- Email: [email protected]
A new method of fluorescence polarization measurement
BIOCHIMIE, 1972, 54, 161-165.
A new method of fluorescence polarization measurement. J. LAVOREL, C. VERNOTTE. Laboratoire de Photosynth~se du C.N.R.S., 91- Gi[-sur-Yvette, France. B. ARRIO a n d F. RODmR.
Laboratoire de Biologie Physico-Chimique, Universit~ de Paris-Sud, Centre d'Orsay, 91- Orsay, France. (16/2/1972). Summary. - - Two a p p a r a t u s for m e a s u r i n g p o l a r i z a t i o n fluorescence are described ; they are b u i l t according to the principle t h a t aH t h e i n f o r m a t i o n needed to calculate p can be o b t a i n e d vrith a r o t a t i n g p o l a r i z e r on the exciting b e a m in t e r m s of successive m a x i m a a n d m i n i m a of fluorescence i n t e n s i t y emitted at r i g h t angles to the direction of excitation. The essential a d v a n t a g e s of t h i s m e t h o d are its simplicity and c o n s e q u e n t l y its easy a d a p t a t i o n on simple fluorimetersg its sensitivity, its a p t i t u d e to p e r f o r m kinetic measur e m e n t s a n d the possil~ility to give a n a u t o m a t i z e d measure of p. The measurement of t h e p o l a r i z a t i o n of t h e light emitted by a fluorescent compound in solut i o n g i v e s i n f o r m a t i o n s a b o u t i t s m o l e c u l a r geom e t r y a n d t h e o r i e n t a t i o n of its e l e c t r o n i c o s c i l l a t o r s . E x t e n s i v e t r e a t m e n t s of t h e t h e o r y , i n t r u m e n t a t i o n a n d a p p l i c a t i o n s of f l u o r e s c e n c e p o l a r i z a tion can be found in the following references: [1, 2, 3, 4, 5, 6, 7, 8, 9, 10]. H e r e , w e d e s c r i b e a n o r i g i n a l m e t h o d of m e a s u r e m e n t .
The polarization may be obtained by combining F I and F 2 : p
_
F 1 -- F 2
(4)
Fx
~z
~z
t
Ii
Iv T h e s t a t e of p o l a r i z a t i o n of t h e f l u o r e s c e n c e e m i t t e d n o r m a l to t h e d i r e c t i o n of e x c i t a t i o n is defined by : p =
--
IH
Iv +
IH
I.
(1)
The fluorescence intensity in the direction is : F 1 ~,~ I,. + l H beam F2 ~
is p o l a r i z e d 21 n
ox
(2) along
~x
oz
(3)
w h e n i t is p o l a r i z e d a l o n g o x (Fig. l b ) .
EXCITATION
--
jXz
H
OBSERVATION
fig
w h e r e I v is t h e i n t e n s i t y of t h e c o m p o n e n t of f l u o r e s c e n t l i g h t v i b r a t i n g p a r a l l e l to t h e e l e c t r i c v e c t o r of t h e p l a n e p o l a r i z e d e x c i t i n g l i g h t , a n d I n t h e i n t e n s i t y of t h e c o m p o n e n t v i b r a t i n g i n t h e dir e c t i o n of p r o p a g a t i o n of t h e e x c i t i n g l i g h t .
when the exciting (Fig. l a ) , o r :
i IH •
THEORY.
EX C I T A T I O N __
IH
iv
Y
OBSERVATION
1a
fig
1b
Fie. 1. - - Components of fluorescence excited b y vertical (a) or h o r i z o n t a l (b) polarized light.
T h e r e f o r e all t h e i n f o r m a t i o n n e e d e d to c a l c u lated p may be obtained with a rotating polarizer o n t h e e x c i t i n g b e a m i n t e r m s of s u c c e s s i v e m a x i m a a n d m i n i m a of t h e f l u o r e s c e n c e i n t e n s i t y e m i t t e d at r i g h t a n g l e to t h e d i r e c t i o n of e x c i t a t i o n . T w o t y p e s of a p p a r a t u s h a v e b e e n b u i l t , according to this principle. [NSTRUMENTATON.
Apparatus n ° 1 (Fig. 2a). Ce t r a v a i l reeouvre en p a r t i e la th6se de doctorat d ' E t a t 6s-Sciences de M. B e r n a r d Arrio, enregistr6e au Centre N a t i o n a l ¢Le la Recherche Scientifique avec le N ° AO5425.
a. Description. A p a r a l l e l b e a m of l i g h t is o b t a i n e d t h r o u g h q u a r t z l e n s e s L L a n d Lp f r o m a n O S R A M 200 w a t t s m e r c u r y a r c . A n a r r o w w a v e -
J. Lavorel, C. Vernotte, B. Arrio and F. Rodier.
162
length range is selected by i n t e r p o s i n g interfer e n c e Klters. A r o t a t i n g sector (2 RPM) built as d e s c r i b e d on Fig. 3 a gives in succession the f o l l o w i n g signals and o p e r a t i o n s : 1) F 1 and F 2 are r e c o r d e d w h e n the two polarizers ( p o l a r o i d HNPB) are i n t e r p o s e d on the beam. It is to be noted (see discussion) that the p o l a r i z e r s need not be p o s i t i o n n e d in p r e c i s e l y crossed c o n f i g u r a t i o n ;
T h e light e m e r g i n g f r o m the o b s e r v a t i o n p a r t is f r e e d of stray e x c i t i n g light w i t h WnATTEN KODAK filters and its angular s p r e a d is l i m i t e d w i t h a set of d i a p h r a g m s in o r d e r to a d m i t only light rays as close to the ox d i r e c t i o n as possible. T h e fluo-
exciting
exciting
2) total f l u o r e s c e n c e i n t e n s i t y F t w i t h the t h i r d e m p t y hole ; 3) d u r i n g the h a l f - p e r i o d of the sector w h e n the b e a m is i n t e r r u p t e d , s t i r r i n g of the sample occurs
switch for stirring
fig 3a
S LF
FIG. 3.
