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Influence of adventitious light scattering on fluorescence anisotropy measurements with front-face excitation. Simulation and correction
.I. Phmchem.
PhotobioL A: Chem,
78 (1994) 193-200
193
Influence of adventitious light scattering measurements with front-face excitation. Angel
P. Dorado,
Depatrat?~~~t~ de Qukca
Miguel
A. Llorente
Ftiica, F~dtod
and
In&
on fluorescence anisotropy Simulation and correction
F. Pierola
de Ciencias, Chiversid~d National de Educocih
A DiWmcia (UNED), 28040 Madrid
&ah)
(Received June, 9, 1993; accepted October 26, 1993)
Abstract Fluorescence anisotropy spectra of a generic chromophore have been simulated for different proportions of adventitious light scattering with respect to fluorescence. The spectral overlap of Rayleigh light scattering and stray light with fluorescence causes an increase in the apparent anisotropy with TCSpeCt to the real fluorescence anisotropy and curvature of the anisotropy spectra. In the presence of up to about 10% fluorescence contamination, the largest effect of these artefacts occurs for true anisotropies close to 0.25 and the least for anisotropies close to -0.2. The apparent anisotropy, determined at the fluorescence maximum or at the anisotropy minimum, increases almost linearly with f (the fraction of stray light with respect to the maximum fluorescence intensity) for lowfvalues cf< 15%). In the light of these results, a method is proposed to correct the influence of adventitious light scattering on anisotropy measurements. This method has been applied to determine the fluorescence anisotropy of o- and m-xylene in a polymeric matrix with front-face excitation.
1. Introduction Light scattering plays an important role in fluorescence anisotropy measurements. Biochemical samples (e.g. aqueous suspensions of membranes) are often turbid and this produces an artefact in fluorescence depolarization measurements in the usual right-angle cell configuration [l-6]. The polarized incident light can be scattered before absorption and the emitted can be scattered before detection. Both phenomena give rise to partial depolarization and decrease the observed anisotropy (r). These purely optical effects must be corrected if the rotational diffusion of the fluorescent molecule is to be quantified adequately. A concentration-based correction (better than a correction based on turbidity measurements) has been proposed [6] with the argument that a fluorometer’s photodetector generally has a different acceptance angle from an absorption spectrophotometer, a technique which is usually employed to perform turbidity measurements. It has also been suggested [l, 21 that intense light scattering may affect the concentration-dependent extrapolation method proposed by Teale [43 and it is thus recommended that the correction is applied to sufficiently dilute samples, whenever possible_ If this is not possible, light-scattering depolarization
lOlO-6030/94/$07.00 @ 1994 Elsevier Sequoia. All rights reserved SSDZ 1010-5030(93)03733-W
can be avoided by performing anisotropy measurements with front-face excitation instead of with the usual right-angle geometry. Front-face excitation is also a requirement for non-transparent solid samples [7, 81. This instrumental geometry is not free of light-scattering artefacts. The technical spectrum of any chromophore is formed by the spectral overlap of the long-wavelength part of Rayleigh light scattering and reflected light (centred at the excitation wavelength), the peak of Raman scattering, stray light and the fluorescence itself. The contribution to the observed spectrum of each of these terms depends predominantly on the fluorescence quantum yield of the chromophore and on its wncentration in the medium, but also on the experimental conditions which may become very important in front-face excitation experiments. Rayleigh scattering of polarized incident light observed in the plane~perpendicular to its electric vector, at 90” to the excitation direction, is totally polarized (r= l), whereas the fluorescence of a randomly (isotropically) oriented ensemble of fluorophores is partially depolarized ( - 0.2
194
A.P. Dorodo et al. / Infknce
of light scattering on fluorescence anisotropy
complicated and it can be experimentally avoided with chromophores of large Stokes shift or with smaller excitation wavelengths; in fact, in frontface excitation experiments the Rayleigh lightscattering band is broad, and often includes the much less intense Raman peak. In this paper we attempt to show the influence of the overlap of light scattering on the fluorescence anisotropy spectra in different situations and propose a method which can be used to obtain true anisotropy values from the apparent values determined experimentally. To check the validity of the correction method proposed here, we have chosen the worst possible experimental situation: chromophores of low fluorescence quantum yield and anisotropy close to zero. The chromophores (xylenes) were dissolved in a vitreous polymer matrix (polymethylmethacrylate) and their fluorescence anisotropies were measured by front-face excitation at room temperature. The fluorescence anisotropy technique has been found to be very useful in determining the microenvironmental properties of solid or swollen polymer samples [lO-131 but, in view of the simulation results presented here, caution should be exercised when both making and interpreting such measurements.
