Scanned laser fluorescence line narrowing spectroscopy of

Anal. Chem. 1990, 62, 1989-1994

1989

Scanned Laser Fluorescence Line Narrowing Spectroscopy of Photosensitive Organic Chromophores Bradford B. Price and John C. Wright* Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706

Inhomogeneous broadenlng typically Interferes wlth the observation of sharp featured fluorescence from organic chromophores dkpersed In glasses and polymers. Fluorescence llne narrowlng (FLN) and nonllnear spectroscopic techniques can be used to obtain sRe seiectlve spectra in photochemlcally stable systems but produce ambiguous r e d s when the Incldent laser Induces frequency domain hole burning. The effects of hole burnlng on the observed llne narrowed fluorescence slgnal are descrlbed by introducing a tlme-dependent site distribution. To overcome the lhttatlons of hole burnlng, FLN spectra are acquired whlle scanning both the laser and monochromator wavelengths. The requislte laser scan rate Is predicted from the klnetlcs of the hole burning. FeaslbllRy of the technique Is demonstrated In the system of octaethytporphlne In pdy(methy1 methacrylate). I t Is postulated that unllke previous methods of clrcumventlng site depletion, this approach should be readily applicable to our experlments In nonlinear spectroscopy.

INTRODUCTION Fluorescence line narrowing (FLN) and hole burning are two widely used techniques for site selective spectroscopy in inhomogeneously broadened systems where chromophores are dispersed in organic glasses and polymers (1-5). The single site spectrum consists of a sharp, purely electronic transition of the chromophore (zero phonon line) and a relatively broad phonon sideband. The sideband is the result of the interaction of the chromophore electronic transition with the host material lattice dynamics and will vary in intensity relative to the zero phonon feature for different host-guest systems. Both FLN and hole burning techniques rely on the static site distribution in low-temperature systems but are complementary both in the method of site selection and in the resulting information. In FLN a subset of sites is selectively excited and the fluorescence of this subset is monitored. In hole burning a subset of photolabile chromophore sites is excited, producing a site-selective depletion. The resulting change in absorption is site specific assuming the zero phonon condition of Burland ( 6 )is fulfilled. The complementary nature of the two techniques leads to conflict when FLN is applied to systems where hole burning can occur. Since hole burning processes deplete resonant sites, fluorescence signal intensities are dramatically decreased. This decrease distorts the observed spectrum and ultimately eliminates the sharp-featured fluorescence. Several methods have been developed to circumvent the problems of site depletion during a fluorescence scan. By increasing the sample temperature, Friedrich and Haarer (7) decreased site depletion at the cost of less intense, broader fluorescence signals. In the case of reversible hole burning, Bykovskaya (8) applied intense white light pulses between laser pulses to reverse the selective depletion. More recently, Hoftstradt et al. (9) and Cooper et al. (10) have used a sol-

* Author to whom correspondence should be sent.

id-state array detector to perform spectral acquisition on a time scale faster than the rate of hole burning. Methods involving synchronous scanning have been developed to perform spectral scans in both fluorescence spectroscopy (11)and nonlinear spectroscopy (12). In their application of FLN to the ultratrace level detection of DNAPAH adducts, Cooper et al. (10) and Jankowiak et al. (13)have suggested and demonstrated that synchronously scanning the laser and monochromator enhances the sensitivity of FLN in systems which exhibit nonphotochemical hole burning. Further enhancements in spectral discrimination against broad phonon sideband emission can be achieved by acquiring both brief (minimal hole burning) and extended (severe hole burning) excitation time spectra and performing a spectral subtraction (10, 14). Both site restoration and array detectors are successful for the majority of fluorescence experiments but require a reasonable investment to modify the standard laser fluorescence system. In addition, neither approach has proven feasible in our efforts to extend site-selective four-wave mixing (15)to systems that undergo hole burning. The four-wave mixing technique uses intense optical fields in a small sample volume with a single output frequency defined by the input frequencies, conditions which decrease the effectiveness of site restoration and remove the advantages of multichannel detection. In this paper we discuss the distortions observed in FLN spectroscopy of photolabile systems and modify the practice of synchronous scanning to circumvent the problems of hole burning in FLN and, presumably, four-wave mixing. The approach we have developed is to scan both the excitation laser and the detection monochromator, but to scan them at different rates. With the laser scanning at a rate faster than the rate of hole burning, new unburned sites are the source of the line-narrowed fluorescence which is detected by the monochromator. The scan rate of the monochromator is selected to cover the spectral region of interest as in conventional fluorescence line narrowing. The utility of the scanned laser fluorescence (SLF) approach is demonstrated on the system of octaethylporphine in poly(methy1 methacrylate), a system which undergoes rapid photochemical hole burning (4,16).Vibrational energies are consistent with previously reported values, demonstrating the applicability of the technique to the fluorescence line narrowing spectroscopy of photolabile systems.