~
Lp
~
RECORDER CELL S LF
D
fig2b
) SHUTTER
Lp
'
;
LIGHT GUIDE AMPLIFIER
P
RECORDER
CELL
-
Schemes of the two polarizing devices (see text).
r e s c e n c e is d e t e c t e d by a p h o t o m u l t i p l i e r (RADIOTECHNIQUE XP 1118) o p e r a t e d at 1 21)0.-1 750V. T h e signal is r e c o r d e d on a TACUSEL EPLI r e c o r d e r w i t h a v a r i a b l e voltage p l u g in unit.
PM
[] ~
-
)
F
P
fig 3b
fig 2 a
[]
D
PM
M2
b. Operations. A t y p i c a l sample of r e c o r d i n g in s h o w n on Fig. 4a. T h e speed of the c h a r t b e i n g l o w as c o m p a r e d to the a n g u l a r speed of the sector, the t h r e e signals a p p e a r as successive v e r t i c a l spikes. A c o r r e c t i o n f a c t o r K k a l l o w i n g for the t r a n s m i s s i o n of p o l a r i z e r s as a f u n c t i o n of w a v e length must be defined in o r d e r to e x p r e s s a n e c e s s a r y r e l a t i o n b e t w e e n F1, F 2 and F t : F t = I v q- 311=I = K), (F 1 q- F 2) (5) T h e n the total fluorescence flux is p r o p o r t i o n n a l to : 1 I v q- 215 ---- F t - -K), F 2 2
Apparatus n ° ? (Fig. 2b). Fro. 2. - - Schemes of the two apparatuss (S : source, Lr, and L, : lens, F : filters, M1 and M,~ : monochromators, P : polarizing device, D : diaphragms, P M : photomultiplier).
w i t h an e l e c t r i c s w i t c h actuated by a cam at the c o r r e s p o n d i n g edge of the sector. This p e r m i t s good t h e r m a l e q u i l i b r a t i o n , m i x i n g of any a d d i t i v e to the sample and r e c o r d i n g of the dark current. T h e fluorescent sample is p l a c e d in a quartz cuvette (4 ml, 10 m m × 10 m m section) housed in a brass h o l l o w block w i t h a p p r o p r i a t e holes for passage of light, w h e r e a l i q u i d is c i r c u l a t e d for t e m p e r a t u r e c o n t r o l (__+0,02°).
BIOCHIMIE, 1'972, 54, n ° 2.
a. Description. T h e outline is quite sinfilar to that of the f o r m e r a p p a r a t u s e x c e p t for the following features. T h e e x c i t a t i o n and o b s e r v a t i o n of fluorescence are m a d e m o r e n e a r l y m o n o c h r o m a t i c by using t w o m o n o c h r o m a t o r s (Bausch & Lomb, h i g h intensity, slit w i d t h for 5 nm). T h e light source is a high p r e s s u r e x e n o n a r c (OsRAra X BO 75 Watts). M o n o c h r o m a t o r s o b v i o u s l y i m p r o v e the v e r s a t i l i t y of the set up, but t h e y m a y i n t r o d u c e some trouble. Even w h e n using n a t u r a l w h i t e light at the input, the e x c i t a t i o n m o n o c h r o m a t o r delivers a p a r t i a l l y p o l a r i z e d light. T h e degree of p o l a r i z a t i o n is a f u n c t i o n of w a v e l e n g t h and it w o u l d be quite urn-
Fluorescence polarization measurement. p r a c t i c a l to t r y to c o r r e c t it at each w a v e l e n g t h w i t h a classical d e v i c e such as a pile of glass plates. Instead, w e h a v e p u t a short segment (50 m m ) o f a 4 m m d i a m e t e r i n c o h e r e n t light guide (made up of 60 I~ glass fibers in a r i g i d b u n d l e ) ; this c o m p l e t e l y d e p o l a r i z e s the monoc h r o m a t i c light w i t h o u t too large a loss of intensity. Light, after b e i n g m a d e parallel w i t h lens Lv, goes t h r o u g h the c e n t e r of a r o t a t i n g p o l a r i z e r (2rpm, P o l a r o i d HN 32). This slightly c o m p l i c a t e s the m e c h a n i c a l device, but o v e r c o m e s a serious difficulty w h i c h w e h a v e e x p e r i e n c e d in selecting large pieces of P o l a r o i d sheet of u n i f o r m transmission. T h e same p o l a r i z i n g effect as d e s c r i b e d above should be c o n s i d e r e d and c o r r e c t e d for w i t h the o b s e r v a t i o n m o n o c h r o m a t o r . Calculation and exper i e n c e s h o w that the e r r o r is s m a l l e r in this case. H o w e v e r , since it is not negligible a n d d e p e n d e n t on the m a g n i t u d e of p, it was n e c e s s a r y to p l a c e a n o t h e r P o l a r o i d in v e r t i c a l position before the e n t r a n c e of the o b s e r v a t i o n m o n o c h r o m a t o r (not s h o w n in Fig. 2b). W i t h this modification, the s i m p l i c i t y of the p r i n c i p l e is lost and :
163
plified (Multimesureur (SERVOTRACE SEFRAM).