2. Experimental
details
The xylene isomers (o- and m-) were purchased from Fluka (GC Standard). The polymer matrix was made of polymethylmethacrylate (PMMA) of high molecular weight (8.5 x 10’ Da), synthesized by radical polymerization. Films about 100 pm thick were cast from a solution of the chromophore and the polymer in a good solvent of both (chloroform). The composition of the solid solution was about 5% w/w of xylene, a concentration low enough to avoid plastification but high enough to yield an observable fluorescence intensity [14]. The chromophore concentration in the solid sample was determined spectrophotometrically after fluorescence measurements by redissolving a known mass of the film in a known volume of tetrahydrofuran (THF); the extinction coefficients of xylenes in dilute solution were taken from ref. 15. Measurements were performed at room temperature by front-face excitation using a Hitachi F4000 spectrofluorometer provided with film polarizers. The excitation wavelength was 260 nm. The anisotropy r(h) was calculated at each wavelength h as
r(h) =
fll(4 -~~ww) I,,(4 + 21~ (AMA)
where I,, and I, represent the intensities measured when the transmission axis of the polarizer is oriented vertically and that of the analyser is parallel (vertical) or perpendicular (horizontal) respectively. G is a correction factor which takes into account the different efficiencies of registration of the vertically or horizontally polarized emission; it is calculated for each wavelength as the ratio of the intensities when the transmission axis of the polarizer is horizontal and that of the analyser is vertical or horizontal. 3. Results
and discussion
3.1. Fluorescence measurements The fluorescence spectra of xylene isomers in a PMMA matrix were run with front-face excitation at room temperature. Different ratios of fluorescence and spurious light were obtained on changing the slit widths in both the incident and emergent beams. Spectra run with blanks made of PMMA film without chromophore (see Fig. 1) or with scatter show that the stray light intensities are fairly constant (C) with wavelength and that they increase with increasing excitation and emission bandpass. The anisotropy of both Rayleigh light scattering and stray light is in all cases about unity. A typical anisotropy spectrum (Fig. 2) shows a minimum in the spectral region corresponding to the maximum fluorescence intensity and at both sides it reaches a value close to unity. The minimum anisotropy corresponds to the apparent fluores-
0 260
I
280
300
I.
320
Wavelength
,__r__r340
360
------A 380
400
(nm)
Fig. 1. Experimental spectra of a PMMA film without chromophore, measured with several bandwidths, under the same conditions as the samples. Excitation and emission band passes: (-.-.), S/5 nm; (-), 10/5 nm; and (-.-) S/10 nm.
A.P. Donado et al. / In@ence of lightscattehg on fluorescenceaniFotropy
336
Wovelength
Fig. 2. Experimental anisotropy spectrum of m-xylem! in a PMMA matrix with front-face excitation, showing the two limiting values of the anisotropy at the excitation wavelength and on the red side of the spectrum.
195
374
(nm)
Fig. 3. Experimental emission spectra of o-xylene in a Ph4MA film run with three different bandpasses, which give rise to different levels of stray light. Excitation and emission band passes in nm: (-), 5/S; (. . . . ), 10/5; and (-.-) 5/l&
anisotropy and the anisotropy values corresponding to the excitation wavelength region and the red side of the spectrum can be ascribed to Rayleigh light scattering and stray light respectively. The fraction f of stray light with respect to the fluorescence spectral peak (A& is defined on the spectrum run with vertical polarizer and analyser transmission axes (IC(A,))
cence
C f=
I,, (A,) - c
c ZZG&4)
For the xylene samples under study, A,= 290 nm and the constant C was obtained at 450 nm, sufficiently far to the red to ensure that fluorescence is negligible in comparison. In Fig. 3, the fluorescence spectra of o-xylene with and without stray light are shown and it can be seen that at 450 nm, the fluorescence intensity is about zero in the second case, but the baseline is shifted by a quantity C in the first case, with larger slits. These experimental results support the method of simulation employed to generate the anisotropy spectrum of a generic chromophore. 3.2. Simulation method Fluorescence spectra was simulated as gaussian functions of an independent variable which represents the wavelength or spectral position (Fig. 4). A single gaussian N(258, aRLs), centred at 258 a.u., with a bandwidth at half-maximum a(5.2 a.u. in most simulations) is taken in general to represent Rayleigh light scattering J-(h). The full width at half-maximum (FWHM) of this band
4 750
270
290
310
330
350
Wavelength
370
(nm)
390
410
430
450
Fig. 4. Simulated emission spectrum with three components: fluorescence, Rayleigh light scattering and stray light with+0.01 (see text). The halfwidths of the gaussian functions representing fluorescence and Rayleigb light scattering are the standard values given in the text.
was increased in a few calculations to simulate the opening of the slits. The maximum intensity is normalized to unity. The agreement between the wavelength range of the experimental spectra (see Fig. 3) and the range of values of the independent variable (in a.u.) in the simulated spectra (see Fig. 4) was good. The units of the independent variable of the simulated spectra are given as nanometres in the discussion below. The sum of two gaussian functions IF(A) = N(290, 16) +N(300,24) has been used to generate a band similar to a fluorescence spectrum centred at 291 nm. One function would be enough, but in this way we can assign the same or different anisotropies to each component to investigate the resolution
196
A.P. Dorado et aL I Influence
of light scattering on ~uorescence animtrqy
of the anisotropy spectra with respect to the resolution of the fluorescence spectra [14]. The maximum value of IF(h) has also been normalized to unity. We have observed experimentally (see Fig. 1) that the stray light spectrum is featureless and approximately constant with wavelength and thus, to simulate this component of the spectrum, we have added a constant C to the gaussian functions representing fluorescence and/or Rayleigh light scattering. The intensity Z(A) of the final spectrum shown in Fig. 4 is thus I(A) =N(258, G-)
+N(290, 16)
+ N(300, 24) + C
(3) As already indicated, the component C represents a fraction f of the maximum fluorescence intensity
1 .cJo
0.80
f
0.m
-;
L
040
0.2D
I, oOO250
(5) Spectra in the two relative positions of the polarizers were generated and the apparent anisotropy r_(h) was calculated as a function of A
(6) Equation (6) was applied to different f values, different extents of overlap of Rayleigh light scattering and fluorescence and different values of r(A), the true fluorescence anisotropy, to simulate experimental conditions of greater interest. Raman scattering and fluorescence impurities were not taken into account. Figure 5 shows the calculated anisotropy spectra for several relative values of stray light and fluorescence for r=0.2, independent of wavelength. Without stray light cf= 0), the anisotropy spectrum is flat at r==0.2 in the fluorescence wavelength region and increases sharply to unity at wavelengths
290 Wavelength
I
310 (nm)
330
3 0
Fig. 5. Simulated anisotropy spectra r&AM) for different f values. Rayleigh light scattering is centred at 258 nm and the emission maximum at A=291 nm. The real anisotropy value was taken as 0.20. The halfwidths of the gaussian functions representing fluorescence and Rayleigh light scattering are the standard values given in the text.