EXPERIMENTAL SECTION Sample Preparation. 2,3,7,8,12,13,17,18-Octaethyl21H,23H-porphine (OEP) was obtained from Aldrich Chemical Co. and used without further purification. 2,2'-Azobis(2methylpropionitrile) (AIBN) (Alfa Products, Danvers MA) was recrystallized from ethanol prior to use. Methyl methacrylate monomer (Alfa Products) was vacuum distilled immediately prior to use to remove inhibitors. Sample preparation involved the polymerization of methyl methacrylate in the presence of the initiator AIBN and the desired chromophore. Methyl methacrylate monomer is vacuum distilled and combined with 1%(w/w) AIBN and an appropriate amount of OEP to give a concentration of 10" M. The resulting solution

0003-2700/90/0362-1989$02.50/0 0 1990 American Chemical Society

1990

ANALYTICAL CHEMISTRY, VOL. 62, NO. 18, SEPTEMBER 15, 1990 Incident Pulses ( * l o a ) 0

I )

5

10

I

I

15

‘ I

-t 1

I

I

I

800

I

I

1000

0,-

I

I

I

1200

o,

I

-.

1400

I

I.

I 1600

I

(cm-’)

Figure 1. Static excitation fluorescence of porphine in PMMA at 2 K. Excitation at wL = 16375 cm-’, scan emission, we,,,.

is degassed and sealed under vacuum prior to polymerization at 40 O C . After 3 h, the viscous solution is transferred to a template held between two glass plates and polymerization is continued at 7 5 “C for 8 h. Resulting samples can be several millimeters thick and have excellent optical quality. There is no significant alteration of the OEP absorbance spectrum as a result of the polymerization although less robust porphyrins, such as porphine, do exhibit some degradation. Experimental Apparatus. Fluorescence is excited by a XeCl excimer pumped home-built dye laser operating in ninth order at a 13 Hz repetition rate with DCM dye (Exciton Chemical Co.). Typical output characteristics include a 5-11s pulse duration, 0.2-cm-’ (0.07 A) bandwidth (fwhm), and 100 J per pulse at 620 nm. The excitation laser is attenuated and directed to a 2 mm diameter spot on the sample which is immersed in superfluid liquid helium. Fluorescence is collected from the face of the sample and monitored by a 0.85-m double monochromator with an EM1 9658R photomultiplier and associated electronics. The wavelength position of both the dye laser and the monochromator is monitored and controlled by a computer-based data acquisition system. Both components are scanned linearly in wavelength using a traditional sine-bar drive. For all the experiments discussed the monochromator (we,) is scanned at 0.1 A/s while the excitation laser (q,) scan rates varied over the range of 0-0.02 A/s. A typical fluorescence scan requires 10-15 min with 1 cm-l resolution. The experiments have been performed with comparable success using the laser system, omitting the computer-based acquisition and control.

THEORY Excitation of a photolabile chromophore will ultimately lead to site depletion. As expected, the total fluorescence intensity decreases as a result of resonant site depletion. However, in FLN this depletion has the observed effect of preferentially removing the sharp spectral features with broad, phononlike features dominating the spectrum after prolonged irradiation a t a fixed wavelength. Figure 1 illustrates the distortions typically observed when hole burning interferences occur in FLN and spans the range from short to prolonged illumination. At short times sharp peaks corresponding to the chromophore vibrational energies are observed superimposed on a relatively intense background. At longer times both the background and sharp feature intensities decrease. Once the background broad-band emission reaches a stable value, no vibrational lines are observed. Most noteworthy is the absence of the 1316-cm-’ line and 1600-cm-’ multiplet, which are the dominant features in the absence of hole burning (8). Throughout the scan, sharp feature intensities are decreasing, prohibiting accurate estimates of relative line intensities. While line-narrowed features decay rapidly, phonon sideband emission features show a slow decay (9) and dominate the fluorescence after prolonged irradiation. Although these observations have been previously documented (7-9),models for FLN have not been expanded to include the effects of site depletion. To explain the features

