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A Modified Phase-Modulation Spectrofluorometer for Lifetime-Resolved Fluorescence-Detected Circular Dichroism

K A R E N W U and L I N D A B. M c G O W N * Department of Chemistry, P. M. Gross Chemical Laboratory, Duke University, Durham, North Carolina 27706

The first demonstration of lifetime resolution in fluorescence-detected circular dichroism (FDCD) is described, performed on a modified phase- modulat ion spectrofluorometer in which a Babinet-Soleil compensator is used to generate the circularly polarized excitation. Measurements of optically inactive solutions indicate the absence of significant instru- mental or optical art ifacts for steady-state and lifetime-resolved FDCD. Advantages of the instrumental approach are discussed and early results are presented for a system of benzo(a)pyrene and benzo(k)fluoranthene in 'y-cyclodextrin, demonstrating the accuracy of the lifetime determi- nations with circularly polarized excitation as well as the applicability of lifetime-resolved FDCD for studies of induced chirality in asymmetric microenvironments.

Index Headings: Fluorescence-detected circular dichroism; FDCD; Flu- orescence lifetime detection; Polarizat ion techniques; Spectroscopic tech- niques.

INTRODUCTION

Fluorescence-detected circular dichroism (FDCD) 1-s is used to study the chirality of fluorescent molecules. The chirality may be an intrinsic property of the molecule, or it may be induced or modified by association of the molecule with a chiral microenvironment in a macro- molecule or molecular assembly. Fluorescence detection provides greater selectivity and sensitivity than do ab- sorption circular dichroism (CD) techniques, making it particularly attractive for molecular probe studies as well as chemical analysis? Unfortunately, both CD and FDCD only provide information about the average chirality of a system, which may contain several different contri- butions to the total difference observed in the CD or FDCD spectrum.

I N S T R U M E N T DESCRIPTION

We describe here a modified frequency-domain fluo- rescence lifetime instrument for lifetime-resolved FDCD measurements, which will allow us to resolve the indi- vidual contributions to the emission signals resulting from right-handed and left-handed circularly polarized exci- tation. Fluorescence lifetimes are calculated from the phase-shift and demodulation 5 of the emission signals

Received 11 Sep tember 1990. * Au tho r to whom correspondence should be sent .

produced by right-handed and left-handed circularly po- larized excitation that is intensity modulated. Hetero- geneity analysis of multifrequency lifetime data 6 pro- vides the lifetime distributions and fractional intensity contributions of individual chiral and achiral compo- nents to the total FDCD signal. This is the first dem- onstration of dynamic lifetime measurements in FDCD.

We recently completed the modification of a frequen- cy-domain fluorescence lifetime spectrofluorometer for FDCDY The basic instrument is a commercially available frequency-domain fluorescence lifetime apparatus (SLM 48000S, SLM Instruments, Inc.). In the FDCD-modified instrument, a xenon arc lamp is used to produce the excitation beam, which is passed through a monochro- mator for wavelength selection; a Pockels cell to create the intensity-modulated component of the excitation beam for the frequency-domain lifetime measurements; and a linear polarizer to select horizontally or vertically polarized light. A Babinet-Soleil compensator (Karl Lambrecht Corp.) is used in combination with the linear polarizer to produce right-handed or left-handed circu- larly polarized light. The circularly polarized excitation beam is then directed into the sample cell, and the re- sulting emission is passed through an optional linear polarizer for magic angle correction of photoselection, an emission wavelength selector (monochromator or inter- ference filter), a P M T detector, system electronics, and an on-line IBM PC-AT microcomputer for data acqui- sition and analysis. Prior to entering the sample cell, a portion of the circularly polarized excitation beam is diverted to a reference P M T to obtain ratiometric mea- surements and to provide a"s top" reference for the phase measurements.

Our instrument is the first to use a Babinet-Soleil com- pensator (BSC) for FDCD. We chose the BSC over other circular polarizers s for several reasons. Dynamic devices were ruled out because we are already using a dynamic device (a Pockels cell) for the frequency-domain lifetime measurements. A dynamic device for FDCD would mul- tiply the intensity modulation function by an oscillating circular polarization function, which would unnecessarily complicate our experiment by combining two dynamic, t ime-dependent functions in the single excitation beam. In fact, as stated above, the main purpose of dynamic

Volume 45, Number 1, 1 9 9 1 0003-7028/91/4501-000152.00/0 APPLIED SPECTROSCOPY 1 © 1991 Society for Applied Spectroscopy

0.200

.b -~ 0.150 c

2 .E

0 .100

0.050, o

o~ 0.000.