LEMOUZY) and
recorded
b. Operation. T h e speed of the c h a r t is set in such a w a y as to p r o d u c e a well resolved sinusoidal c u r v e w h e r e the m a x i m a and m i n i m a (F x and F~) can be l o c a t e d g r a p h i c a l l y (Fig. 4b). A dark c o n t r o l (light off) is n e e d e d to c o r r e c t for the d a r k current. RESULTS. T h e s e n s i t i v i t y and p r e c i s i o n of the t w o instruments have been tested t a k i n g v a r i o u s solutions of o r g a n i c dyes. W i t h s o d i u m fluorescein salt, meas u r e m e n t s of p h a v e been c a r r i e d d o w n to 10 -8 M, w i t h an absolute e r r o r of 8.10 -a. 1
I
F a -= I v F~ = I u the signal is r e d u c e d by m o r e than 50 p. cent. It should be noted that the o b s e r v a t i o n m o n o c h r o m a t o r m a y be r e p l a c e d by b r o a d b a n d filters in most a p p l i c a t i o n s w h e r e the d e p e n d a n c e of p on w a v e l e n g t h in the e m i s s i o n band is not c o n s i d e r e d as such. T h e glass cuvette, also housed in a t h e r m o s t a t e d block, has t w o a l u m i n i z e d a d j a c e n t faces for inc r e a s i n g the fluorescence signal. F l u o r e s c e n c e is detected w i t h a p h o t o m u l t i p l i e r (RADIOTEEHNIQUE XP 1 002) o p e r a t e d at 1 000 V. T h e signal is am-
IN,TENSITY one
r I
°"~
FT
rotation
- 2 tH ,
1
k ta)
.J (b)
L
TIME
FIt;. 4. - - Examples of recording ; (a) with al)paratus n ° 1 (b) with apparatus n ° '2.
BIOCHIMIE, 1972, 54, n ° 2.
500
1000
1500
FIG. 5. Perrin's plot for fluorescein (10-6M) in various concentrations of glycerol, at 20°C. T/~1 is in °K per poise. The squares correspond to measurements done with the apparatus n ° 1, and the circles, with the apparatus n ° 2. -
-
F i g u r e 5 shows an instance of P e r r i n ' s plot for fluorescein. F r o m the slope of the straight line, a lifetime of 4,3 ns is c o m p u t e d , assuming a m o l a r v o l u m e of 500 cln:~ ; this result agrees w i t h the data of P e r r i n [1, 2]. Po has been d e t e r m i n e d from m e a s u r e m e n t s w i t h v a r i o u s dyes and found to agree w i t h p u b l i s h e d data (Table I).
FLUORESCENCE
rotation
T
DISCUSSION. Besides its i n h e r e n t s i m p l i c i t y w h i c h allows c o n s i d e r i n g its a d a p t a l i o n to almost any fluorimeter, this m e t h o d has several a d v a n t a g e s : 1) In c o n v e n t i o n a l i n s t r u m e n t s w i t h t w o fixed p o s i t i o n s (<( v e r t i c a l >>, <( h o r i z o n t a l >>) of the pola-
J. Lavorel, C. Vernotte, B. Arrio and F. Rodier.
164
rizer, the t w o c o r r e s p o n d i n g d i r e c t i o n s s h o u l d be p r e c i s e l y at 90 ° a n d f r u t h e r the axis of the polar i z e r must be p r e c i s e l y set to c o i n c i d e w i t h one of these d i r e c t i o n s [see Weber, 7~. This is automatically o b t a i n e d w i t h the r o t a t i n g polarizer. Other sources of e r r o r (non p a r a l l e l i s m of beams, n o n n o r m a l i t y of o p t i c a l axis) o b v i o u s l y m u s t be minimized.
r e m e n t by q u i c k l y g a t h e r i n g a large n u m b e r of values of F 1 and F e on the r e c o r d e r chart. One m i g h t i m a g i n e several w a y s of getting a b e t t e r effic i e n c y f r o m this m e t h o d . In all case, the f r e q u e n c y of the r o t a t i n g p o l a r i z e r could be i n c r e a s e d at advantage. One obvious w a y of using best the r e p e t i t i v e p r o p e r t y w o u l d be to pass the signal to a s a m p l i n g a n d a v e r a g i n g amplifier.
TABLE I.
P, determinations with various dyes. Substances Fluorescein . . . . . . . . . . . . . . . . . . . . . . . . . . F l u o r e s c e i n isothiocyanate . . . . . . . . . . . . .
Rhodamin B . . . . . . . . . . . . . . . . . . . . . . . . Quinine sulfate . . . . . . . . . . . . . . . . . . . . . . Dimethyl amino naphtalene 5 sulfonate Dimethyl amino naphtalene 7 sulfonate
2) W i t h a u t o m a t i c operation, although m o d e rately fast in its p r e s e n t versions, this t y p e of a p p a r a t u s is w e l l suited for m a n y k i n e t i c as well as s p e c t r o s c o p i c studies. 3) B e i n g r e p e t i t i v e , this t y p e of i n s t r u m e n t a l r e a d y h e l p s to i m p r o v e the p r e c i s i o n of measu-
iI
Chalnnel-ch@~~li2F2
:
(IF 1 + ~F 2
i2 :
~ F 1 + o. 5
commutation il lv. <'- Input signals 9 ~ •
Output signal
-i 1
I F,
1
"~ U a ~ l O Fla. 6. - - Computation network for p (see text).
BIOC.HIMIE, 1'972, 54, n ° 2.