f-C/1:(291)
(4) The spectrum generated by eqn. (3) is taken as that observed with vertically polarized excitation and observation (I,,). When the transmission axis of the polarier is vertical and that of the analyser horizontal (I,), no Rayleigh light scattering or stray light is observed in an ideal experiment since the anisotropy is unity (see Fig. 2 for measured values of Rayleigh light-scattering and stray light anisotropy spectra); in these conditions, the intensity of the fluorescence component (If) depends on the emission anisotropy r(A) in such a way that, given G = 1 for any wavelength
I
270
0.50
I
0.30
-
2 -*010 ,”
-0.10
-0.30
1 e-
ee
-
0 DD
I D.06
1 0 12 f
0
8
Fig. 6. Simulated anisotropy r&AM) at the fluorescence maximum (A,=291 nm) as a function of the fraction f of stray light, for several real anisotropy values r=0.4, 0.2, 0.0 and -0.2. The halfwidths of the gaussian functions representing fluorescence and Rayleigh light scattering are the standard values given in the text.
just below that at which the intensity of the Rayleigh light-scattering tail is about equal to the fluorescence intensity. With even small proportions of stray light the anisotropy shows a minimum centred at the position of the fluorescence maximum. The minimum may appear as a plateau which extends over a shorter range, the larger the contributions of stray light; r,,(A) determined for the plateau increases with respect to the true Y value with increasing stray light. The anisotropy r_(A,) determined at the fluorescence peak wavelength (A, = 291 nm) increases almost linearly with f for low f values, as shown in Fig. 6. The slope S, of the plots in Fig. 6 depends on thefrange taken to apply the linear regression due to curvature, but is at a maximum for r equal to about 0.25 and at a minimum for r- -0.2 (Fig.
197
A.P. Dorado et al. / Influence of light scatiering on fluorescence anisotmp 7). Changes in S, are, nevertheless, very small because the curvature of the plots in Fig. 6 is small for low f values; the scale of Fig. 7 is overexpanded. Table 1 shows how rq changes due to the effect of stray light for selected values of r and a low f value (0.01). The overlap of Rayleigh light scattering and fluorescence is often a greater problem than stray light, Figures 8(a) and 8(b) illustrate how the observed spectra and the anisotropy spectra are modified when the FWHM of the Rayleigh lightscattering band increases (referred to a constant maximum intensity). The influence of Rayleigh light scattering on the anisotropy spectra is qualitatively the same as that of stray light (decreasing the width of the anisotropy plateau and increasing its minimum value) but, in addition, Rayleigh light scattering shifts the minimum of the anisotropy spectrum to the red with respect to the fluorescence emission maximum. It is interesting to note that, even when the overlap of the fluorescence emission and the Rayleigb light-scattering bands is very extensive (Fig. 8(a)), the anisotropy spectrum resolves the fluorescence band much more clearly (Fig. 8(b)).
Wavelength
(4
1.o 0.8 z:,
0.6
r 0.4 0.2 o.oI
I
250 @)
040
c
0
I
0
O
I
0
x
I
270
1
I
I
290
310 (nm)
Wavelength
I
330
350
Fig. 8. Simulated fluorescence spectra (a) and anisotropy spectra (b) for different full width at half-maxima (FWHhQ of the Rayleigh light-scattering band. In all casesf=O.Ol and the maxima of the fluorescence and Rayleigb light scattering were normalized to unity. In (b), increasing FWHM shifts the minimum up and to the red side.
cooco f=O mm0 MAAA
(nm)
010 f=O.O48
0 50
f=O 09,
00000 f=O
160 0 40
020 1-p -0 30
-0.10
0.10 Anisotropy
030
0 50
Fig. 7. Slopes S, of the hear regression of simulated rev(&) f ranges) plotted as a function of the real anisotropy r.
‘; 5
030
j
VS. f (for different
TABLE 1. Anisotropy values r,(A,,,) of the effect of 1% stray light cf=O.Ol) pies r
obtained by simulation on several real anisotro-
r
r&M)
b
- 0.20 0.00 0.20 0.25 0.40
-0.1976 0.0033 0.2037 0.2537 0.4036
0.00240 0.00333 0.00373 0.00375~ 0.00360
~Maximum increment.
0 20
0 10
0 00
0.08
016
f
Fig. 9. Simulated anisotropyrq(hM) at the fluorescence maximum (Am=291 nm) as a function of the fraction f of stray light, for r=O.Z and several relative values of the FWHM of Rayleigh light scattering. Tbe lines corresponding to FWHM of 4, 12 and 20 overlap.
