Absorbance-Corrected Synchronous Fluorescence with a Fiber-Optic-Based Fluorometer

Absorbance-Corrected Synchronous Fluorescence with a Fiber-Optic-Based Fluorometer

MARK A. VICTOR and STANLEY R. CROUCH* Department of Chemistry, Michigan State University, East Lansing, Michigan 48824

Synchronous fluorescence spectroscopy is frequently used to resolve multiple components in mixtures without separation. However, uncompen- sated inner-filter effects can lead to unexpected and/or large quantitative errors. An instrument and a method that correct for primary and secondary inner-filter effects in synchronous fluorescence spectroscopy have been designed and characterized. A bifurcated fiber-optic-based diode array fluorometer that simultaneously measures front-surface fluorescence and absorbance was constructed. A transfer function that math- ematically describes the coupling efficiency between the excitation and emission legs of the fiber optics is defined and characterized. Once characterized, such a fluorometer is capable of calculating what the fluorescence measurement would be in the absence of inner-filter effects. Several experimental procedures for determining the instrument's transfer function are compared and discussed. Applications of the various procedures to experimental fluorescence inner-filter corrections to problematic samples are demonstrated. Index Headings: Fluorescence; Synchronous fluorescence; Absorption- corrected fluorescence.

INTRODUCTION

This paper describes an instrument that measures synchronous fluorescence while simultaneously correcting for primary and secondary inner-filter effects. The ability of synchronous fluorescence spectroscopy to resolve multiple fluorophores simultaneously has been used success- fully for quantifying and fingerprinting mixtures of po- lyaromatic hydrocarbons (PAHs) when prior physical separation has not been carried out ~-6 or when separations are incomplete. 7-9 However, with increasing complexity of the sample matrix, the advantages of synchronous fluorescence are quickly overcome by quenching and inner- filter effects. Because of the complexity of the matrices to which synchronous fluorescence techniques are applied, reliable quantitative synchronous fluorescence data have been elusive.

Synchronous fluorescence is primarily used for mixture analysis and is particularly prone to inner-filter effects. Other investigators have observed dramatic signal reductions for mixtures of PAHs. ~0-12 Although ambiguities between quenching and inner-filter effects in synchronous fluorescence have been discussed in the literature, 1o-16 dilution studies offered evidence that signal attenuation was attributable to inner-filter effects. ~° It has also been ex- plained empirically how fluorophores whose synchronous fluorescence signals are observed at shorter wavelengths are more likely to be suppressed. 13 Without methodology to correct for non-quenching effects, 14.15 the source of signal reductions from their expected values can be ambig-

Received 30 January 1995; accepted 20 March 1995. * Author to whom correspondence should be sent.

uous. 16 Inner-filter effects can be compensated for, by sample dilution or the standard addition method. ~7 This approach may result in lowering the concentrations of weak fluorophores below the limits of detection. In the case of strong absorbance by the analyte itself, the standard addition method will not produce a linear calibra- tion curve.

The quantitative and fingerprinting capability of synchronous fluorescence would be markedly enhanced if matrix-dependent inner-filter anomalies could be recognized. An appropriate mathematical model that compensates for inner-filter effects in the synchronous fluorometer's response can then be applied. A technique that can deal with absorbances as high as 3.0 is as analytically useful as a 1000-fold dilution. Dynamic correction and a fiber-optic interface are preferable for monitoring dynamic systems or flowing streams where removing ali- quots for dilution is not always feasible.

Previous investigators have described mathematical corrections and/or instrumentation to explain and com- pensate for absorption effects in fluorometry. ~8-z2 Most of this work deals with conventional instrumentation based on standard 1-cm-square cuvettes and requires spatial displacement of either the cell or the optical path in time. This requirement precludes such a method from mea- suring small volumes, flowing streams, or kinetic or tran- sitory effects.

A mathematical model and an instrument useful for absorption correction with fiber-optic fluorometers were developed by Ratzlaff et al. 23 The model incorporates an empirical transfer function describing the coupling efficiency between the excitation and emission elements of a front-surface fiber-optic-based fluorometer. Once ade- quately characterized, the transfer function completes a mathematical description in the front-surface fluorometer capable of deconvolving the effect of primary and secondary inner-filter effects in real time. 23

To establish the stability of the transfer function across the broad wavelength ranges common in synchronous fluorescence spectroscopy, a rapid means of determining the function at all excitation/emission wavelengths is es- sential. This paper deals with a rapid method for char- acterization of the transfer function as a function of exci tat ion/emission wavelength. The instrument, once completely calibrated, can then be used for absorbance- corrected synchronous fluorescence measurements.