Po given in references
Po measured 0,435 -t- 0,008 0,435 0,440 0,460 0,270
0,440
[Weber. 7]
0,444
[Weber, 71 [Perrin, 1]
0,2651 [Weber, 7]
H o w e v e r , some g r a p h i c a l o p e r a t i o n w o u l d still be needed. A solution e l i m i n a t i n g this step is prop o s e d on Fig. 6. It is best a p p l i e d to v e r s i o n n ° 2 (with the o b s e r v a t i o n m o n o c h r o m a t o r r e p l a c e d by filters) w h i c h in p r i n c i p l e gives a l a r g e r a m o u n t of i n f o r m a t i o n p e r cycle of operation. T h e signal is s y n c h r o n o u s l y c o m m u t e d so as to send the t w o successive half w a v e s into separate chanels. Both signals are filtered and c o m b i n e d in an analog c o m p u t a t i o n n e t w o r k w h i c h solves the equations i 1 = aF 1 + ~F2 i 2 -~ ~F 1 -1- a F 2 for F 1 a n d F~ 01 and i 2 are the output of the channels, a and [3 are n u m e r i c a l factors r e s u l t i n g f r o m filtering). A f u r t h e r i m p r o v e m e n t in autom a t i z a t i o n could be the analog c o m p u t a t i o n of F 1 - F 2 and f e e d - b a c k c o n t r o l of the i n p u t signals (e. g. by v a r y i n g t h e voltage of the p h o t o m u l t i plier) l o c k i n g F 1 to a r e f e r e n c e value t a k e n as unity. E q u a t i o n (4) shows that in this case : m e a s u r e of p = F 1 - F
2
W h e n the o b s e r v a t i o n m o n o c h r o m a t o r is used w i t h the P o l a r o i d at the e n t r a n c e , e q u a t i o n (4) should be r e p l a c e d by : F 1 --F 2 p -F 1 -t- F2 and an a d d i t i o n a l c o n n e c t i o n (dashed line on fig. 0) is used. R~suM~. Deux appareils destines & la mesure des taux de polarisatiou de fluorescence sont d~crits ; ils ont ~t~ construits suivant le prineipe que toute l'information
Fluorescence polarization measurement. n6cessaire p o u r calculer le t a u x de p o l a r i s a t i o n p e u t ~tre o b t e n u e en m o d u l a n t le f a i s c e a u d ' e x c i t a t i o n avec u n p o l a r i s e u r . Les a v a n t a g e s de cette m ~ t h o d e s o n t sa simplieit~ et p a r c o n s e q u e n t la possibilit~ de l ' a d a p t e r f a c i l e m e n t s u r u n fluorim~tre, sa sensibilrit~, son aptit u d e a u x m e s u r e s cindtiques, enfin l ' a u t o m a t i s a t i o n p e r m e t t a n t d ' o b t e n i r d i r e c t e m e n t la v a l e u r du t a u x de polarisation. ZUSAMMENFASSUN,G.
Zwei Geriite z u r M e s s u n g der F l n o r e s z e n z p o l a r i s a tion w e r d e n b e s c h r i e b c n ; sie w e r d e n a u f G r u n d des P r i n z i p s gebaut, w o n a c h die g e s a m t e , z u r B e r e c h n u n g yon p b e n 6 t i g t e I n f o r m a t i o n m i t Hilfe eines i m a n r e genden S t r a h l e n g a n g befindlichen r o t i e r e n d e n P o l a r i s a t o r s e r h a l t e n w e r d e n k a n n , u.z. als eine Folge yon M a x i m a u n d M i n i m a der Fluoreszenzintensit~it, welche s e n k r e c h t z u r R i c h t u n g des e i n f a l l e n d e n S t r a h l e n b i i n dels e m i t t i e r t vcird. Die w i c h t i g s t e n Vorteile dieser Methods s i n d ihre E i n f a c h h e i t u n d folglich der leichte E i n b a u in die
BIOCHIMIE, 1972, 54, n ° 2.
165
h e r k 6 m m l i c h e n F l u o r i m e t e r , ihre E m p f i n d l i c h k e i t , ihre Fiihigkeit z u r k i n e t i s c h e n M e s s u n g u n d die M6glichkeit der a u t o m a t i s i e r t e n E r f a s s u n g yon p. REFERENCES. 1. P e r r i n , F. (1929') Ann. Phys. Fr., 12, 169. 2. P e r r i n , F. (1926) J. Phys. Radium, 7, 390. 3. Feofilov, P. P. << T h e P h y s i c a l b a s i s of p o l a r i z e d e m i s s i o n >> E n g l i s h T r a n s l a t i o n , C o n s u l t a n t s B u r e a u , New-York, 1961. 4. W e b e r , G. (1953,) Ado. Protein Chem., 8, 415. 5. P r i n g s h e i m , P. << F l u o r e s c e n c e a n d P h o s p h o r e s cence >> I n t e r s c i e n c e P u b l i s h e r s , New-York, 1949. 6. W e b e r , G. in ¢ F l u o r e s c e n c e a n d P h o s p h o r e s c e n c e A n a l y s i s >>, p. 217, Interscience P u b l i s h e r s , J. W i l e y a n d Sons, New-York, 1967. 7. Weber, G. (195~6) J. Opt. Soc. Am., 46, 962. 8. Monnerie, L. ~ N.eels, J. (1965) J. Chim. phys. Phys. Chim. biol. Ft., 62, 504. 9. D e r a n l e a u , D. A. (19,66) Anal. Biochem., 16, 438. 10. Mc Kay, R. (1969) Arch. of Biochem. and Biophys., 135, 218.