of the time varying emission spectrum, we will combine the time-dependent behavior of spectral hole burning as developed by Friedrich (7)with the convolution model for fluorescence line narrowing, initially proposed by Abram (17). The resulting equation demonstrates a decay rate that is strongly dependent on both the Debye-Waller factor and the breadth of the spectral feature and is in good agreement with the observations associated with Figure 1. This approach, while lacking the generality to permit rigorous extension to systems which undergo nonphotochemical hole burning, permits a qualitative understanding of the interfering effects of hole burning on FLN spectra and quantitatively is in good agreement with our data. In addition, the implicit assumptions of Friedrich (7)regarding weak, linear electron phonon coupling have been adopted since this approach provides a tractable, analytic solution. Recently Hayes and Lee (16, 18) have generalized the description of hole burning spectra to systems with arbitrarily strong electron phonon coupling. Incorporating the strong coupling model may be appropriate for many biologically significant systems but is not necessary to demonstrate the efficacy of scanned laser excitation on the enhancement of FLN spectral features. Since porphyrins dispersed in polymers generate a broad distribution of antiholes (18), effects due to site restoration will be minimal and are not included in the analysis. A single site in the inhomogeneous distribution with a zero phonon energy of wo will have the intrinsic peak normalized absorption and emission given by Sab(a,uO)

= aZab(u,uO) + [1 - a I P a b ( ~ , w ~ ) (la)

S e m ( ~ , a O ) = ( ~ Z e m ( a 0 , ~+) f l- ~ l f ‘ e m ( o o , o )

(1b)

where 2 represents the homogeneous line shape of the zero phonon line, P the spectral profile of the associated phonon sideband, and (Y the Debye-Waller factor for the site (17). The relative shift of the phonon sideband is implicit in the functionality of P (19). For a sample volume containing N molecules, we can express the number of sites centered a t wo as N(uo)and will represent the inhomogeneously broadened site distribution as an area normalized probability distribution function, D(wo) = N(wo)/N. When a photostable sample is excited with a laser with an area normalized frequency profile L(w’,wL) centered a t wL, the fluorescence spectrum can be written as the convolution S(W) =

JI:-

JI:-Sab(u’,uo)

DtwO) L(w’,~L) Sem(u,uO)dw’ duo (2)

In a photolabile system the site distribution is altered by the incident laser and frequency domain hole burning occurs. The rate of change of N(wo)over the interval d s is given by

(3) where I , u, and v h b are the laser intensity, absorption cross section, and hole burning quantum efficiency, respectively. Friedrich (7) considered a laser with a 6 function frequency dependence to derive eq 4 for the relative number of molecules remaining at wo after an irradiation time r

C, = expl-r(I/hwL)uvhbSab(wL,wg)}and No(wo)is the number

of sites centered a t

wo

at time

T

= 0. Equation 2 must be

ANALYTICAL CHEMISTRY, VOL. 62, NO. 18, SEPTEMBER 15, 1990

modified for a time-dependent site distribution since N(uo)/N is now time dependent. The time dependent analogue to eq 2 is SAW) =

l ~ m l ~ m s a b ( u ' C, r~D(u0) O ) L(W',~L.J Sem(a,uO) du' duo

(5) where the laser frequency uL induces the site depletion and % is the laser frequency generating the fluorescence spectrum. For brevity we will consider the typical case of ub = wL where the excitation source simultaneously induces hole burning and fluorescence. The form of C, prohibits the analytic solution of eq 5 without approximation. To generate a useful form, we consider the case of a laser with a &function frequency dependence and the short burn time limit where C, is expanded in a Taylor series, truncating after the linear term. These approximations transform eq 5 into

Considering the two terms in eq 6, the first term is equivalent to eq 2 in the limit of an infinitely narrow laser, a case which has been previously addressed (17, 19). The second term represents a first-order correction for resonant site depletion and is accurate to a few percent for C, I0.8. To evaluate the depletion term (6b), we substitute eq la-b for the absorption and emission features, expand the products, and combine terms of comparable spectral breadth. In the case of weak electron-phonon coupling we can consider D(uo) to be broad enough relative to the single site features to be considered a constant and 6b becomes

