Hevesi, János

Marek, Nándor

Vass, I.

English

Vass I.

Marek Nándor

Hevesi János

University of Szeged

A POLARIZATION SPECTROFLUORIMETER WITH ON-LINE DATA PROCESSING* By . • I. VASS, N. MAREK** and J. HEVES1*** Department of Theoretical Physics, Jözsef Attila University, Szeged (Received November 1. 1980) Semi-automatic set up adaptable for series measurements is described for determination of polarization of fluorescence emission with high precision. Measurements of fluorescence intensities are carried out in a spectrofluorimeter equipped with' polarizers. The control of the measurements and on-line data processing is made by a, minicomputer WANG-600-14. Examination of fluorescence polarization is a useful tool for studying the nature of electric transitions associated with absorption and emission of fluorescing mole-cules [1]. Fluorescence polarization technique is often used for the investigation of intermolecular energy transfer [2], molecular motions in solutions, microviscosity of solvents [3], and for a number of biological problems [4]. The degree of fluorescence polarization p is defined by the following relationship : \ W , . ( i ) h+h where /„ is the fluorescence intensity' polarized in the same plane as the exciting, beam, and I x is the fluorescence intensity polarized at right angels thereto. Although several methods were elaborated for determination of p [5—11], computer data handling offers new possibilities for fast and very precise determination of lumi-nescence polarization. A usual method is to measure the fluorescence intensities Iw and I ± either subsequently or simultaneously [6, 8, 10, 12]. By this simple method, however, due to instability in the intensity of exciting light and fluctuations in photo-multiplier dark current confident results can only be obtained by statistical data evaluation from a great number of repetitions of the measurement. Especially at low fluorescence intensities this makes the measurements rather tedious. The aim of the present work was to construct an equipment which connects the simplicity of the direct method with the controlling and. on-line data processing facilities of computers. * * * * * * Presented at the 3rd Summer School on Luminescence, Budapest, August 25—26, 1980. Department of Medical Chemistry, Medical University, Szeged. Department of Biophysics, József Attila University, Szeged. 1 1 2 I. VASS, N. MAREK and J. HEVESI Fig. 1 shows the block diagram of the instrument. Exciting light is provided by an 500 W Xe arc lamp L. The polarized monochromatic light incident on the sample, located in a termostated cuvette C, is obtained by a Hilger double grating monochromator M (Typ. D331), and by polarizer Pu mounted on a hand rotating disc. Before collection at the photomultiplier PM (EMI 9558 QB), the fluorescence light passes through a filter F, to block the scattered light, and polarizer P2, with Fig. 1. Block diagram of the equipment fixed position. Arrows on Fig. 1 indicate the electric vectors of the polarizers P1 and P2, respectively. For measuring the intensity of the exciting light a mirror Mr, and-a photodiode PD is used. After suitable amplification the signals of photo-multiplier and photodiode are transferred separatly to two analog-to-digital con-verters AjD, which are synchronized to each other with a maximal sampling rate of 50 s-1 . The digital data proportional to the exciting and the fluorescent light enter simultaneously the minicomputer WANG—600—14, across a digital microinter-face INT. The semi-automatic measurement is processed by a laboratory-built unit DU, constructed from digital integrated circuits. This unit makes possible to start the computer program at the optical unit — located in about 50 m distance from the minicomputer — and gives information about the run of the program by a con-trolling board CB. During the measurement the electric vector of the exciting light passing through the polarizer P1 was brought first into parallel, then into perpendicular position with respect to the electric vector of fluorescence light passing through the fixed polarizer P2. The partially polarized light from monochromator M, that also depends on the wavelength X, has generally different intensity in the two positions of Px. Taking into consideration this fact in determiningp, the quotient c(A)=/Xje(A)//,i>e(A) has to be measured. To carry out this, a scrambler was put into the sample holder and in this way P2 obtained always depolarized light. 