A new method of fluorescence polarization measurement. J. LAVOREL, C. VERNOTTE. Laboratoire de Photosynth~se du C.N.R.S., 91- Gi[-sur-Yvette, France. B. ARRIO a n d F. RODmR.
Laboratoire de Biologie Physico-Chimique, Universit~ de Paris-Sud, Centre d'Orsay, 91- Orsay, France. (16/2/1972). Summary. - - Two a p p a r a t u s for m e a s u r i n g p o l a r i z a t i o n fluorescence are described ; they are b u i l t according to the principle t h a t aH t h e i n f o r m a t i o n needed to calculate p can be o b t a i n e d vrith a r o t a t i n g p o l a r i z e r on the exciting b e a m in t e r m s of successive m a x i m a a n d m i n i m a of fluorescence i n t e n s i t y emitted at r i g h t angles to the direction of excitation. The essential a d v a n t a g e s of t h i s m e t h o d are its simplicity and c o n s e q u e n t l y its easy a d a p t a t i o n on simple fluorimetersg its sensitivity, its a p t i t u d e to p e r f o r m kinetic measur e m e n t s a n d the possil~ility to give a n a u t o m a t i z e d measure of p. The measurement of t h e p o l a r i z a t i o n of t h e light emitted by a fluorescent compound in solut i o n g i v e s i n f o r m a t i o n s a b o u t i t s m o l e c u l a r geom e t r y a n d t h e o r i e n t a t i o n of its e l e c t r o n i c o s c i l l a t o r s . E x t e n s i v e t r e a t m e n t s of t h e t h e o r y , i n t r u m e n t a t i o n a n d a p p l i c a t i o n s of f l u o r e s c e n c e p o l a r i z a tion can be found in the following references: [1, 2, 3, 4, 5, 6, 7, 8, 9, 10]. H e r e , w e d e s c r i b e a n o r i g i n a l m e t h o d of m e a s u r e m e n t .
The polarization may be obtained by combining F I and F 2 : p
_
F 1 -- F 2
(4)
Fx
~z
~z
t
Ii
Iv T h e s t a t e of p o l a r i z a t i o n of t h e f l u o r e s c e n c e e m i t t e d n o r m a l to t h e d i r e c t i o n of e x c i t a t i o n is defined by : p =
--
IH
Iv +
IH
I.
(1)
The fluorescence intensity in the direction is : F 1 ~,~ I,. + l H beam F2 ~
is p o l a r i z e d 21 n
ox
(2) along
~x
oz
(3)
w h e n i t is p o l a r i z e d a l o n g o x (Fig. l b ) .
EXCITATION
--
jXz
H
OBSERVATION
fig
w h e r e I v is t h e i n t e n s i t y of t h e c o m p o n e n t of f l u o r e s c e n t l i g h t v i b r a t i n g p a r a l l e l to t h e e l e c t r i c v e c t o r of t h e p l a n e p o l a r i z e d e x c i t i n g l i g h t , a n d I n t h e i n t e n s i t y of t h e c o m p o n e n t v i b r a t i n g i n t h e dir e c t i o n of p r o p a g a t i o n of t h e e x c i t i n g l i g h t .
when the exciting (Fig. l a ) , o r :
i IH •
THEORY.
EX C I T A T I O N __
IH
iv
Y
OBSERVATION
1a
fig
1b
Fie. 1. - - Components of fluorescence excited b y vertical (a) or h o r i z o n t a l (b) polarized light.
T h e r e f o r e all t h e i n f o r m a t i o n n e e d e d to c a l c u lated p may be obtained with a rotating polarizer o n t h e e x c i t i n g b e a m i n t e r m s of s u c c e s s i v e m a x i m a a n d m i n i m a of t h e f l u o r e s c e n c e i n t e n s i t y e m i t t e d at r i g h t a n g l e to t h e d i r e c t i o n of e x c i t a t i o n . T w o t y p e s of a p p a r a t u s h a v e b e e n b u i l t , according to this principle. [NSTRUMENTATON.
Apparatus n ° 1 (Fig. 2a). Ce t r a v a i l reeouvre en p a r t i e la th6se de doctorat d ' E t a t 6s-Sciences de M. B e r n a r d Arrio, enregistr6e au Centre N a t i o n a l ¢Le la Recherche Scientifique avec le N ° AO5425.
a. Description. A p a r a l l e l b e a m of l i g h t is o b t a i n e d t h r o u g h q u a r t z l e n s e s L L a n d Lp f r o m a n O S R A M 200 w a t t s m e r c u r y a r c . A n a r r o w w a v e -
J. Lavorel, C. Vernotte, B. Arrio and F. Rodier.
162
length range is selected by i n t e r p o s i n g interfer e n c e Klters. A r o t a t i n g sector (2 RPM) built as d e s c r i b e d on Fig. 3 a gives in succession the f o l l o w i n g signals and o p e r a t i o n s : 1) F 1 and F 2 are r e c o r d e d w h e n the two polarizers ( p o l a r o i d HNPB) are i n t e r p o s e d on the beam. It is to be noted (see discussion) that the p o l a r i z e r s need not be p o s i t i o n n e d in p r e c i s e l y crossed c o n f i g u r a t i o n ;
T h e light e m e r g i n g f r o m the o b s e r v a t i o n p a r t is f r e e d of stray e x c i t i n g light w i t h WnATTEN KODAK filters and its angular s p r e a d is l i m i t e d w i t h a set of d i a p h r a g m s in o r d e r to a d m i t only light rays as close to the ox d i r e c t i o n as possible. T h e fluo-
exciting
exciting
2) total f l u o r e s c e n c e i n t e n s i t y F t w i t h the t h i r d e m p t y hole ; 3) d u r i n g the h a l f - p e r i o d of the sector w h e n the b e a m is i n t e r r u p t e d , s t i r r i n g of the sample occurs
switch for stirring
fig 3a
S LF
FIG. 3.