THEORETICAL

The transfer function of the fiber-optic-based front- surface fluorometer 23 is illustrated for a multifiber instrument in Fig. 1. At zero distance into the cell, the radiant

Volume 49, Number 7, 1995 0003.7028/95/4907.104152.00/0 APPLIED SPECTROSCOPY 1041 © 1995 Society for Applied Spectroscopy

Pathlength for absorption end Front surface emission

FRONT ( ) ] ~ ×

b

I

x = 0

() i i ,

I

b

REAR

-4J I m

oo

G)

] w

0 b distance

J

excitation fiber ineff icient

" excitat ion

emission fiber

excitation fiber

" ineff icient col lection

emission fiber of emission

I I I

minimum penetrat ion depth

FIG. 1. Representation of fluorescent intensity observed vs. distance from an infinitely thin slice for a front-surface, bifurcated, fiber-optic fluorometer. This figure also demonstrates the spatial relationship between the minimum penetration depth and the fibers' transfer function.

power directly in front of the excitation beam is greatest. However, directly in front of any excitation fiber, the finite thickness of the fiber's cladding places the fluoresc- ing portion of the solution out of view of the emission fibers. An empirical mathematical description of the effect predicts an exponential rise in fluorescence intensity at-

tributable to increased coupling efficiency, offset by an exponential decay with increasing distance into the ce11.~2,23,24 A suitable mathematical description of the above process is

T ( x ) = G ( e l~x _ e - r ~ ) (1)

1042 Volume 49, Number 7, 1995

bifurcated excitation fiber optic

front surface emission

flow cell in out

I i I

micrometer drive

immobilized phosphor

FIG. 2. Modified flow cell to allow the measurement of the fluorescence from an infinitely thin slice as a function of distance.

where T(x) is the transfer function, which is a combi- nation of the efficiencies of excitation generation and emission observation as a function of distance. The transfer function is dictated by the fiber geometry and is a measurable but nonadjustable instrumentation parameter. A normalization constant, G, compensates for source and detector wavelength dependence and fluorescent quantum efficiency; H describes the signal's initial exponential rise; and K describes the exponential decay. Although this model is empirical, experimental evidence by Smith et al/4 and modeling studies by Plaza et al. 12 provide support for this description.

A correction factor (CF) was calculated from Eq. 2 (see Ref. 23 for details). The CF value is a multiplier relating the observed to the corrected fluorescence in terms of the solution's absorptivities [(eC)ex and (eC)om] and the fiber's transfer function descriptors (H and K).

(1 - e - ' b ) - 1(1 - e -Kb)

CF = ( 2 )

1 ( 1 - - e - M b ) - - l ( 1 - - e - N b )

Values of H and K can also be obtained from Eq. 1 by nonlinear regression of the fluorescence observed from simulated infinitely thin slices at varying depths into the solution.

EXPERIMENTAL

A front-surface, bifurcated, fiber-optic-based fluorometer similar to the previous instrument 23 was constructed with several modifications. The flow cell was altered to allow the micrometer-controlled incremental advance- ment of a brass rod from the rear of the cell (Fig. 2). In order to simulate the measurement of the fluorescence from successive infinitely thin layers, the end of a brass rod was coated with an immobilized surface phosphor. The solid phosphor was constructed by grinding and mix- ing blue, yellow, orange, and red fluorescent chalks (We- ber CosteUo Inc., Troy, MO). The experimentally determined emission spectrum of the individual luminophores and the broad-band output are shown in Fig. 3. In a procedure to ensure sampling only from the front surface of the phosphor, the mixture was finely ground with de-

c

4000 -

3500,

30001

2500-

2000 ~

1500

1000-

500-

mixture emission le

' '"i .... i',,,i,',,i .... i,,,,i ,,'i,',,i, ,,,i .... i',

415 440 465 490 515 540 565 590 615 640

Wavelength (nm)

Fro. 3. Individual emission curves for the luminophores comprising the solid phosphor plotted underneath the net emission (solid line).

colorizing charcoal and then mixed with an epoxy resin. The presence of large amounts of charcoal in the resin/ phosphor mixture was necessary to render the transparent epoxy resin matrix opaque and prevent depth profiling, light piping, and scattering.