Figure 9 shows rSXP(hM) as a function of f for different overlap of Rayleigh light scattering and fluorescence, with r= 0.2 in all cases. The approximately linear relationship is conserved for
A.P. Lhrado et al. I InpUence
198
of lightscattering on @orescence- aniwtropy
f
intensity (291 nm) when the transmission axes of both the polarizer and the analyser are vertical. A linear regression of the minimum anisotropy r&AM) ~3. f yields, as the intercept, the real r value. To render the determination more reliable, a double extrapolation to a common intercept can be performed. We assume that two chromophores of real anisotropy rl and rZ have emission bands which overlap partially at the wavelength AMI of the
maximum
emission
of
chromophore 1. If at this wavelength, and fz is the ratio of the emission at AM1 corresponding to chromophore 2 to that corresponding to chromophore 1, when the transmission axes of both polarizer and analyser are vertically oriented, then
rexp(AMI)is the observed anisotropy
(7) It can be shown that
In the particular case considered light)
here, r,= 1 (stray
r = 3~cxP(b41)-fD + kxP&dl 1 3 -fi[l +~ex&dl Equation (9) can be used to obtain the real anisotropy r for each background f value, and the
plot of values given by eqn. (8) US. f, being independent, must give a horizontal line except for dispersion due to experimental errors. Figures 11 and 12 depict the simultaneous plot of rup(AM1) and rl w. f for o-xylene and m-xylene. They show a considerable dispersion of data due to the low emission intensity and to the difficulties inherent
0001,
,
270
280
I
290
300
Wavelength
310
320
4 3x
(nm)
Fig. 10. Experimental anisotropy spectra of m-xylene for different f values corresponding to different bandwidths: f=0.093 and f-0.189.
0.00
0.00
0 02
0.04
0.06
f
0.08
0.10
0 12
Fig. 11. Double extrapolation of experimental tap(&) (0) and calculated rl (0) vs. the fraction of stray light f for o-xylene.
A.P. Dorado et al. I Injluence of right scattering on flmrescence
-0.02 -0.04
anisotyy
199
I 0 00
’ 0 04
0.08
0 12
0.16
’ 0 20
0 24
f
Fig. 12. Double extrapolation of experimental r_+,(,&) (0) and calculated r, (a) ~8. the fraction of stray light f for m-xylene. TABLE 2. Experimental anisotropy values corrected for the influence of light scattering (rf_o(hM)) and slopes (S,) of the plot of measured values vs. f, the fraction of stray light Sample
+Ll(U
Sf
o-Xylene m-Xylene
0.029 f0.005 0.022 f 0.006
0.27 * 0.02 0.36*0&t
in front-face excitation experiments. To fit the data, a graphical method was employed: a horizontal line was drawn through the r, values and the line corresponding to rup(AM1) was forced to take the same intercept, which is the corrected anisotropy. The slopes of this last line for the two xylene isomers (S, in Table 2) are coincident with the calculated S, values shown in Fig. 7 and r is about the same for the two isomers (Table 2). There is an alternative method which can be used to correct the apparent anisotropy spectra for the artefacts caused by the spectral overlap of Rayleigh light scattering and stray light: the spectra obtained with a blank are subtracted for any position of the polarizer and analyser. However, this is not always possible because it is difficult to prepare a film of the same thickness and turbidity as the matrix of the sample. Even if it is possible, the light absorbed by the chromophore decreases the intensity of Rayleigh light scattering and stray light in the sample with respect to the blank, in such a way that the film without chromophore overestimates the adventitious light of the sample. Figure 13 compares the anisotropy spectra of a sample containing m-xylene determined with and without subtraction of the spectrum of a PMMA film prepared under the same conditions as the sample. The real anisotropy spectrum of the chromophore is wavelength independent, but the anisotropy spectrum corrected with the blank is not
Fig. 13. JZxperirnental anisotropy spectra of m-xylene in a PMhM film with (a) and without @) subtraction of the blank (a PMMA film without chromophore obtained under the same conditions as the sample).
and this indicates that the correction is not totally reliable. The correction method proposed here is much simpler since it requires much less experimental manipulation.
4. Conclusions The spectral overlap of light scattering and stray light with fluorescence modifies its anisotropy spectrum, increasing the anisotropy directly observed with respect to the true value. In view of the simulation results presented here, it is recommended that anisotropy measurements are taken over a range of wavelengths covering the fluorescence region to detect the presence of artefacts. Even if a plateau is observed, there is no guarantee that the minimum anisotropy is the true value unless f, the fraction of stray light (which can be measured on the red side of the spectrum), is low with respect to the maximum fluorescence intensity. For low proportions of spurious light, the observed anisotropy minimum increases approximately linearly with f- This makes it possible to use linear regression to calculate r from several measurements performed with different levels of light scattering controlled by opening the excitation and emission slits to different degrees. This method is convenient, using only one sample and requiring no additional manipulation. The correction is particularly relevant to frontface excitation measurements since, in the usual right-angle geometry, there are other mechanisms related to light scattering which give rise to depolarization, rather than spectral overlap.
200
A.P. Lbndo
et al. I Influence of light scattering on jIuomscence mubtrofl
Acknowledgment
This work was supported by Dirrecih General de Investigacih Cientifica y Tecnica (DGICYT) (Spain) under grant PB89/0198. 9
References 1 B.F. Dickens, T.R. Snow, Ce!I Biochem., 79 (1988) 2 H. Kutchai, V.H. Huxley (1982) 229. 3 B.R. Lena, B.M. Moore (1979) 489. 4 P.W.J. Teale, Photochem
V. Green and W.B. Wegiicki, Mol. 91. and L.H. Chandler, Bioghys. 1, 39 and D.A. Barrow,
Biophys. I., 25
Photobiol., 10 (1969) 363.