~
Lp
~
RECORDER CELL S LF
D
fig2b
) SHUTTER
Lp
'
;
LIGHT GUIDE AMPLIFIER
P
RECORDER
CELL
-
Schemes of the two polarizing devices (see text).
r e s c e n c e is d e t e c t e d by a p h o t o m u l t i p l i e r (RADIOTECHNIQUE XP 1118) o p e r a t e d at 1 21)0.-1 750V. T h e signal is r e c o r d e d on a TACUSEL EPLI r e c o r d e r w i t h a v a r i a b l e voltage p l u g in unit.
PM
[] ~
-
)
F
P
fig 3b
fig 2 a
[]
D
PM
M2
b. Operations. A t y p i c a l sample of r e c o r d i n g in s h o w n on Fig. 4a. T h e speed of the c h a r t b e i n g l o w as c o m p a r e d to the a n g u l a r speed of the sector, the t h r e e signals a p p e a r as successive v e r t i c a l spikes. A c o r r e c t i o n f a c t o r K k a l l o w i n g for the t r a n s m i s s i o n of p o l a r i z e r s as a f u n c t i o n of w a v e length must be defined in o r d e r to e x p r e s s a n e c e s s a r y r e l a t i o n b e t w e e n F1, F 2 and F t : F t = I v q- 311=I = K), (F 1 q- F 2) (5) T h e n the total fluorescence flux is p r o p o r t i o n n a l to : 1 I v q- 215 ---- F t - -K), F 2 2
Apparatus n ° ? (Fig. 2b). Fro. 2. - - Schemes of the two apparatuss (S : source, Lr, and L, : lens, F : filters, M1 and M,~ : monochromators, P : polarizing device, D : diaphragms, P M : photomultiplier).
w i t h an e l e c t r i c s w i t c h actuated by a cam at the c o r r e s p o n d i n g edge of the sector. This p e r m i t s good t h e r m a l e q u i l i b r a t i o n , m i x i n g of any a d d i t i v e to the sample and r e c o r d i n g of the dark current. T h e fluorescent sample is p l a c e d in a quartz cuvette (4 ml, 10 m m × 10 m m section) housed in a brass h o l l o w block w i t h a p p r o p r i a t e holes for passage of light, w h e r e a l i q u i d is c i r c u l a t e d for t e m p e r a t u r e c o n t r o l (__+0,02°).
BIOCHIMIE, 1'972, 54, n ° 2.
a. Description. T h e outline is quite sinfilar to that of the f o r m e r a p p a r a t u s e x c e p t for the following features. T h e e x c i t a t i o n and o b s e r v a t i o n of fluorescence are m a d e m o r e n e a r l y m o n o c h r o m a t i c by using t w o m o n o c h r o m a t o r s (Bausch & Lomb, h i g h intensity, slit w i d t h for 5 nm). T h e light source is a high p r e s s u r e x e n o n a r c (OsRAra X BO 75 Watts). M o n o c h r o m a t o r s o b v i o u s l y i m p r o v e the v e r s a t i l i t y of the set up, but t h e y m a y i n t r o d u c e some trouble. Even w h e n using n a t u r a l w h i t e light at the input, the e x c i t a t i o n m o n o c h r o m a t o r delivers a p a r t i a l l y p o l a r i z e d light. T h e degree of p o l a r i z a t i o n is a f u n c t i o n of w a v e l e n g t h and it w o u l d be quite urn-
Fluorescence polarization measurement. p r a c t i c a l to t r y to c o r r e c t it at each w a v e l e n g t h w i t h a classical d e v i c e such as a pile of glass plates. Instead, w e h a v e p u t a short segment (50 m m ) o f a 4 m m d i a m e t e r i n c o h e r e n t light guide (made up of 60 I~ glass fibers in a r i g i d b u n d l e ) ; this c o m p l e t e l y d e p o l a r i z e s the monoc h r o m a t i c light w i t h o u t too large a loss of intensity. Light, after b e i n g m a d e parallel w i t h lens Lv, goes t h r o u g h the c e n t e r of a r o t a t i n g p o l a r i z e r (2rpm, P o l a r o i d HN 32). This slightly c o m p l i c a t e s the m e c h a n i c a l device, but o v e r c o m e s a serious difficulty w h i c h w e h a v e e x p e r i e n c e d in selecting large pieces of P o l a r o i d sheet of u n i f o r m transmission. T h e same p o l a r i z i n g effect as d e s c r i b e d above should be c o n s i d e r e d and c o r r e c t e d for w i t h the o b s e r v a t i o n m o n o c h r o m a t o r . Calculation and exper i e n c e s h o w that the e r r o r is s m a l l e r in this case. H o w e v e r , since it is not negligible a n d d e p e n d e n t on the m a g n i t u d e of p, it was n e c e s s a r y to p l a c e a n o t h e r P o l a r o i d in v e r t i c a l position before the e n t r a n c e of the o b s e r v a t i o n m o n o c h r o m a t o r (not s h o w n in Fig. 2b). W i t h this modification, the s i m p l i c i t y of the p r i n c i p l e is lost and :
163
plified (Multimesureur (SERVOTRACE SEFRAM).