The immobilized phosphor mixture was excited at wavelengths from 280 to 400 nm with emission occurring in the region from 400 to 600 nm. The solid phosphor was advanced toward the front of the cell while direct intensity vs. distance data were collected. This pattern was repeated at 2.0-nm intervals for all possible excitation/emission wavelength combinations. Nonlinear regression of the weighted intensity vs. distance data provided parameter estimates for H and K in Eq. 1.

The soluble fluorophores dimethylaminonaphthalene- sulphonylchloride (dansyl chloride) 98%, perylene 99 ÷%, and 1,1,4,4-tetraphenylbutadiene 99% (Aldrich Chemi- cals of Milwaukee Wisconsin), as well as pyrene 99+%, fluorene 98%, and phenanthrene 98% (Sigma Chemical Supply), anthracene 99.9 + % (Eastman Kodak Co., Roch- ester, NY), and spectrophotometric-grade cyclohexane (Mallincrodt, Paris, KY), were used without further pu- rification. Solutions were prepared and stored in the dark to prevent photodecomposition.

The simultaneous determination of the fluorescence and absorbance for most of the fluorophores in Table I was performed as described previously. 23 The perylene fluorescence was measured with an intensified diode array detector. Nonlinear regression was performed with the programs Plotit (Scientific Programming Enterprises, Okemos, MI) and Kinfit, 25 which provided estimated values for the parameters H and K.

A mixture of five PAHs, similar to that of Latz and Winefordner, t6 was prepared from stock reagents. Four of the PAHs were present at constant concentrations: fluorene, 144.0 ~M (24.0 ~g/mL); phenanthrene, 225.0 ~M (40 ~g/mL); anthracene, 5.625 ~tM, (1.0/~g/mL); and perylene, 0.238 #g/mL (0.06 ug/mL). The concentration of pyrene was varied from 0.0 to 200.0 tzM (0.0--40 t~g/ mL). A simultaneous determination of the synchronous fluorescence and absorbance was performed.

APPLIED SPECTROSCOPY 1043

TABLE I. Regression fit parameters for various soluble fluorophores.

Fluorescent probe exci tat ion/ Regression fit parameters ~ C o m m e n t s (max. A U and cone. emiss ion wavelengths T ( x ) = G(e "* - e - K 9 for Beer 's law adherence)

1,1,4,4-Tetraphenylbutadiene (in cyclohexane) X~, = 348, Xom = 450 n m

Dansyl chloride (in ethanol) X0. = 322, X0m = 460 n m

Dansyl chloride (in ethanol) Xo, = 322, Xom = 522 n m

Dansyl chloride (in ethanol) ~x = 322, )kem= 600 n m

Quin ine sulfate (in 0.1 M HC104) ~o, = 365, Xom = 450 n m

Quin ine sulfate (in 0.1 N H2SO4) Xox = 365, hem = 450 n m

Perylene (in cyclohexane) Xo~ = 388, X0~ = 434 n m

Perylene (in cyclohexane) H = 1.12 ± 0.25 X~ = 388, hem = 468 n m K = 9.24 ± 1.19

Perylene (in cyclohexane) H = 1.05 ± 0.26 Xo, = 388, X~m = 502 n m K = 9.36 ± 0.39

H = 1.28 ± 0.16 This invest igat ion K = 9.72 ± 0.74 (2.88 A U at 30.0 uM)


H = 1.24 ± 0.20 This invest igat ion K = 9.86 _+ 0.62 (2.45 A U at 70.0 uM)



H = 1.11 ± 0.05 Earlier work 23 K = 9.60 ± 0.33

H = 0.67 ± 0.32 This invest igat ion K = 7.82 ± 1.69 (diode array detection)

(2.67 A U at 8.1 uM)

This investigation (diode array detection) (2.67 A U at 8.1 uM)

This investigation (diode array detection) (2.67 A U at 8.1 uM)

" Extra insignificant figure retained for H and K to reduce round-offer rors .

