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Dual time scale measurements of the multiexponential fluorescence decay of quinacrine mustard
Volume 37, number 1
OPTICS COMMUNICATIONS
1 April 1981
DUAL TIME SCALE MEASUREMENTS OF THE MULTIEXPONENTIAL FLUORESCENCE DECAY OF QUINACRINE M U S T A R D S. De SILVESTRI, F. DOCCHIO, P. LAPORTA, A. LONGONI and F. ZARAGA Centro di Studio per l'Elettronica Quantistica e la Strumentazione Elettronica del CNR, Istituto di Fisica del Politecnico, Milano, Italy
Received 12 November 1980
The fluorescence decay of Quinacrine Mustard has been measured with good accuracy by means of a dual time scale digitizer-averag©r. Least-squares analysis of the experimental data with appropriate statistical weights has been performed. Values of the time constants for three exponential components have been obtained.
1. Introduction
The excited state dynamics of a great number of aromatic molecules involves complex reactions, such as photon transfer [ 1 - 2 ] , exciplex or excimer formation [3], conformational changes [4], etc. These excited state reactions are usually faster than the fluorescence lifetime of the aromatic molecules and can produce new excited species, which are different from the ground state molecules. When these new species are fluorescent, their formation kinetics in the excited state gives rise to a multiexponential fluorescent emission. A three-exponential fluorescence decay has recently been observed for an acridine dye, Quinacrine Mustard (QM) in this laboratory [5]. The acridine molecules undergo a marked change in the properties of their basic and acid groups, upon excitation [1,6]. The three-exponential decay measured for the QM molecules hasbeen explained in terms of a multiproton transfer in the excited state. The considerable difference in the values of the time constants of the decay of the QM fluorescence [5] demanded a choice of an observation time such that it would be possible to measure with good accuracy the fast initial fluorescence transient, and to follow the tail of the decay for at least one time constant of the slowest exponential component. This procedure, however, does not enable the slowest time constant to be determined 20
with precision, or the risetime of the signal to be measured. The purpose of this paper is to present a measurement of the multiexponential fluorescence decay of QM, using a dual time scale signal digitizer-averager.
2. Experimental The experimental setup, as regards excitation and detection of the fluorescence, is similar to that previously described in ref. [5]. The excitation light at 430 nm, produced by a nitrogen-pumped dye laser with pulses of 150 ps duration, was sent through a cell containing a solution of 3 X 10 -6 M of QM in acetate buffer (pH = 4.6). The fluorescence signal was detected by a Varian 154 M photomultiplier (400 ps FWHM) placed at the exit slit of a monochromator. The photomultiplier output was then sent to a microprocessorcontrolled digitizer-averager [7]. The signal was sequentially sampled by a sampling oscilloscope (Tektronix 7904, sampling head $4) driven by the microprocessor unit. The vertical output of the oscilloscope was digitized, stored in the RAM memory, and averaged over many sampling cycles. The averager has a programmable dual time scale that makes it possible to measure the slowest decay for a long enough time while maintaining a high temporal resolution for the initial fast decay. This feature is obtained by increasing
0 030-4018/81/0000--0000/$ 02.50 © North-Holland Publishing Company
Volume 37, number 1
OPTICS COMMUNICATIONS
1 April 1981
the sample number per unit time in the region of the fast transient. tu
A ~ A A A
3. Results and discussion
A
r dP
A typical QM fluorescence decay observed at 470 nm is shown by the dots in fig. 1. The fluorescence signal was averaged over 100 scan cycles. Samples are taken every 150 ps in the initial part ( 6 - 2 4 ns) of the curve and every 1.2 ns in the slow tail. An expanded view of the rise of this fluorescence curve is shown in fig. 2. The rise-time turns out to be of the order of 500 ps. The fluorescence decay was analyzed by using the non-linear least-squares method. This method requires the knowledge of the standard deviations o i of the experimental points Yi in order to weight appropriately the squares of the residuals. Measurements [8] of the statistical distribution of the noise and fluctuations found in the sampling technique have provided the following relationship o 2 = K y i which indicates a predominant Poisson statistics. The constant K depends 35e98 r-'l
38898 u
>.