1 . A POLARIZATION SPECTROFLUORIMETER WITH ON-LINE DATA PROCESSING 113 Taking into account the dark current of the photomultiplier Jd, the following formula was used for the calculation of p „ = (.h-h)cW-{Ix-Id) P ( / „ - / , ) c ( l ) + ( / , . - / , ) • W The flow diagram of the measurement is shown in Fig. 2. The random errors of this method originate mainly from two sources. The first is the fluctuation of the dark current and the electric noise of the whole equipment. The distribution of a great number of intensities was found to be Gaussian-type, so the relative error of the mean value is AIre,=(rJI ~fn, where n is the number of the measurements, an is the OPT. UNIT DATA TRANSFER MINICOMPUTER Fig. 2. Flow diagram of the measurement standard deviation and I is the mean value. To reduce the error due to the fluctua-tion of the dark current, the computer program continously makes the sampling and compute AJrel. When AIrel is less than a given-error limit, <5, the sampling stops. The great advantage of this sampling method is that the sampling number is not fixed, but is always optimal for the given error limit <5. The other source of the errors is the instability of the exciting lamp. To avoid this difficulty, before the measurement of / j and J±, we determine the intensity of the exciting light and this, value is consi-114 I. VASS, N. MAREK'-and J. HEVESI . dered as reference I r e f , in the following. In determination of /„ and I ± the sampling procedure is similar to that of the dark current. Except that in this case, together with the intensity of the polarized fluorescence, the intensity of the exciting light Ig is measured, too. If \Ig—/„y|<e, where e is programmed error limit, the measured /D or I± value is accepted, if \Ig—Iref\>e the measurement is repeated..The sampling continues until a J I in <<5. Because of the continuous computations during the measurement the maximail sampling rate was reduced from 50 s - 1 to 12.5 s - 1 . Fig 3 shows the absolute and relative error of p calculated from -i, ten test measurements at ¿=£=1%. It can be seen that the absolute error Ap is nearly constant indepen-dently on the value of p, and so the relative error Ap/p is steeply rising at low values of p. With appropriate choice of the error limits <5 and e, the desired absolute or relative error for a certain p can be obtained even at low fluorescence intensities. Sett-ing low error levels the time necess-ary for the measurement consider-ably increases. For the degree of polarization of 10~4 M Na-fluor-escein solution we obtain p= = 0.0016±0.0012, which is similar to earlier results [8]. In conclusion, we may say that our set up connecting the direct determination of p with an up-to-date data processing method makes possible the fast determination of fluorescence polarization with high precision. The errors due to the instability of the exciting light and fluctuations of the dark current can be pushed under a given level by the optimal choice of the number of measurements to these error limits. The p values obtained can be saved on magnetic casette for later evaluation. Fig. 3. Absolute and relative error of 10 p values as a result of test measurements 5=e= 1%, O — Ap (absolute error), —x—/}p/p (relative error) References 11] Vavilov. Sz. I.: ZhETF 10, 1363 (1940). 12] Förster, Th.: Ann. Physik 2, 55 (1948). {3] Nishijama, Y„ G. Osier: Bull. Chem. Soc. Japan 33, 1649 (1960). [4] Goedheer, J. C., J. C. van der Commen: Polarized Light Spectroscopy of Biological, Systems. Proceedings of Spring School, Kolobrzeg, (1980). 15] Wille. H.: Optik 9, 84 (1952). 16] Ketskemety, I., L. Gargya, E. Salkovits: Acta Phys. et Chem. Szeged 3, 16 (1957). (7] Dehler, J., F. Dörr: Z. angew. Phys. 19, 147 (1965). A POLARIZATION SPECTROFLUORIMETER WITH ON-LINE DATA PROCESSING 115 [8] Vize, L.: Acta Phys. et Chem. Szeged 15, 27 (1969). [9] Bauer, R. K.: J. of Phys. E: Sci. Inst. 3, 965 (1970). [10] Teale, F. W.: J. of Phys. E: Sci. Inst. 3, 555 (1970). [11] Clout, P. N.. M. A. Haque, D. W. O. Heddle: J. of Phys. E: Sci. Inst. 4, 893 (1971). [12] Teissié, J., B. Valeur, L. Monnerie: J. of Phys. E.: Sci. Inst. 8, 700 (1975). ПОЛЯРИЗАЦИОННЫЙ СПЕКТРОФЛУОРИМЕТР С НЕПОСРЕДСТВЕННОЙ ОБРАБОТКОЙ ДАННЫХ И. Вашш, Н. Марек и Й. Хевеши Описан полуавтоматический аппарат применимый к серийным измеремиям поляризации флуоресценции с большой точностю. Проведены измерения интенсивности флуоресценции в спектрофлуориметре с поляризаторами. Контроль измерения и непосредственную обра-ботку данных проводили на настольном вычислителе типа WANG—600—14. 

A polarozation spectrofluorimeter with on-line data processing

http://acta.bibl.u-szeged.hu/24152/1/phys_chem_026_fasc_003_004_111-115.pdf

A polarozation spectrofluorimeter with on-line data processing

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