RESULTS AND DISCUSSION

Determination of Correction Factor. Equation 2 is valid only in samples where adherence to Beer's law is observed? 3 Adherence was confirmed by the simultaneous measurement ofabsorbance and fluorescence data for the fluorophore 1,1,4,4-tetraphenylbutadiene. An absorption maximum at 348 nm was chosen for excitation, while fluorescence was measured at the emission maximum of 450 nm. For solutions of concentrations ranging from 0.0 to 30.0 #M, linearity to 2.88 absorbance units was observed. The parameters H and K were determined by a nonlinear regression of the absorbance [(~c)o~] vs. fluorescence data values with Eq. 2. The parameters determined at various wavelengths with the other soluble fluorophores (dansyl chloride, quinine sulfate, and perylene)

P~

g g

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E

1 0 0 0 . 0 0 ~

1 0 0 . 0 0

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0.01 . . . . . . . . , . . . . . . . . , . . . . . . . . F . . . . . . . . ~ . . . . . . . . , 0 .01 0 . 1 0 1 . 0 0 1 0 . 0 0 1 0 0 . 0 0 1 0 0 0 . 0 0

1 , 1 , 4 , 4 - T e t r G p h e n y l b u t o d i e n e (/~M)

Fro. 4. Corrected and uncorrected fluorescence intensi ty vs. concentrat ion curve o f 1,1,4,4-tetraphenylbutadiene. Correct ion technique of- fers a lO0-fold increase in linear range.

returned values consistent with work performed earlier in our laboratory. 23

Figure 4 shows the absorbance-corrected vs. the uncorrected fluorescence curves for 1,1,4,4-tetraphenylbutadiene. It is significant to note that the only piece of equipment common to this and to the earlier investigation 23 was the bifurcated fiber optic. The dissim- ilarities included solvents (aqueous H2SO4 vs. cyclohexane), monochromators, PMTs, flow cells, lamps, data ac- quisition systems, curve-fitting software, sources, ampli- fiers, and fluorophores. The independence of the transfer curve descriptors H and K from any other experimental variable except the fiber optic gives the technique a measure of independence from the normal experimental vail-

c

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Z

1 . 0 - 0

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0 . 8 - - o °

0 . 7 2

0 . 6 2

0.5-

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0 . 0 ; . . . . , . . . . , . . . . , . . . . , . . . . , . . . . , . . . . , . . . . , . . . . , . . . . ' 0 0 . 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 0 . 6 0 . 7 0 . 8 0 . 9 1.

D i s t a n c e ( c m )

FIG. 5. A plot o f the solid-state phosphors emiss ion intensi ty vs. distance; Xe, = 290 nm, hem = 500 rim.


0

D

L L _

o

1 2 . 0 - •

11.0 -i

10.0

9.0

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7.0

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v, v v Absorbance = 3.0

o ::i ' ~ J ~ ' , -v w V , - ~ ~7ZX V 0 4 .04 Absorbance = 2.0

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0 3.o

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1 . 0 . . . . , , , , , [ . . . . I . . . . I . . . . [ ' ' ' 1 [ . . . . I ' ' ' ' I . . . . I

425 450 475 500 525 550 575 600 625 650

W a v e l e n g t h ( n m )

Fie. 6. Comparison of correction factors determined by soluble fluorophores and immobilized phosphors for several simulated absorb- anees. Quinine sulfate (©), perylene (*), phosphor mixture (v), dansyl chloride (zx), 1,1,4,4-tetraphenylbutadiene (rq).

ations encountered in fluorometry. The empirical mathematical function describing the interaction of the excitation/emission arms of the bifurcated fiber optic is a constant for a particular fiber optic. Table I gives values determined for H and K along with the experimental conditions and the maximum concentration for Beer's law adherence.

Fortunately the wavelength dependence of the transfer function is that of the smallest numerical aperture (NA) of the system. The transfer function depends entirely on the wavelength-independent NA of the f/3.5 monochromators 26 and appears independent of the wavelength-dependent NA of the fiber opt ic . 27 The f /# of the fiber is related to the numerical aperture by f /# = NA/[2tan(00)].26 Because the fibers are immersed in the cyclohexane sol- vent with a refractive index of 1.420, the effective numerical aperture is reduced to 0.2/1.42 = 0.14. This con- dition puts the wavelength-dependent range of the fibers' effective f /# between 1.7 and 3.8 over the wavelength region from 350 to 650 nm. 27 When compared to the monochromators' wavelength-independentJT# of 3.5 over the same wavelength region, the limiting aperture is that of the monochromators. Other investigators have described an effective N A , 20,24,28-31 a measured quantity that is noticeably smaller than the calculated NA based on the manufacturer's literature. This value is attributed to the measured emission efficiency of the fiber decreasing from a maximum value near the center of the fiber to near z e r o . 2°,28,29 The Gaussian beam profile concentrates over 96% of the emitted radiation in roughly the center one half of the field. This feature has the effect of reducing the fibers' effective numerical aperture to roughly 1.18 (or f /# ~ 4.0).