1991

The enhanced decay of zero phonon features relative to phonon sideband features can be explained by eq 7. By expanding the convolution integrals analogous to those in eq 7, Friedrich (7) examined the rate of hole formation in absorption experiments and demonstrated an inverse relationship between the width of the absorbing feature and the rate of hole burning. Since the dominant perturbation is due to site depletion, Friedrich's analysis can be applied directly. Taking the homogeneous line width of porphine in PMMA of 0.016 cm-' ( 4 ) and a the phonon sideband width of 30 cm-' as representative values, we would initially predict the depletion due to zero phonon absorption occurs 2000 times faster than that expected for phonon sideband absorption. Since 7a-b will be affected by zero-phonon hole burning, these terms will cause depletion at a rate 3 orders of magnitude faster than the depletion due to 7c-d. Term 7a will deplete the entire zero-phonon feature (ref 15, eq 4a) while 7b will only be capable of rapidly depleting one-third of the total observed phonon sideband feature (ref 15, eq 7b-d) which is observed in FLN. In addition, the dependence of each term on the DebyeWaller factor will further enhance the sharp feature's relative depletion. Taking the estimate of Kador et al. (20) of a = 0.77 f 0.1 for phthalocyanine in PMMA to be comparable to that expected for OEP in PMMA, we calculate the f ( a ) for terms 7a-d to be 0.45 f 0.2,0.13 f 0.04,0.04 f 0.01 and 0.012 f 0.002, respectively, further enhancing the relative depletion due to term 7a. This effect is less dramatic than the line breadth contribution from G(Z,P), especially considering the fact that there are three contributions from each term 7b and 7c due to the summation of the three convolution integrals. In situations of weak electron-phonon coupling as CY 1, the contributions from f ( a ) will become more significant. Given the assumptions of eq 7 and the estimates for the line widths and the Debye-Waller factor, we would expect the depletion of zero phonon features to be approximately 3 orders of magnitude faster than that observed for the phonon sideband features. This discrepancy in decay rates adequately explains the observations in Figure 1 where sharp features are short lived under prolonged irradiation at a fixed frequency.

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SCAN RATE DEPENDENCE To predict the effect of scanned laser excitation on the observed spectra, eq 3 is evaluated for the case of a time varying aL.By considering a &function laser scanned linearly with a scan rate r, the energy at any time r can be written as uL = uLo+ rr. Under these conditions, eq 3 can be rewritten in the time-dependent form

Each term in eq 7 takes the general form -Zuqrf(a)g(Z,P) where g(Z,P) indicates the breadth of the spectral feature being observed. In the situation where the zero phonon line is sharp relative to the phonon sideband, the breadth of the depleted features becomes apparent. All terms other than 7a have a significant phononlike contribution to their FLN breadth and represent the depletion of relatively broad spectral features while 7a represents the decay of the zero phonon fluorescence. The nature of g(2,P) is defined by the breadth of the individual features involved in the hole burning, absorption, and emission processes. Each term contains the product of two absorption profiles and an emission profile. The fluorescence intensity is determined by the absorption and emission profile and the hole burning is determined by the absorption profile. The different terms result because the zero phonon feature and the phonon sideband can participate in all combinations.

Since eq 7 indicates that Zab(uL,WO)is the dominant line shape affecting sharp feature depletion, we can consider S a b to be a single Lorentzian with a half width at half maximum of Au, Sab(uL,uo) = Au2/ [ (uLo rr - uo)2+ A d ] . In this discussion, we have only considered the laser with a &function frequency dependence where Au reflects the homogeneous line width of the zero phonon line sab(W,Wo). An equivalent result would be achieved by treating Sab(u,uO) as a S function in frequency space. In practice, Au reflects the width of the convolution of the zero phonon line and the laser profile. To perform the integration of eq 8, we assume that the photon flux, ZL = I/huL, is a slowly varying function over the laser scan range and treat it as a constant. This approximation is reasonable considering the 200-400 cm-' tuning range of typical laser dyes which emit in the vicinity of 16000-20000 cm-'. If the excitation laser were scanned across the entire tuning curve, the change in ZL would be predominantly due

+

1992

ANALYTICAL CHEMISTRY, VOL. 62, NO. 18, SEPTEMBER 15, 1990 16250~ m-'

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Incident Pulses (*IO2)

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Flgure 2. Comparison of static (lower) and scanned laser excited fluorescence of OEP in PMMA at 2 K. Excitation at two positions in the inhomogeneous absorption profile. Laser scan rate of 1.6 cm-' per minute gives upper spectra.