10 11 12 13 14 15
J.R. Lakowia, 13inciples of Ffuorescence Spectmscopy, Plenum, New York, 1983. J. Eisinger and J. Fiores, Biophys. J., 48 (1985) 77. M. Hennecke and J. Fuhrrnann, Makronwl. Chem. Mukromol. Symp., 5 (1986) 181. M. Hennecke, K. Kurz and J. Fuhtmann, MacromolecuIes, 2.5 (1992) 6190. J. Michl and E.W. Thuistrup, Specbosco~ wifft PolarizedLight, VCH, New York, 1986. J. Parreiio and LF. Pierola, Polymer, 31 (1990) 1768. J. Parrefio and I.F. Pidrola, PO+. Net.Blends, I (1991) 181. A.D. Stein, D.A. HolIman, C.W. Frank and M.D. Fayer,L Cfiem. phys., 96 (4) (1992) 3269. A. Watanabe and M. Matsuda, Macromolecules, 19 (1986) 2253. A.P. Dorado and I.F. Pitrola, to be published. LB. Berhnan, Handbook of Fluorescence Spectra of Aromatic Molecules, Academic Press, New York, 1979.
PhotobioL A: Chem,
78 (1994) 193-200
193
Influence of adventitious light scattering measurements with front-face excitation. Angel
P. Dorado,
Depatrat?~~~t~ de Qukca
Miguel
A. Llorente
Ftiica, F~dtod
and
In&
on fluorescence anisotropy Simulation and correction
F. Pierola
de Ciencias, Chiversid~d National de Educocih
A DiWmcia (UNED), 28040 Madrid
&ah)
(Received June, 9, 1993; accepted October 26, 1993)
Abstract Fluorescence anisotropy spectra of a generic chromophore have been simulated for different proportions of adventitious light scattering with respect to fluorescence. The spectral overlap of Rayleigh light scattering and stray light with fluorescence causes an increase in the apparent anisotropy with TCSpeCt to the real fluorescence anisotropy and curvature of the anisotropy spectra. In the presence of up to about 10% fluorescence contamination, the largest effect of these artefacts occurs for true anisotropies close to 0.25 and the least for anisotropies close to -0.2. The apparent anisotropy, determined at the fluorescence maximum or at the anisotropy minimum, increases almost linearly with f (the fraction of stray light with respect to the maximum fluorescence intensity) for lowfvalues cf< 15%). In the light of these results, a method is proposed to correct the influence of adventitious light scattering on anisotropy measurements. This method has been applied to determine the fluorescence anisotropy of o- and m-xylene in a polymeric matrix with front-face excitation.
1. Introduction Light scattering plays an important role in fluorescence anisotropy measurements. Biochemical samples (e.g. aqueous suspensions of membranes) are often turbid and this produces an artefact in fluorescence depolarization measurements in the usual right-angle cell configuration [l-6]. The polarized incident light can be scattered before absorption and the emitted can be scattered before detection. Both phenomena give rise to partial depolarization and decrease the observed anisotropy (r). These purely optical effects must be corrected if the rotational diffusion of the fluorescent molecule is to be quantified adequately. A concentration-based correction (better than a correction based on turbidity measurements) has been proposed [6] with the argument that a fluorometer’s photodetector generally has a different acceptance angle from an absorption spectrophotometer, a technique which is usually employed to perform turbidity measurements. It has also been suggested [l, 21 that intense light scattering may affect the concentration-dependent extrapolation method proposed by Teale [43 and it is thus recommended that the correction is applied to sufficiently dilute samples, whenever possible_ If this is not possible, light-scattering depolarization
lOlO-6030/94/$07.00 @ 1994 Elsevier Sequoia. All rights reserved SSDZ 1010-5030(93)03733-W
can be avoided by performing anisotropy measurements with front-face excitation instead of with the usual right-angle geometry. Front-face excitation is also a requirement for non-transparent solid samples [7, 81. This instrumental geometry is not free of light-scattering artefacts. The technical spectrum of any chromophore is formed by the spectral overlap of the long-wavelength part of Rayleigh light scattering and reflected light (centred at the excitation wavelength), the peak of Raman scattering, stray light and the fluorescence itself. The contribution to the observed spectrum of each of these terms depends predominantly on the fluorescence quantum yield of the chromophore and on its wncentration in the medium, but also on the experimental conditions which may become very important in front-face excitation experiments. Rayleigh scattering of polarized incident light observed in the plane~perpendicular to its electric vector, at 90” to the excitation direction, is totally polarized (r= l), whereas the fluorescence of a randomly (isotropically) oriented ensemble of fluorophores is partially depolarized ( - 0.2
194
A.P. Dorodo et al. / Infknce
of light scattering on fluorescence anisotropy
complicated and it can be experimentally avoided with chromophores of large Stokes shift or with smaller excitation wavelengths; in fact, in frontface excitation experiments the Rayleigh lightscattering band is broad, and often includes the much less intense Raman peak. In this paper we attempt to show the influence of the overlap of light scattering on the fluorescence anisotropy spectra in different situations and propose a method which can be used to obtain true anisotropy values from the apparent values determined experimentally. To check the validity of the correction method proposed here, we have chosen the worst possible experimental situation: chromophores of low fluorescence quantum yield and anisotropy close to zero. The chromophores (xylenes) were dissolved in a vitreous polymer matrix (polymethylmethacrylate) and their fluorescence anisotropies were measured by front-face excitation at room temperature. The fluorescence anisotropy technique has been found to be very useful in determining the microenvironmental properties of solid or swollen polymer samples [lO-131 but, in view of the simulation results presented here, caution should be exercised when both making and interpreting such measurements.