-0.050 350 460 4so 500

Wavelength (nm)

FIG. 1. Steady-state FDCD emission spectrum of (1S)-(+)-10-cam- phorsulfonic acid (CSA), obtained on the modified instrument.

devices in CD and FDCD is to facilitate spectral scan- ning. Since the purpose of our experiments is to acquire fluorescence lifetime data, which are measured under single wavelength conditions, dynamic devices offer no benefits and would only serve to increase the complexity of our measurements.

Static devices include quarter-wave plates and ach- romatic Fresnel rhombs, as well as the BSC. These are the devices of choice for single wavelength measurements because they are simpler to align and are generally more free of optical artifacts than are dynamic devices. All of the static devices have high throughput, although they must be coupled to linear polarizers if the incident beam is not inherently polarized. The rhomb has the disad- vantage of physically displacing the emergent beam rel- ative to the incident beam. Quarter-wave plates have the disadvantage of wavelength specificity, so that a different plate is required for each excitation wavelength. The BSC is a variable-wave plate that operates similarly to a quarter-wave plate but can be adjusted to produce circularly polarized light over a wide range of wave- lengths. In fact, a single BSC covers the entire UV-visible region of our instrument.

RESULTS AND DISCUSSION

In order to establish that the FDCD signals obtained on our instrument are real, and not the result of optical or other instrumental artifacts, several optically inactive solutions of achiral fluorophores were measured. Aque- ous solutions of sodium fluorescein (NaF) and of pyrene yielded negligible FDCD signals, indicating the absence of significant artifacts in our measurements. An FDCD emission spectrum of (1S)-(+)-10-camphorsulfonic acid (CSA)--a commonly used chiral reference compound-- obtained on the FDCD-modified instrument is shown in Fig. 1. Note that the emission spectrum is collected at a fixed excitation wavelength and therefore requires only a single setting for the BSC. The spectrum in Fig. 1 agrees with published spectra.

In order to evaluate the feasibility of measuring flu- orescence lifetimes on the FDCD-modified frequency- domain instrument, we used a solution of NaF in water. Results obtained from heterogeneity analysis of the mul- tifrequency lifetime data are shown in Table I. Clearly, as expected for the optically inactive solution, there is

TABLE I. Fluorescence lifetimes ('r A and ~ , in nanoseconds) and frac- tional intensity contributions (fA andfB) of components A and B, obtained from two-component heterogeneity analysis of multifrequency lifetime data.

Excitation polarization

Unpolarized Left-handed Right-handed

Fluorescein (1.5 #M) in water TA(YA ) 3.72 (1.00) 3.74 (1.00) 3.70 (1.00) ~-~(f~) . . . . . . . . . .

BaP (10 ttM) in ~CDx (15 mM) rA(/A) 36 (1.00) 35 (1.00) ~-~(f.) . . . . . . .

BkF (10 #M) in ~CDx (15 mM) I"A(fA ) 9.7 (i.00) 9.7 (1.00) ~.~(f~) . . . . . . .

BaP (10 tiM) and BkF (10 ttM) in ~CDx (15 mM) b rA(/A) 35 (0.78) 35 (0.74) rB(/B) 9.7 (0.22) 9.7 (0.26)

Only one component found. h Lifetimes fixed in heterogeneity analysis to values obtained for so-

lutions of individual components.

no significant difference between the lifetimes obtained with unpolarized excitation, right-handed circularly po- larized excitation, and left-handed circularly polarized excitation. The close agreement between the results for right-handed and left-handed circularly polarized exci- tation confirms the accuracy of the lifetime measure- ments with circularly polarized excitation.

Further studies of lifetime-resolved FDCD focused on solut ions of benzo(a)pyrene (BaP) and ben- zo(k)fluoranthene (BkF) in ~-cyclodextrin (~CDx). A formation complex of 6.5 × 104 has been reported for the BaP-~CDx complex, 9 which should produce a sig- nificant induced chirality in the BaP. We obtained a positive signal of 0.014 _+ 0.004 for the induced FDCD intensity (calculated as 2(IL - I,)/(IL + IR), where IL and IR are the emission intensities for left-handed and right-handed excitation, respectively1°), which is rela- tively large for induced chirality. A value of -0.019 _+ 0.003 was calculated for the FDCD of BkF in ~-CDx.