25888
*" Q
29809
H 41 O C 0 0 v g
t~eela
loe88
L.
0 5~e
.,...' 25 O -25 8
Ie
2e
38
48
58
~
78
r'~l;
Fig. 1. Experimental (dots) and computed (unbroken line) fluorescence decay curves of QM observed at 470 nm. Below is the plot of the weighted residuals from the difference in the computed and experimental curves.
8e
c
1-t
e
A
N
E
4e
A
L
0 29
A A
0
A
A,
A
I
ps
Fig. 2. Enlarged-scaleview of the rise of the QM fluorescence curve observed at 470 rim. on parameters of the experimental apparatus such as the gain of the photomultiplier, the vertical sensitivity (mV/div) of the sampling oscilloscope, and the analogto-digital conversion factor. A least-squares analysis of the fluorescence decay curve (see fig. 1) shows a three-exponential decay with the constants r I = 0.76, 1"2 = 2.91, r 3 = 17.94 and corresponding initial amplitudes A 1 = A 2 = 48%, A 3 = 4%. The interpolating curve calculated with the above parameters is shown in the unbroken line in fig. 1. The goodness of the fit has been judged by inspection of the autocorrelation function of the residuals. The weighted residuals shown in fig. 1 are equally distributed around zero, indicating the good quality of the fit. The autocorrelation function of the residuals, relating to the fast portion (6--24 ns, see fig. 1) of the fluorescence decay, shown in fig. 3a, confirms the three-exponential analysis. For comparison, fig. 3b shows the autocorrelation function of the residuals of the same portion that would be obtained assuming only two exponential components, indicating the poor quality of the fit in this case. The results previously reported in ref. ['5] for the fluorescence decay of QM showed a decay time for the slow tail (r2) of ~ 16.4 ns. The present measurement gives a value o f t 3 ~ 18 ns, which is about 10% longer. This value appears to be more reliable because the dual time scale enables the slowest exponential 21
Volume 37, number 1
(o)
OPTICS COMMUNICATIONS
e
-I
0
g
I@
i,-I<: I
(b)
@
-I
\ @
Fig. 3. Autocorrelation functions of the residuals relating to the fast portion (6-24 ns) of the fluorescence decay: (a) three-component analysis, Co) two-component analysis. decay to be followed for at least three time constants. In the previous measurement, this decay was followed for about one time constant. This difference in the value o f r 3 does not appreciably affect the value o f r 1 or r 2 reported in ref. [5], due to the small amplitude A 3 .
22
1 April 1981
In conclusion, the measurement of the fluorescence decay of QM by means of dual-time scale signal digitizer-averager made possible a better determination o f the time constants. The fast rise-time o f the signal was also measured. A three-exponential component decay was again found, which confirms the previous interpretation of the experimental results in terms of an excited-state proton transfer.
References
[1] S.G. Schulman, in: Modern fluorescence spectroscopy, Vol. 2, ed. E.L. Wehry (Plenum Press, New York and London, 1976) p. 239. [2] W.R. Laws and L. Brand, L Phys. Chem. 83 (1979) 795. [3] P. Froehlich and E.L. Wehry, in: Modern fluorescence spectroscopy, Vol. 2, ed. E.L. Wehry (Plenum Press, New York and London, 1976) p. 319. [4] T.C. Werner, ibidem p. 277. [5] A. Andreoni, R. Cubeddu, S. De Sflvestri and P. Laporta, Optics Comm. 33 (1980) 277. [6] A. Albert, The acridines (E. Arnold Ltd, London, 1966) p. 155.
[7] F. Docchio, A. Longoni and F. Zaraga, submitted to J. Phys. E: Sci. Instrum. [8] A. Andreoni, R. Cubeddu, S. De Silvestri and P. Laporta, submitted to Rev. Sci. Instrum.