2. Experimental
details
The xylene isomers (o- and m-) were purchased from Fluka (GC Standard). The polymer matrix was made of polymethylmethacrylate (PMMA) of high molecular weight (8.5 x 10’ Da), synthesized by radical polymerization. Films about 100 pm thick were cast from a solution of the chromophore and the polymer in a good solvent of both (chloroform). The composition of the solid solution was about 5% w/w of xylene, a concentration low enough to avoid plastification but high enough to yield an observable fluorescence intensity [14]. The chromophore concentration in the solid sample was determined spectrophotometrically after fluorescence measurements by redissolving a known mass of the film in a known volume of tetrahydrofuran (THF); the extinction coefficients of xylenes in dilute solution were taken from ref. 15. Measurements were performed at room temperature by front-face excitation using a Hitachi F4000 spectrofluorometer provided with film polarizers. The excitation wavelength was 260 nm. The anisotropy r(h) was calculated at each wavelength h as
r(h) =
fll(4 -~~ww) I,,(4 + 21~ (AMA)
where I,, and I, represent the intensities measured when the transmission axis of the polarizer is oriented vertically and that of the analyser is parallel (vertical) or perpendicular (horizontal) respectively. G is a correction factor which takes into account the different efficiencies of registration of the vertically or horizontally polarized emission; it is calculated for each wavelength as the ratio of the intensities when the transmission axis of the polarizer is horizontal and that of the analyser is vertical or horizontal. 3. Results
and discussion
3.1. Fluorescence measurements The fluorescence spectra of xylene isomers in a PMMA matrix were run with front-face excitation at room temperature. Different ratios of fluorescence and spurious light were obtained on changing the slit widths in both the incident and emergent beams. Spectra run with blanks made of PMMA film without chromophore (see Fig. 1) or with scatter show that the stray light intensities are fairly constant (C) with wavelength and that they increase with increasing excitation and emission bandpass. The anisotropy of both Rayleigh light scattering and stray light is in all cases about unity. A typical anisotropy spectrum (Fig. 2) shows a minimum in the spectral region corresponding to the maximum fluorescence intensity and at both sides it reaches a value close to unity. The minimum anisotropy corresponds to the apparent fluores-
0 260
I
280
300
I.
320
Wavelength
,__r__r340
360
------A 380
400
(nm)
Fig. 1. Experimental spectra of a PMMA film without chromophore, measured with several bandwidths, under the same conditions as the samples. Excitation and emission band passes: (-.-.), S/5 nm; (-), 10/5 nm; and (-.-) S/10 nm.
A.P. Donado et al. / In@ence of lightscattehg on fluorescenceaniFotropy
336
Wovelength
Fig. 2. Experimental anisotropy spectrum of m-xylem! in a PMMA matrix with front-face excitation, showing the two limiting values of the anisotropy at the excitation wavelength and on the red side of the spectrum.
195
374
(nm)
Fig. 3. Experimental emission spectra of o-xylene in a Ph4MA film run with three different bandpasses, which give rise to different levels of stray light. Excitation and emission band passes in nm: (-), 5/S; (. . . . ), 10/5; and (-.-) 5/l&
anisotropy and the anisotropy values corresponding to the excitation wavelength region and the red side of the spectrum can be ascribed to Rayleigh light scattering and stray light respectively. The fraction f of stray light with respect to the fluorescence spectral peak (A& is defined on the spectrum run with vertical polarizer and analyser transmission axes (IC(A,))
cence
C f=
I,, (A,) - c
c ZZG&4)
For the xylene samples under study, A,= 290 nm and the constant C was obtained at 450 nm, sufficiently far to the red to ensure that fluorescence is negligible in comparison. In Fig. 3, the fluorescence spectra of o-xylene with and without stray light are shown and it can be seen that at 450 nm, the fluorescence intensity is about zero in the second case, but the baseline is shifted by a quantity C in the first case, with larger slits. These experimental results support the method of simulation employed to generate the anisotropy spectrum of a generic chromophore. 3.2. Simulation method Fluorescence spectra was simulated as gaussian functions of an independent variable which represents the wavelength or spectral position (Fig. 4). A single gaussian N(258, aRLs), centred at 258 a.u., with a bandwidth at half-maximum a(5.2 a.u. in most simulations) is taken in general to represent Rayleigh light scattering J-(h). The full width at half-maximum (FWHM) of this band
4 750
270
290
310
330
350
Wavelength
370
(nm)
390
410
430
450
Fig. 4. Simulated emission spectrum with three components: fluorescence, Rayleigh light scattering and stray light with+0.01 (see text). The halfwidths of the gaussian functions representing fluorescence and Rayleigb light scattering are the standard values given in the text.