LEMOUZY) and
recorded
b. Operation. T h e speed of the c h a r t is set in such a w a y as to p r o d u c e a well resolved sinusoidal c u r v e w h e r e the m a x i m a and m i n i m a (F x and F~) can be l o c a t e d g r a p h i c a l l y (Fig. 4b). A dark c o n t r o l (light off) is n e e d e d to c o r r e c t for the d a r k current. RESULTS. T h e s e n s i t i v i t y and p r e c i s i o n of the t w o instruments have been tested t a k i n g v a r i o u s solutions of o r g a n i c dyes. W i t h s o d i u m fluorescein salt, meas u r e m e n t s of p h a v e been c a r r i e d d o w n to 10 -8 M, w i t h an absolute e r r o r of 8.10 -a. 1
I
F a -= I v F~ = I u the signal is r e d u c e d by m o r e than 50 p. cent. It should be noted that the o b s e r v a t i o n m o n o c h r o m a t o r m a y be r e p l a c e d by b r o a d b a n d filters in most a p p l i c a t i o n s w h e r e the d e p e n d a n c e of p on w a v e l e n g t h in the e m i s s i o n band is not c o n s i d e r e d as such. T h e glass cuvette, also housed in a t h e r m o s t a t e d block, has t w o a l u m i n i z e d a d j a c e n t faces for inc r e a s i n g the fluorescence signal. F l u o r e s c e n c e is detected w i t h a p h o t o m u l t i p l i e r (RADIOTEEHNIQUE XP 1 002) o p e r a t e d at 1 000 V. T h e signal is am-
IN,TENSITY one
r I
°"~
FT
rotation
- 2 tH ,
1
k ta)
.J (b)
L
TIME
FIt;. 4. - - Examples of recording ; (a) with al)paratus n ° 1 (b) with apparatus n ° '2.
BIOCHIMIE, 1972, 54, n ° 2.
500
1000
1500
FIG. 5. Perrin's plot for fluorescein (10-6M) in various concentrations of glycerol, at 20°C. T/~1 is in °K per poise. The squares correspond to measurements done with the apparatus n ° 1, and the circles, with the apparatus n ° 2. -
-
F i g u r e 5 shows an instance of P e r r i n ' s plot for fluorescein. F r o m the slope of the straight line, a lifetime of 4,3 ns is c o m p u t e d , assuming a m o l a r v o l u m e of 500 cln:~ ; this result agrees w i t h the data of P e r r i n [1, 2]. Po has been d e t e r m i n e d from m e a s u r e m e n t s w i t h v a r i o u s dyes and found to agree w i t h p u b l i s h e d data (Table I).
FLUORESCENCE
rotation
T
DISCUSSION. Besides its i n h e r e n t s i m p l i c i t y w h i c h allows c o n s i d e r i n g its a d a p t a l i o n to almost any fluorimeter, this m e t h o d has several a d v a n t a g e s : 1) In c o n v e n t i o n a l i n s t r u m e n t s w i t h t w o fixed p o s i t i o n s (<( v e r t i c a l >>, <( h o r i z o n t a l >>) of the pola-
J. Lavorel, C. Vernotte, B. Arrio and F. Rodier.
164
rizer, the t w o c o r r e s p o n d i n g d i r e c t i o n s s h o u l d be p r e c i s e l y at 90 ° a n d f r u t h e r the axis of the polar i z e r must be p r e c i s e l y set to c o i n c i d e w i t h one of these d i r e c t i o n s [see Weber, 7~. This is automatically o b t a i n e d w i t h the r o t a t i n g polarizer. Other sources of e r r o r (non p a r a l l e l i s m of beams, n o n n o r m a l i t y of o p t i c a l axis) o b v i o u s l y m u s t be minimized.
r e m e n t by q u i c k l y g a t h e r i n g a large n u m b e r of values of F 1 and F e on the r e c o r d e r chart. One m i g h t i m a g i n e several w a y s of getting a b e t t e r effic i e n c y f r o m this m e t h o d . In all case, the f r e q u e n c y of the r o t a t i n g p o l a r i z e r could be i n c r e a s e d at advantage. One obvious w a y of using best the r e p e t i t i v e p r o p e r t y w o u l d be to pass the signal to a s a m p l i n g a n d a v e r a g i n g amplifier.
TABLE I.
P, determinations with various dyes. Substances Fluorescein . . . . . . . . . . . . . . . . . . . . . . . . . . F l u o r e s c e i n isothiocyanate . . . . . . . . . . . . .
Rhodamin B . . . . . . . . . . . . . . . . . . . . . . . . Quinine sulfate . . . . . . . . . . . . . . . . . . . . . . Dimethyl amino naphtalene 5 sulfonate Dimethyl amino naphtalene 7 sulfonate
2) W i t h a u t o m a t i c operation, although m o d e rately fast in its p r e s e n t versions, this t y p e of a p p a r a t u s is w e l l suited for m a n y k i n e t i c as well as s p e c t r o s c o p i c studies. 3) B e i n g r e p e t i t i v e , this t y p e of i n s t r u m e n t a l r e a d y h e l p s to i m p r o v e the p r e c i s i o n of measu-
iI
Chalnnel-ch@~~li2F2
:
(IF 1 + ~F 2
i2 :
~ F 1 + o. 5
commutation il lv. <'- Input signals 9 ~ •
Output signal
-i 1
I F,
1
"~ U a ~ l O Fla. 6. - - Computation network for p (see text).
BIOC.HIMIE, 1'972, 54, n ° 2.