Synchronous scans of the phosphor and nonlinear regression of the resultant curves produced values for H and K. These values produced a series of correction factors that were found to agree with the values predicted

> ,

4-' 1,0 @

g } 0,5

8 ~ m ~ 0,1 th 4~

K " eQ c 0,05

g

0 o ~ 0,01

cu -~ 0,005 .>_ ,~

0,001 0,01

[ ]

[] [] Corrected

0 0 Uncorrected

' ' 'I .... I ' ' '~ .... I . . . . . . . . . . . . . . I

0,050.1 0.5 1 ,5 1~0 5~0 100200

P y r e n e (/l,M)

Fro. 7. Synchronous fluorescence de te rmina t ion o f fluorene intensi ty vs. pyrene concentrat ion. Previous invest igators measu red ~ 2% o f the fluorene signal when [pyrene] = 12.5 #M at 306 nm.

by indirect measurements with the soluble fluorophores. The correction factors obtained by regression of the immobilized phosphor data were found to agree within experimental error with the correction factors obtained via the soluble fluorophore by regression of the fluorescence vs. absorbance data. One of the intensity vs. distance curves for the phosphor emission is shown in Fig. 5 along with the regression-determined fit of the data to Eq. 1. Although the mathematical description of the transfer function is based on an empirical model, our experimental observations validate the predictions of the transfer curve shape.

We can obtain nearly identical values for the correction factors when compared to the earlier investigation 23 but not for the coefficients that determine the transfer function. The correction factors determined by the direct method (phosphor scattering vs. distance) and the indirect method (soluble fluorophores) are nearly identical. The coefficient of determination for the curve in Fig. 5 was r 2 = 0.995, and the sum of the squares of the residuals (SSR) was 0.0026. However, the magnitudes of the determined coefficients are H = 3.15 ± 0.17 and K = 5.48 ± 0.22. This range is well beyond the experimental limits o f H = 1.24 ± 0.20 and K = 9.86 ± 0.62 obtained via the soluble fluorophore method. The correction factor depends not so much on the absolute values of H or K, but rather on the relative magnitudes of the pair (Eq. 2). Figure 6 shows that both methods arrive at virtually identical values for the correction factor.

Absorption-Corrected Synchronous Fluorescence. The ability of the instrument to provide absorption-corrected

T A B L E II. Absorbance precision of the corrected fluorene fluorescence signal vs. pyrene concentration.

Mixture n u m b e r Pyrene concentra t ion (#M) Percent RSD

1 0.0 1.16 2 9.89 x 10 2 0.75 3 4.94 0.83 4 9.89 1.05 5 49.4 3.72 6 98.8 4.91 7 197 17.5


synchronous fluorescence data was determined with a mixture of five PAHs identical to the mixture of Latz et al. 16 The fluorescence signal from fluorene was determined in the presence of varying amounts of pyrene, as shown in Fig. 7. Table II shows the precision of the fluorene absorbance signal. Signal recovery ranged from 112% for mixture 7 to 98% for mixture 2. Mixture 7 had a primary absorbance of 2.49 AU at 301 nm, and secondary absorbance of 2.53 AU at 306 nm (fluorene emission maximum). Other investigators have noted that the wide difference in the linearity range of synchronous fluorescence bands in a mixture is wavelength dependent) 3 They observed the linearity ranges for the high-energy synchronous fluorescence bands of fluorene (304 nm), acetanapthene (323 nm), and 2,3-benzofluorene (342 nm) extended over less than three orders of magnitude, where- as the linearity ranges of the lower-energy synchronous bands of anthracene (378 nm), benzo-[a]-pyrene (405 nm), and perylene (438 nm) extended over three to four orders of magnitude.

