission maximum could be shifted 6 nm to 448 nm. by
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I
I
% FIG. 5. The ac signal ethanol concentration from shift was 6 nm. Fluctuations size of the data circles. See
AND STEINBERG I
I
Ethanol
from solutions of NADH obtained upon decreasing the 50 to 40% in a water-ethanol solvent. The total wavelength in the ac voltage were maximally kO.2 V, as indicated by the text for experimental details.
reducing the ethanol concentration to 40%. To follow the shift, we prepared 10 /..LM NADH solutions in 2 mM Tris-HCl, pH 7.5, containing various ethanol concentrations from 40% to 50%. The samples, in 1.O-cm fused silica cells (Hellma QS), were excited at 365 nm through a 5840 Corning glass filter (F,). The filter for the emitted light (F,) consisted of 2 M aqueous NaNO, of a l.O-cm optical path, transmitting light only above 400 nm. The ac signal was zeroed using 10 PM NADH in 50% ethanol with the emission monochromator set to 442 nm and the modulator power supply voltage adjusted to give &, = 7r at 442 nm. The monochromator slits were fully open (12 mm). The lock-in amplifier sensitivity was set to 1 .O mV and the time constant to 12.5 s. The ac response to the decreasing ethanol concentration was linear (Fig. 5). Shifts of 0.2 nm in the emission maximum could be detected. 3. Quinine
and 9-Aminoacridine
Information concerning the environment of a fluorescent probe may be lost due to interference from a second fluorescent species in solution whose emission properties are similar to those of the first. This situation may represent two populations of the same fluorophore, for example a bound form and a form free in solution, or two bound forms in different environments, perhaps resulting from a conformational change experienced by a portion of the molecules. To test the resolving ability of the selective modulation technique, two blue fluorescing compounds, quinine and 9aminoacridine, were mixed in various proportions and studied.
DETECTION
OF FLUORESCENCE
SHIFTS
497
-12-lO- -8 r 0 2 -6:: -4-
CQuininel
(pM)
FIG. 6. The ac signal from solutions containing a fixed concentration of 9-aminoacridine and increasing amounts of quinine. The contribution of 9-aminoacridine to the total fluorescence intensity was 5 times that of the lowest (nonzero) quinine concentration used. Fluctuations in the ac voltage were maximally 20.1 V, as indicated by the size of the data circles. See text for experimental details.
The samples were excited at 365 nm. Filter F, was a Corning 5840 glass filter and F2 was a Schott KV cutoff filter transmitting only light above 390 nm. In 0.1 N HzS04, the emission maximum of quinine was obtained at 447 nm and that of 9-aminoacridine at 438 nm. Fused silica spectrophotometer cells (0.2 cm; Hellma QS) were used. The emission monochromator was set at 438 nm and the modulator power supply voltage adjusted so as to achieve +,, = rr at 438 nm. The monochromator slits were open as in the previous experiments. The lock-in amplifier sensitivity was set to 0.3 mV and a time constant of 4.0 s was chosen. The photomultiplier voltage employed was 820 V. A stock 3.75 x 10P5 M quinine solution was prepared in 0.1 N H,SO,. The concentration of the 9-aminoacridine stock solution was adjusted such that the dc signal at 438 nm was equal to that of the quinine stock solution (within a few percent). Samples were prepared by taking l.O-ml aliquots of the 9-aminoacridine stock, adding increasing amounts of the quinine solution and bringing the total sample volume to 3.0 ml with 0.1 N H,SO,. The samples contained a fixed 9-aminoacridine concentration and increasing levels of quinine. The ac output was set to zero, as described previously, against a solution containing 1.0 ml of the 9-aminoacridine stock and 2.0 ml of 0.1 N H,SO,. Figure 6 shows that as the quinine concentration increased, the ac signal became proportionally more negative, suggesting a red shift in the emission of the sample mixture. Emission from micromolar concentrations of
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quinine could be detected successfully in the presence of a fivefold higher emission from the 9-aminoacridine. A variation of the above experiment was also performed in which the concentrations of both quinine and 9-aminoacridine were changed. The principles of the selective modulation technique suggest that the concentration of the substance about whose emission maximum the wavelength is modulated should not be an important factor in measuring the ac signal due to the second fluorescing component. In practice it was found that the ac zero could be set most precisely against a concentration of the fluorophore approximately equal to the maximum concentration to be encountered in the experiment. Portions of a 2.2 x 10e5 M stock solution of quinine were mixed with portions of a stock 9-aminoacridine solution of the same fluorescent intensity at 438 nm such that the final sample volume was constant at 2.0 ml. The ac zero was set using the stock 9-aminoacridine solution with the emission monochromator set at 438 nm. All instrumental settings were as described for the previous experiment in this section, except that the photomultiplier voltage employed was 780 V. Again, the ac response was directly proportional to the quinine concentration. The emission from micromolar levels of quinine could be detected in the presence of more than tenfold higher light intensities due to 9-aminoacridine emission. DISCUSSION