to the decreased laser intensity, I, rather than the change in photon energy, hw,. Considering the case where u, and ?& are constant over the scan range, it is possible to analytically solve eq 8. Integrating eq 8 from 7 = 0 to 7 = (wo - wLo)/rwhen the laser will be centered on the site at wo gives

Since C, is directly related to the number of molecules remaining at wo, the fluorescence signal intensity is proportional to eq 9. For a rapid laser scan rate, C, 1 indicating the absence of hole burning and an unaltered fluorescence spectrum. Similarly for a laser scan rate which is slow compared to the rate of hole burning ILuvhb, c, 0 and the sharp features due to selective excitation at wo will not appear. For the case of pulsed laser excitation in the absence of saturation effects, ZL can be rewritten as the product of the pulse energy per cm2,I,, and the repetition rate, R, where I L = IpR/hwL, and we are able to consider the scan rate of the dye laser not just in terms of laser bandwidths scanned per second but can introduce the units of bandwidths scanned per pulse.

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RESULTS AND DISCUSSION The improvements in sharp featured signal intensity which result from scanned laser FLN are shown in Figure 2. The lower spectra demonstrate the fluorescence obtained with static excitation a t the indicated frequency. Broad (-50 cm-I) emission features are seen over the entire range with a single, sharp feature at 762 cm-' in the case of 16 125-cm-' excitation. In contrast, the upper spectra were obtained by scanning the excitation laser a t 1.6 cm-'/min while the monochromator is scanned a t a rate 10 times faster. The vibrational energies indicated are corrected for the scanning of the laser and are within a few wavenumbers of the values reported by Bykovskaya for OEP in a polystyrene host. This agreement indicated at most a weak dependence of the porphyrin vibrational energy on the choice of host material. Comparable features are seen for the two excitation regions with the exception of the peak enhancement for the 762-cm-' vibration in the case of the red-shifted excitation. The persistence of the 762-cm-' feature in both static and scanned laser excitation indicates that it is associated with a relatively photostable molecule, presumably an impurity or aggregate which absorbs a t a lower energy than the majority of OEP molecules. Spectral contributions from stable impurities are readily removed by a spectral subtraction in a manner analogous to that of Hoftstradt (9) and Zamzow (14), revealing the spectrum of the photolabile chromophore. In experiments where the excitation was varied across the entire inhomogeneous absorption profile, no significant

0

5 10 15 20 Photons/cm* (*lo1')

25

Flgure 3. Signal decay of 1214-cm-' Vibrational feature under 1.5 /.LJ per pulse excitation.

static

changes in the OEP fluorescence spectrum were observed other than overall intensity, indicating that the shape S,(w,wo) is not a strong function of wo. In systems with stronger electron-phonon coupling, the intensity of phonon sidebands relative to zero phonon features will typically increase as the excitation energy is increased (blue shifted). In addition, it is possible that different sites within an inhomogeneous distribution may show an alteration in the relative line intensities. These complications are not expected to diminish the analytical utility of the scanned laser technique since the excitation energy is scanned over a small interval ( < l o cm-' in this work) relative to the inhomogeneous width and the anticipated weak energy dependence of the two effects. By use of a fixed frequency a t 16 175 cm-', the time-dependent fluorescence to a vibrational level can be monitored. Figure 3 is typical of the signal depletion caused by prolonged irradiation a t a fixed frequency where the ordinate is the cumulative intensity and the abscissa is the relative signal intensity measured a t 14961 cm-', corresponding to the 1214-cm-' vibrational level. A single exponential decay is observed, reaching a constant, low intensity within 2000 pulses a t 1.5 pJ per pulse. Fitting the data to a single exponential decay, we can extract an effective decay cross section of 3 x cm2. Considering eq 3 and 4, the decay cross section corresponds to u?hb summed over the frequency spread of the laser and the homogeneous line shape. One can estimate the quantum efficiency of hole burning once the absorption cross section is determined. Considering the comments of Moerner et al. (21) regarding estimating u in inhomogeneously broadened systems, the laser bandwidth defines an effective homogeneous line width and we apply eq 3 of ref 21 directly, inserting 1.50 for the refractive index of PMMA (20). The oscillator strength for the Q(o,o) transition of porphine and etioporphine has been previously calculated (f = 0.019) and estimated from experimental data (f = 0.0058-0.025) by Gouterman (22). The average of the experimental values corresponds to an absorption cross section of 1.2 X cm2. Thus, one would estimate v h b is ca. 2 X W5. While this method of measuring the quantum efficiency is dramatically different than the more conventional technique of measuring the change in transmittance extrapolated to zero time and in the low power limit, it is encouraging that the value is comparable to that observed for other systems which undergo rapid photochemical hole burning (20). Since the product q h b will depend on both the chemical and instrumental systems, in further discussions we will treat it as a single parameter and use the observed value to illustrate the effects of scan rate and laser power on the observed spectra. The laser scan rate required to observe sharp-featured spectra with a laser of given power and bandwidth is determined by eq 8 which depends on the product 1 L q h b P . w and the arctangent function which describes the cumulative la-