OPTICS COMMUNICATIONS
1 April 1981
DUAL TIME SCALE MEASUREMENTS OF THE MULTIEXPONENTIAL FLUORESCENCE DECAY OF QUINACRINE M U S T A R D S. De SILVESTRI, F. DOCCHIO, P. LAPORTA, A. LONGONI and F. ZARAGA Centro di Studio per l'Elettronica Quantistica e la Strumentazione Elettronica del CNR, Istituto di Fisica del Politecnico, Milano, Italy
Received 12 November 1980
The fluorescence decay of Quinacrine Mustard has been measured with good accuracy by means of a dual time scale digitizer-averag©r. Least-squares analysis of the experimental data with appropriate statistical weights has been performed. Values of the time constants for three exponential components have been obtained.
1. Introduction
The excited state dynamics of a great number of aromatic molecules involves complex reactions, such as photon transfer [ 1 - 2 ] , exciplex or excimer formation [3], conformational changes [4], etc. These excited state reactions are usually faster than the fluorescence lifetime of the aromatic molecules and can produce new excited species, which are different from the ground state molecules. When these new species are fluorescent, their formation kinetics in the excited state gives rise to a multiexponential fluorescent emission. A three-exponential fluorescence decay has recently been observed for an acridine dye, Quinacrine Mustard (QM) in this laboratory [5]. The acridine molecules undergo a marked change in the properties of their basic and acid groups, upon excitation [1,6]. The three-exponential decay measured for the QM molecules hasbeen explained in terms of a multiproton transfer in the excited state. The considerable difference in the values of the time constants of the decay of the QM fluorescence [5] demanded a choice of an observation time such that it would be possible to measure with good accuracy the fast initial fluorescence transient, and to follow the tail of the decay for at least one time constant of the slowest exponential component. This procedure, however, does not enable the slowest time constant to be determined 20
with precision, or the risetime of the signal to be measured. The purpose of this paper is to present a measurement of the multiexponential fluorescence decay of QM, using a dual time scale signal digitizer-averager.
2. Experimental The experimental setup, as regards excitation and detection of the fluorescence, is similar to that previously described in ref. [5]. The excitation light at 430 nm, produced by a nitrogen-pumped dye laser with pulses of 150 ps duration, was sent through a cell containing a solution of 3 X 10 -6 M of QM in acetate buffer (pH = 4.6). The fluorescence signal was detected by a Varian 154 M photomultiplier (400 ps FWHM) placed at the exit slit of a monochromator. The photomultiplier output was then sent to a microprocessorcontrolled digitizer-averager [7]. The signal was sequentially sampled by a sampling oscilloscope (Tektronix 7904, sampling head $4) driven by the microprocessor unit. The vertical output of the oscilloscope was digitized, stored in the RAM memory, and averaged over many sampling cycles. The averager has a programmable dual time scale that makes it possible to measure the slowest decay for a long enough time while maintaining a high temporal resolution for the initial fast decay. This feature is obtained by increasing
0 030-4018/81/0000--0000/$ 02.50 © North-Holland Publishing Company
Volume 37, number 1
OPTICS COMMUNICATIONS
1 April 1981
the sample number per unit time in the region of the fast transient. tu
A ~ A A A
3. Results and discussion
A
r dP
A typical QM fluorescence decay observed at 470 nm is shown by the dots in fig. 1. The fluorescence signal was averaged over 100 scan cycles. Samples are taken every 150 ps in the initial part ( 6 - 2 4 ns) of the curve and every 1.2 ns in the slow tail. An expanded view of the rise of this fluorescence curve is shown in fig. 2. The rise-time turns out to be of the order of 500 ps. The fluorescence decay was analyzed by using the non-linear least-squares method. This method requires the knowledge of the standard deviations o i of the experimental points Yi in order to weight appropriately the squares of the residuals. Measurements [8] of the statistical distribution of the noise and fluctuations found in the sampling technique have provided the following relationship o 2 = K y i which indicates a predominant Poisson statistics. The constant K depends 35e98 r-'l
38898 u
>.