Perylene seemed unsuitable for determining cell parameters and correction factors at wavelengths shorter than 450 nm (Fig. 6), which we attribute to significant self-absorption. 32 The solid phosphor method of cell char- acterization is immune from self-absorption because the measurement is made from the phosphor surface. The phosphor/carbon matrix renders the mixture opaque and reduces the possibility of depth profiling.

For the correction technique to prove useful in synchronous fluorescence, corrections for both primary and secondary inner-filter effects must be made on the basis of aggregate absorbances at a single ~k. By stepping the excitation, emission, and transmission monochromators synchronously, we simultaneously measure the emission and absorbance at both the excitation and emission wavelengths. Another requisite is that the fluorescence from only one analyte be observed at a time. Earlier investi- gations, 23 which corrected for primary inner-filter effects in quinine sulfate (QS) due to the presence of varying amounts of 1,5-dihydroxybenzoic acid (DHBA), required multiple corrections. The corrected fluorescence of QS with DHBA present was obtained by subtracting the corrected fluorescence of DHBA from that of the QS/DHBA mixture. For fingerprinting unknown mixtures, the earlier method 23 is impractical because it requires the prepara- tion of solutions containing the correct concentrations of the interfering chromophore to subtract from the unknown. Accurate corrections would thus require total knowledge of the composition of the "unknown". Syn- chronous fluorescence has its own built-in safeguards against observing spurious fluorophores. Judicious selec- tion of a narrow scanning interval (Ak) greatly reduces the likelihood of more than one fluorophore contributing to the fluorescence signal.

Synchronous fluorescence measurements based on the Stokes shift are performed in the wings of the excitation and emission bands (Ak = 3-5 nm). The fluorophore's low absorption cross section and poor quantum efficiency complicate obtaining high signal-to-noise ratios with our front-surface fiber-optic fluorometer. Our detection limit for perylene is over 400 times greater than with our com- mercial right-angle-geometry fluorometer.

At high pyrene concentrations, additional signal av-

eraging became necessary to recover the fluorene signal with a satisfactory signal-to-noise ratio. At 200/zM (40.4 ug/mL), the measured absorbance at the excitation wavelength of fluorene was (W)e3~ °1 = 2.49 AU, while the absorbance at the emission wavelength was (WL3°m 6 = 2.53 AU. The total solution transmittance from primary and secondary inner-filter effects relative to an optically clear solution was 10 (2.49+2.53) = 10 5.02 _ ~ 9.56 × 10 -6.

CONCLUSION

The assumption that the observed fluorescence is pro- portional to fluorophore concentration is valid for a single fluorophore at low concentration. In the complicated matrices encountered in synchronous luminescence measurements, absorbance may be high at the excitation wavelength (primary absorbance) or at the emission wavelength (secondary absorbance) or both, because of multiple fluorophores of unknown concentrations in the sample matrix. If the solution demonstrates significant absorbance at either wavelength, the experimenter must be aware of these matrix effects and the quantitative un- certainties they introduce.

We have demonstrated that primary and secondary inner-filter effects can be corrected when measurements are made with an adequate signal-to-noise ratio. The addition of the transmittance monochromator to reject stray fluorescence makes this instrument an excellent spec- trometer for strong fluorophores. Absorbances that are linear to 3 AU can be measured reliably despite a high fluorescence background. The source of most of the error in correcting a fluorescence measurement for absorbance arises from a large RSD in the fluorescence measurement due to low light levels at high absorbances. However, problematic samples are easily recognized by noting the total (primary and secondary) absorbance.

Clearly our instrument is somewhat over-extended for determining the synchronous fluorescence of fluorene in the presence of > 100 0zM) pyrene. However, even for mixture 6, where pyrene is present at 100 uM, the RSD of our measurement Falls to a respectable 4.9% (Table II). Without the benefit of any correction, the original investigators could recover only ~ 2% of the expected fluorene fluorescence. 1 6 , 2 4

ACKNOWLEDGMENTS

The authors thank the support team at Michigan State University Department of Chemistry for lending their mechanical, electrical, and computer expertise to this research. Financial support for this work was provided by the National Science Foundation through Grant CHE- 8705069.

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Absorbance-Corrected Synchronous Fluorescence with a Fiber-Optic-Based Fluorometer