ANALYTICAL CHEMISTRY, VOL. 62, NO. 18, SEPTEMBER 15, 1990

1903

Table I. Scan-Rate Dependence of Normalized Signal scan rate,

pulses

(A/s) X lo3

(hwhm)

m, A

calcd eq 10

exptl

0.33 0.55 1.1 3.3 6.7 17.0

11000 6400 3200 1100 520 210

0.12 0.20 0.40 1.2 2.4 6.1

0.20 0.35 0.57 0.82 0.91 0.96

0.27 f 0.13 0.35 f 0.18 0.56 f 0.21 0.89 f 0.15 0.91 f 0.09 1.00

0-1.0

-0.5 0.0 0.5 Log(O.OOBB*~J/scanrate)

1.0

Flgure 5. Enhancement of 1214-cm-' vibrational feature as a function of laser power and scan rate. . . n

El 0

5.2

6.7

11 m

vl 1.1 0.55

1 ' 1

'

500

0,-

1

1 ' 1000

w,,

'

1

'

1 ' 1500

I

(cm-')

Figure 4. Line narrowed fluorescence with scanned laser excitation at various laser scan rates, 0.5 pJ per pulse.

ser-line-shape overlap caused by scanning across a Lorentzian line shape. Considering typical experimental conditions and a pulse energy of 0.5 CJ,eq 9 for the predicted normalized peak intensity will take the form

Evaluating eq 10 for the case of r T = wo - wLo >> Aw and with Aw = 0.035 cm-', we expect the sharp-featured fluorescence to increase with laser scan rate following IF = exp[ (-6.6 X 10-4)/r], where the scan rate r is expressed in angstroms per second. For slow scan rates, where r7 becomes comparable to the bandwidth Aw, the arctangent term must be explicitly included to correctly represent the signal growth. Without the arctangent correction, an anomalously low signal would be predicted. In essence, for small values of r7, the time integrated intensity a t wo has not attained the steady-state value before the site fully resonant with the laser. T o avoid distortions in the relative peak intensities in long spectral scans, it is important to scan greater than two bandwidths prior to resonant excitation so that peaks which ap.pear a t the beginning of the scan are not enhanced relative to those at the end of the scan. T o display the data in a format compatible with eq 10, the peak intensity at the maximum laser scan rate is assigned the value 1.0and all other intensities are normalized to this value. Table I indicates good agreement between the predicted and experimentally observed data where the normalized intensity from three sets of scans is averaged to yield the experimental data. The large relative uncertainties associated with the low intensity signals are predominantly caused by baseline noise. In all cases the calculated intensities are within the experimental error. Examples of fluorescence spectra acquired with various laser scan rates are shown in Figure 4. With the initial excitation position at 16 150 cm-l, the scan rates chosen cover the span from 0.0005 to 0.02 8, s-l. This range is sufficient to demonstrate the behavior from the case of extensive site depletion

to that of minimal depletion. At a scan rate of 0.0005 8, s-l, the spectra are indistinguishable from those obtained under static excitation. A continuous enhancement of the sharp features is seen with increasing scan rate until 0.0067 8, s-l where scanning has reduced the site depletion to the level of the pulse to pulse fluctuations in signal intensity. This general trend is predicted by eq 10 where, for small scan rates, the number of resonant centers approaches 0. Similarly, as r / A w becomes greater than the decay rate, the number of centers asymptotically approaches the no hole burning case where C, = 1. One of the concerns about scanned laser excitation is the influence of exciting sites with different intrinsic spectra and incorporating the information from these various sites into a single spectrum. At 0.02 the laser has scanned