Po given in references
Po measured 0,435 -t- 0,008 0,435 0,440 0,460 0,270
0,440
[Weber. 7]
0,444
[Weber, 71 [Perrin, 1]
0,2651 [Weber, 7]
H o w e v e r , some g r a p h i c a l o p e r a t i o n w o u l d still be needed. A solution e l i m i n a t i n g this step is prop o s e d on Fig. 6. It is best a p p l i e d to v e r s i o n n ° 2 (with the o b s e r v a t i o n m o n o c h r o m a t o r r e p l a c e d by filters) w h i c h in p r i n c i p l e gives a l a r g e r a m o u n t of i n f o r m a t i o n p e r cycle of operation. T h e signal is s y n c h r o n o u s l y c o m m u t e d so as to send the t w o successive half w a v e s into separate chanels. Both signals are filtered and c o m b i n e d in an analog c o m p u t a t i o n n e t w o r k w h i c h solves the equations i 1 = aF 1 + ~F2 i 2 -~ ~F 1 -1- a F 2 for F 1 a n d F~ 01 and i 2 are the output of the channels, a and [3 are n u m e r i c a l factors r e s u l t i n g f r o m filtering). A f u r t h e r i m p r o v e m e n t in autom a t i z a t i o n could be the analog c o m p u t a t i o n of F 1 - F 2 and f e e d - b a c k c o n t r o l of the i n p u t signals (e. g. by v a r y i n g t h e voltage of the p h o t o m u l t i plier) l o c k i n g F 1 to a r e f e r e n c e value t a k e n as unity. E q u a t i o n (4) shows that in this case : m e a s u r e of p = F 1 - F
2
W h e n the o b s e r v a t i o n m o n o c h r o m a t o r is used w i t h the P o l a r o i d at the e n t r a n c e , e q u a t i o n (4) should be r e p l a c e d by : F 1 --F 2 p -F 1 -t- F2 and an a d d i t i o n a l c o n n e c t i o n (dashed line on fig. 0) is used. R~suM~. Deux appareils destines & la mesure des taux de polarisatiou de fluorescence sont d~crits ; ils ont ~t~ construits suivant le prineipe que toute l'information
Fluorescence polarization measurement. n6cessaire p o u r calculer le t a u x de p o l a r i s a t i o n p e u t ~tre o b t e n u e en m o d u l a n t le f a i s c e a u d ' e x c i t a t i o n avec u n p o l a r i s e u r . Les a v a n t a g e s de cette m ~ t h o d e s o n t sa simplieit~ et p a r c o n s e q u e n t la possibilit~ de l ' a d a p t e r f a c i l e m e n t s u r u n fluorim~tre, sa sensibilrit~, son aptit u d e a u x m e s u r e s cindtiques, enfin l ' a u t o m a t i s a t i o n p e r m e t t a n t d ' o b t e n i r d i r e c t e m e n t la v a l e u r du t a u x de polarisation. ZUSAMMENFASSUN,G.
Zwei Geriite z u r M e s s u n g der F l n o r e s z e n z p o l a r i s a tion w e r d e n b e s c h r i e b c n ; sie w e r d e n a u f G r u n d des P r i n z i p s gebaut, w o n a c h die g e s a m t e , z u r B e r e c h n u n g yon p b e n 6 t i g t e I n f o r m a t i o n m i t Hilfe eines i m a n r e genden S t r a h l e n g a n g befindlichen r o t i e r e n d e n P o l a r i s a t o r s e r h a l t e n w e r d e n k a n n , u.z. als eine Folge yon M a x i m a u n d M i n i m a der Fluoreszenzintensit~it, welche s e n k r e c h t z u r R i c h t u n g des e i n f a l l e n d e n S t r a h l e n b i i n dels e m i t t i e r t vcird. Die w i c h t i g s t e n Vorteile dieser Methods s i n d ihre E i n f a c h h e i t u n d folglich der leichte E i n b a u in die
BIOCHIMIE, 1972, 54, n ° 2.
165
h e r k 6 m m l i c h e n F l u o r i m e t e r , ihre E m p f i n d l i c h k e i t , ihre Fiihigkeit z u r k i n e t i s c h e n M e s s u n g u n d die M6glichkeit der a u t o m a t i s i e r t e n E r f a s s u n g yon p. REFERENCES. 1. P e r r i n , F. (1929') Ann. Phys. Fr., 12, 169. 2. P e r r i n , F. (1926) J. Phys. Radium, 7, 390. 3. Feofilov, P. P. << T h e P h y s i c a l b a s i s of p o l a r i z e d e m i s s i o n >> E n g l i s h T r a n s l a t i o n , C o n s u l t a n t s B u r e a u , New-York, 1961. 4. W e b e r , G. (1953,) Ado. Protein Chem., 8, 415. 5. P r i n g s h e i m , P. << F l u o r e s c e n c e a n d P h o s p h o r e s cence >> I n t e r s c i e n c e P u b l i s h e r s , New-York, 1949. 6. W e b e r , G. in ¢ F l u o r e s c e n c e a n d P h o s p h o r e s c e n c e A n a l y s i s >>, p. 217, Interscience P u b l i s h e r s , J. W i l e y a n d Sons, New-York, 1967. 7. Weber, G. (195~6) J. Opt. Soc. Am., 46, 962. 8. Monnerie, L. ~ N.eels, J. (1965) J. Chim. phys. Phys. Chim. biol. Ft., 62, 504. 9. D e r a n l e a u , D. A. (19,66) Anal. Biochem., 16, 438. 10. Mc Kay, R. (1969) Arch. of Biochem. and Biophys., 135, 218.