25888
*" Q
29809
H 41 O C 0 0 v g
t~eela
loe88
L.
0 5~e
.,...' 25 O -25 8
Ie
2e
38
48
58
~
78
r'~l;
Fig. 1. Experimental (dots) and computed (unbroken line) fluorescence decay curves of QM observed at 470 nm. Below is the plot of the weighted residuals from the difference in the computed and experimental curves.
8e
c
1-t
e
A
N
E
4e
A
L
0 29
A A
0
A
A,
A
I
ps
Fig. 2. Enlarged-scaleview of the rise of the QM fluorescence curve observed at 470 rim. on parameters of the experimental apparatus such as the gain of the photomultiplier, the vertical sensitivity (mV/div) of the sampling oscilloscope, and the analogto-digital conversion factor. A least-squares analysis of the fluorescence decay curve (see fig. 1) shows a three-exponential decay with the constants r I = 0.76, 1"2 = 2.91, r 3 = 17.94 and corresponding initial amplitudes A 1 = A 2 = 48%, A 3 = 4%. The interpolating curve calculated with the above parameters is shown in the unbroken line in fig. 1. The goodness of the fit has been judged by inspection of the autocorrelation function of the residuals. The weighted residuals shown in fig. 1 are equally distributed around zero, indicating the good quality of the fit. The autocorrelation function of the residuals, relating to the fast portion (6--24 ns, see fig. 1) of the fluorescence decay, shown in fig. 3a, confirms the three-exponential analysis. For comparison, fig. 3b shows the autocorrelation function of the residuals of the same portion that would be obtained assuming only two exponential components, indicating the poor quality of the fit in this case. The results previously reported in ref. ['5] for the fluorescence decay of QM showed a decay time for the slow tail (r2) of ~ 16.4 ns. The present measurement gives a value o f t 3 ~ 18 ns, which is about 10% longer. This value appears to be more reliable because the dual time scale enables the slowest exponential 21
Volume 37, number 1
(o)
OPTICS COMMUNICATIONS
e
-I
0
g
I@
i,-I<: I
(b)
@
-I
\ @
Fig. 3. Autocorrelation functions of the residuals relating to the fast portion (6-24 ns) of the fluorescence decay: (a) three-component analysis, Co) two-component analysis. decay to be followed for at least three time constants. In the previous measurement, this decay was followed for about one time constant. This difference in the value o f r 3 does not appreciably affect the value o f r 1 or r 2 reported in ref. [5], due to the small amplitude A 3 .
22
1 April 1981
In conclusion, the measurement of the fluorescence decay of QM by means of dual-time scale signal digitizer-averager made possible a better determination o f the time constants. The fast rise-time o f the signal was also measured. A three-exponential component decay was again found, which confirms the previous interpretation of the experimental results in terms of an excited-state proton transfer.
References
[1] S.G. Schulman, in: Modern fluorescence spectroscopy, Vol. 2, ed. E.L. Wehry (Plenum Press, New York and London, 1976) p. 239. [2] W.R. Laws and L. Brand, L Phys. Chem. 83 (1979) 795. [3] P. Froehlich and E.L. Wehry, in: Modern fluorescence spectroscopy, Vol. 2, ed. E.L. Wehry (Plenum Press, New York and London, 1976) p. 319. [4] T.C. Werner, ibidem p. 277. [5] A. Andreoni, R. Cubeddu, S. De Sflvestri and P. Laporta, Optics Comm. 33 (1980) 277. [6] A. Albert, The acridines (E. Arnold Ltd, London, 1966) p. 155.
[7] F. Docchio, A. Longoni and F. Zaraga, submitted to J. Phys. E: Sci. Instrum. [8] A. Andreoni, R. Cubeddu, S. De Silvestri and P. Laporta, submitted to Rev. Sci. Instrum.