As shown in Table I, the individual solutions of BaP and BkF show homogeneous lifetimes for each of the components. These lifetimes are in excellent agreement with reported lifetimes of 36 ns and 9 ns for BaP and BkF, respectively, in deoxygenated solution. 11 Since none of the solutions in our experiments were deoxygenated, these lifetime values indicate inclusion of the solubilized compounds in the cyclodextrin cavity. For the solutions of the individual components, the same lifetime values are obtained with right-handed and left-handed exci- tation despite the induced chirality. This is because a single, homogeneous component is contributing 100 % of the fractional intensity to both emission signals.

In the mixture of BaP and BkF in ~CDx, the fractional intensity contribution of BaP is slightly greater with left- handed excitation than with right-handed excitation, which is in agreement with the positive FDCD for BaP and negative FDCD for BkF that we observed. Thus, for this heterogeneous system, we see the dependence of lifetime distribution on the orientation of the circularly polarized excitation beam.

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CONCLUSION

The simplicity of our frequency-domain approach is accomplished without sacrificing the quality of the life- time or CD information; in fact, the Babinet-Soleil com- pensator produces a high-quality circularly polarized ex- citation beam that is easy to align and is demonstrably free of optical artifacts. The use of frequency-domain lifetime instrumentation greatly facilitates the imple- mentation of lifetime-resolved FDCD relative to a time- domain approach, which would require simultaneous pulsing and circular polarization of the excitation beam. Simultaneous correction for anisotropy effects in the combined lifetime and FDCD measurement is easily ac- complished by setting the emission polarizer to the "mag- ic angle. ''~,1° Alternatively, the emission polarizer can be set to vertical and horizontal orientations instead of to the magic angle, in order to exploit photoselection effects for the study of rotational dynamics. 12-14 Early results indicate the accuracy and absence of instrumental arti- facts for both steady-state and lifetime-resolved FDCD measurements, as well as applicability to molecular probe studies of asymmetric microenvironments.

ACKNOWLEDGMENTS

This work was supported by the United States Department of Energy through the Office of Basic Energy Sciences (Grant Number DE-FG05- 88ER13931).

1. D. H. Turner, I. Tinoco, Jr., and M. Maestre, J. Am. Chem. Soc. 96, 4340 (1974).

2. I. Tinoco, Jr., and D. H. Turner, J. Am. Chem. Soc. 98, 6453 (1976). 3. D. H. Turner, in Methods in Enzymology, C. H. W. Hirs and S.

N. Timasheff, Eds. (Academic Press, New York, 1978), Vol. 49G, Chap. 8.

4. M. Thomas, G. Patonay, and I. Warner, Rev. Sci. Instrum. 57, 1308 (1986).

5. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum Press, New York, 1983).

6. D. M. Jameson, E. Gratton, and R. D. Hall, Appl. Spectrosc. Rev. 20, 55 (1984).

7. C. K. Williamson, K. Wu, and L. B. McGown, unpublished results. 8. D. S. Kliger, J. W. Lewis, and C. E. Randall, Polarized Light in

Optics and Spectroscopy (Academic Press, San Diego, 1990), Chap. 3.

9. L. A. Blyshak, I. M. Warner, and G. Patonay, Anal. Chim. Acta 232, 239 (1990).

10. B. Ehrenberg and I. Z. Steinberg, J. Am. Chem. Soc. 98, 1293 (1976).

11. Y. Kawabata, T. Imasaka, and N. Ishibashi, Anal. Chim. Acta 173, 367 (1985).

12. I. Tinoco, Jr., B. Ehrenberg, and I. Z. Steinberg, J. Chem. Phys. 66, 916 (1977).

13. E. W. Lobenstine and D. H. Turner, J. Am. Chem. Soc. 101, 2207 (1979).

14. G. Weber, in Fluorescence and Phosphorescence Analysis, D. M. Hercules, Ed. (John Wiley and Sons, New York, 1966), pp. 217- 240.

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