was increased in a few calculations to simulate the opening of the slits. The maximum intensity is normalized to unity. The agreement between the wavelength range of the experimental spectra (see Fig. 3) and the range of values of the independent variable (in a.u.) in the simulated spectra (see Fig. 4) was good. The units of the independent variable of the simulated spectra are given as nanometres in the discussion below. The sum of two gaussian functions IF(A) = N(290, 16) +N(300,24) has been used to generate a band similar to a fluorescence spectrum centred at 291 nm. One function would be enough, but in this way we can assign the same or different anisotropies to each component to investigate the resolution
196
A.P. Dorado et aL I Influence
of light scattering on ~uorescence animtrqy
of the anisotropy spectra with respect to the resolution of the fluorescence spectra [14]. The maximum value of IF(h) has also been normalized to unity. We have observed experimentally (see Fig. 1) that the stray light spectrum is featureless and approximately constant with wavelength and thus, to simulate this component of the spectrum, we have added a constant C to the gaussian functions representing fluorescence and/or Rayleigh light scattering. The intensity Z(A) of the final spectrum shown in Fig. 4 is thus I(A) =N(258, G-)
+N(290, 16)
+ N(300, 24) + C
(3) As already indicated, the component C represents a fraction f of the maximum fluorescence intensity
1 .cJo
0.80
f
0.m
-;
L
040
0.2D
I, oOO250
(5) Spectra in the two relative positions of the polarizers were generated and the apparent anisotropy r_(h) was calculated as a function of A
(6) Equation (6) was applied to different f values, different extents of overlap of Rayleigh light scattering and fluorescence and different values of r(A), the true fluorescence anisotropy, to simulate experimental conditions of greater interest. Raman scattering and fluorescence impurities were not taken into account. Figure 5 shows the calculated anisotropy spectra for several relative values of stray light and fluorescence for r=0.2, independent of wavelength. Without stray light cf= 0), the anisotropy spectrum is flat at r==0.2 in the fluorescence wavelength region and increases sharply to unity at wavelengths
290 Wavelength
I
310 (nm)
330
3 0
Fig. 5. Simulated anisotropy spectra r&AM) for different f values. Rayleigh light scattering is centred at 258 nm and the emission maximum at A=291 nm. The real anisotropy value was taken as 0.20. The halfwidths of the gaussian functions representing fluorescence and Rayleigh light scattering are the standard values given in the text.
f-C/1:(291)
(4) The spectrum generated by eqn. (3) is taken as that observed with vertically polarized excitation and observation (I,,). When the transmission axis of the polarier is vertical and that of the analyser horizontal (I,), no Rayleigh light scattering or stray light is observed in an ideal experiment since the anisotropy is unity (see Fig. 2 for measured values of Rayleigh light-scattering and stray light anisotropy spectra); in these conditions, the intensity of the fluorescence component (If) depends on the emission anisotropy r(A) in such a way that, given G = 1 for any wavelength
I
270
0.50
I
0.30
-
2 -*010 ,”
-0.10
-0.30
1 e-
ee
-
0 DD
I D.06
1 0 12 f
0
8
Fig. 6. Simulated anisotropy r&AM) at the fluorescence maximum (A,=291 nm) as a function of the fraction f of stray light, for several real anisotropy values r=0.4, 0.2, 0.0 and -0.2. The halfwidths of the gaussian functions representing fluorescence and Rayleigh light scattering are the standard values given in the text.
just below that at which the intensity of the Rayleigh light-scattering tail is about equal to the fluorescence intensity. With even small proportions of stray light the anisotropy shows a minimum centred at the position of the fluorescence maximum. The minimum may appear as a plateau which extends over a shorter range, the larger the contributions of stray light; r,,(A) determined for the plateau increases with respect to the true Y value with increasing stray light. The anisotropy r_(A,) determined at the fluorescence peak wavelength (A, = 291 nm) increases almost linearly with f for low f values, as shown in Fig. 6. The slope S, of the plots in Fig. 6 depends on thefrange taken to apply the linear regression due to curvature, but is at a maximum for r equal to about 0.25 and at a minimum for r- -0.2 (Fig.
197
A.P. Dorado et al. / Influence of light scatiering on fluorescence anisotmp 7). Changes in S, are, nevertheless, very small because the curvature of the plots in Fig. 6 is small for low f values; the scale of Fig. 7 is overexpanded. Table 1 shows how rq changes due to the effect of stray light for selected values of r and a low f value (0.01). The overlap of Rayleigh light scattering and fluorescence is often a greater problem than stray light, Figures 8(a) and 8(b) illustrate how the observed spectra and the anisotropy spectra are modified when the FWHM of the Rayleigh lightscattering band increases (referred to a constant maximum intensity). The influence of Rayleigh light scattering on the anisotropy spectra is qualitatively the same as that of stray light (decreasing the width of the anisotropy plateau and increasing its minimum value) but, in addition, Rayleigh light scattering shifts the minimum of the anisotropy spectrum to the red with respect to the fluorescence emission maximum. It is interesting to note that, even when the overlap of the fluorescence emission and the Rayleigb light-scattering bands is very extensive (Fig. 8(a)), the anisotropy spectrum resolves the fluorescence band much more clearly (Fig. 8(b)).
Wavelength
(4
1.o 0.8 z:,
0.6
r 0.4 0.2 o.oI
I
250 @)
040
c
0
I
0
O
I
0
x
I
270
1
I
I
290
310 (nm)
Wavelength
I
330
350
Fig. 8. Simulated fluorescence spectra (a) and anisotropy spectra (b) for different full width at half-maxima (FWHhQ of the Rayleigh light-scattering band. In all casesf=O.Ol and the maxima of the fluorescence and Rayleigb light scattering were normalized to unity. In (b), increasing FWHM shifts the minimum up and to the red side.
cooco f=O mm0 MAAA
(nm)
010 f=O.O48
0 50
f=O 09,
00000 f=O
160 0 40
020 1-p -0 30
-0.10
0.10 Anisotropy
030
0 50
Fig. 7. Slopes S, of the hear regression of simulated rev(&) f ranges) plotted as a function of the real anisotropy r.
‘; 5
030
j
VS. f (for different
TABLE 1. Anisotropy values r,(A,,,) of the effect of 1% stray light cf=O.Ol) pies r
obtained by simulation on several real anisotro-
r
r&M)
b
- 0.20 0.00 0.20 0.25 0.40
-0.1976 0.0033 0.2037 0.2537 0.4036
0.00240 0.00333 0.00373 0.00375~ 0.00360
~Maximum increment.