The use of a photoelastic light modulator allows the wavelength of light emerging from a monochromator to be modulated without the necessity of moving mechanical parts, making the technique a practical and reliable one. The sensitivity achievable in the detection of small shifts in emission spectra depends on various experimental factors. Over the modulation interval, the slope of the shifted emission spectrum affects the amplitude of the ac signal detected. A broad, flat maximum will yield a lower signal than a sharp, well-defined peak. Two limiting sources of noise exist in the detected ac signal. One is instrumental in nature, arising from the various electronic components or from lamp instability. The second is shot noise due to the quantum nature of light. At this time, we cannot distinguish which contribution is dominant, but their sum ultimately limits the sensitivity of the instrument. In our experiments, fluctuations in detected ac voltages were roughly + 100 to +-200 mV and depended somewhat on the chromophore studied and the solvent used. Increasing the time constant on the phase-sensitive amplifier may further improve the signal-to-noise ratio. In our investigations of the fluorescence of dansyl-glutamic acid and NADH, shifts in the emission maximum of half a nanometer were detected readily. The fluorescence maxima of dansyl-glutamic acid in
DETECTION
OF FLUORESCENCE
499
SHIFTS
methanol and n-propanol, respectively, closely resemble those of dansylcysteine linked covalently to the monomeric and polymerized forms of the muscle protein, actin (9). The concentration of label we employed, however, was one to two orders of magnitude lower than those reported in the above study on actin. Similarly, the emission maxima of NADA in 40 to 50% ethanol resemble those of protein-bound NADH (7,8). The concentrations of NADH we employed resembled those reported in the studies referenced. The sensitivity achieved in these experiments suggests that precise titrations of spectral shifts will be possible with the instrument described. Preliminary results from our laboratory confirm selective wavelength modulation to be useful in detecting shifts in the emission of dansylcysteine linked to a protein through a disulfide bridge. The use of uv polarizers to study emission from protein tyrosine and tryptophan residues is also being investigated. Our data also demonstrate that the emission from micromolar levels of quinine can be quantitated in the presence of a more than tenfold excess intensity of emission from 9-aminoacridine, which possesses similar emission properties. This was achieved by detecting shifts in the total fluorescence emission maximum in solutions containing both fluorophores. The emission maximum of neither component changed during these experiments; only changes in their concentration ratio led to the overall shift detected. Such experiments may be of interest in studies of ligand-macromolecule interactions where ligand binding causes a conformational change in the macromolecule. If the two populations of macromolecules (ligand-bound and ligand-free) emit at slightly different wavelengths, the appearance of the ligand-bound form could be followed as ligand concentration increases. The application of wavelength modulation to the resolution of spectral components or of spectral shifts is not limited to fluorescence emission. Such a method may be as readily applied to absorption spectrometry. The modulation unit also in this case would be inserted at the exit slit of the monochromator. The wavelength modulation technique may also be applied to the measurement of fluorescence excitation spectra, as was demonstrated by O’Haver and Parks (2) by their experimental approach to the problem. Furthermore, the high frequency of wavelength modulation obtained in our case (100 kHz) suggests the possibility of very fast detection of spectral shifts. Kinetics of fast processes occurring in milliseconds or less which are accompanied by spectral shifts may thus be monitored by this technique in conjunction with stop-flow methods. ACKNOWLEDGMENT L.D.B. wishes to thank the Medical in the form of a MRC Fellowship.
Research
Council
of Canada
for personal
support
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BURTNICK
AND STEINBERG
REFERENCES 1. Chen, R. D., and Edelhoch, H. (eds.) (1975) Biochemical Fluorescence: Concepts, Vol. 2, Dekker, New York. 2. O’Haver, T. C., and Parks, W. M. ( 1974) And. Chem. 46, 1886- 1894. 3. O’Haver, T. C., Green, G. L., and Keppler, B. R. (1973)Chem. Instrum. 4, 197-201. 4. Kemp, J. C. (1969) J. Opt. Sot. Amer. 59, 950-954. 5. Steinberg, I. Z., and Gafni, A. (1972) Rev. Sci. Instrum. 43, 409-413. 6. Chen, R. F. (1967) Arch. Biochem. Biophys. 120, 609-620. 7. Yonetani, T., and Theorell, H. (1962) Arch. Biochem. Biophys. 99, 433-446. 8. Scott, T. G., Spencer, R. D., Leonard, N. J., and Weber, G. (1970)3. Amer. Chem. Sot. 92, 687-695. 9. Cheung, H. C., Cooke, R., and Smith, L. (1971)Arch. Biochem. Biophys. 142,333-339.