0 20
0 10
0 00
0.08
016
f
Fig. 9. Simulated anisotropyrq(hM) at the fluorescence maximum (Am=291 nm) as a function of the fraction f of stray light, for r=O.Z and several relative values of the FWHM of Rayleigh light scattering. Tbe lines corresponding to FWHM of 4, 12 and 20 overlap.
Figure 9 shows rSXP(hM) as a function of f for different overlap of Rayleigh light scattering and fluorescence, with r= 0.2 in all cases. The approximately linear relationship is conserved for
A.P. Lhrado et al. I InpUence
198
of lightscattering on @orescence- aniwtropy
f
intensity (291 nm) when the transmission axes of both the polarizer and the analyser are vertical. A linear regression of the minimum anisotropy r&AM) ~3. f yields, as the intercept, the real r value. To render the determination more reliable, a double extrapolation to a common intercept can be performed. We assume that two chromophores of real anisotropy rl and rZ have emission bands which overlap partially at the wavelength AMI of the
maximum
emission
of
chromophore 1. If at this wavelength, and fz is the ratio of the emission at AM1 corresponding to chromophore 2 to that corresponding to chromophore 1, when the transmission axes of both polarizer and analyser are vertically oriented, then
rexp(AMI)is the observed anisotropy
(7) It can be shown that
In the particular case considered light)
here, r,= 1 (stray
r = 3~cxP(b41)-fD + kxP&dl 1 3 -fi[l +~ex&dl Equation (9) can be used to obtain the real anisotropy r for each background f value, and the
plot of values given by eqn. (8) US. f, being independent, must give a horizontal line except for dispersion due to experimental errors. Figures 11 and 12 depict the simultaneous plot of rup(AM1) and rl w. f for o-xylene and m-xylene. They show a considerable dispersion of data due to the low emission intensity and to the difficulties inherent
0001,
,
270
280
I
290
300
Wavelength
310
320
4 3x
(nm)
Fig. 10. Experimental anisotropy spectra of m-xylene for different f values corresponding to different bandwidths: f=0.093 and f-0.189.
0.00
0.00
0 02
0.04
0.06
f
0.08
0.10
0 12
Fig. 11. Double extrapolation of experimental tap(&) (0) and calculated rl (0) vs. the fraction of stray light f for o-xylene.
A.P. Dorado et al. I Injluence of right scattering on flmrescence
-0.02 -0.04
anisotyy
199
I 0 00
’ 0 04
0.08
0 12
0.16
’ 0 20
0 24
f
Fig. 12. Double extrapolation of experimental r_+,(,&) (0) and calculated r, (a) ~8. the fraction of stray light f for m-xylene. TABLE 2. Experimental anisotropy values corrected for the influence of light scattering (rf_o(hM)) and slopes (S,) of the plot of measured values vs. f, the fraction of stray light Sample
+Ll(U
Sf
o-Xylene m-Xylene
0.029 f0.005 0.022 f 0.006
0.27 * 0.02 0.36*0&t
in front-face excitation experiments. To fit the data, a graphical method was employed: a horizontal line was drawn through the r, values and the line corresponding to rup(AM1) was forced to take the same intercept, which is the corrected anisotropy. The slopes of this last line for the two xylene isomers (S, in Table 2) are coincident with the calculated S, values shown in Fig. 7 and r is about the same for the two isomers (Table 2). There is an alternative method which can be used to correct the apparent anisotropy spectra for the artefacts caused by the spectral overlap of Rayleigh light scattering and stray light: the spectra obtained with a blank are subtracted for any position of the polarizer and analyser. However, this is not always possible because it is difficult to prepare a film of the same thickness and turbidity as the matrix of the sample. Even if it is possible, the light absorbed by the chromophore decreases the intensity of Rayleigh light scattering and stray light in the sample with respect to the blank, in such a way that the film without chromophore overestimates the adventitious light of the sample. Figure 13 compares the anisotropy spectra of a sample containing m-xylene determined with and without subtraction of the spectrum of a PMMA film prepared under the same conditions as the sample. The real anisotropy spectrum of the chromophore is wavelength independent, but the anisotropy spectrum corrected with the blank is not
Fig. 13. JZxperirnental anisotropy spectra of m-xylene in a PMhM film with (a) and without @) subtraction of the blank (a PMMA film without chromophore obtained under the same conditions as the sample).
and this indicates that the correction is not totally reliable. The correction method proposed here is much simpler since it requires much less experimental manipulation.
4. Conclusions The spectral overlap of light scattering and stray light with fluorescence modifies its anisotropy spectrum, increasing the anisotropy directly observed with respect to the true value. In view of the simulation results presented here, it is recommended that anisotropy measurements are taken over a range of wavelengths covering the fluorescence region to detect the presence of artefacts. Even if a plateau is observed, there is no guarantee that the minimum anisotropy is the true value unless f, the fraction of stray light (which can be measured on the red side of the spectrum), is low with respect to the maximum fluorescence intensity. For low proportions of spurious light, the observed anisotropy minimum increases approximately linearly with f- This makes it possible to use linear regression to calculate r from several measurements performed with different levels of light scattering controlled by opening the excitation and emission slits to different degrees. This method is convenient, using only one sample and requiring no additional manipulation. The correction is particularly relevant to frontface excitation measurements since, in the usual right-angle geometry, there are other mechanisms related to light scattering which give rise to depolarization, rather than spectral overlap.
200
A.P. Lbndo
et al. I Influence of light scattering on jIuomscence mubtrofl
Acknowledgment
This work was supported by Dirrecih General de Investigacih Cientifica y Tecnica (DGICYT) (Spain) under grant PB89/0198. 9
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