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15 Use of the OMA for Analyzing Light Intensity Gradients

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from the Absorption Optical System of the Ultracentrifuge DARREL L. ROCKHOLT and E. GLEN RICHARDS Pre-Clinical Science Unit, Veterans Administration Medical Center and Department of Biochemistry, University of Texas Health Science Center, Dallas, TX 75216 In this symposium on Image Detectors in Chemistry there have been described a number of physical techniques that have been greatly aided by the use of the Optical Multichannel Analyzer (OMA) for the recording of light intensity patterns. We have used the OMA with the absorption optical system of the ultracentrifuge (1,2). Obtaining data from any desired cell in a multicell spinning rotor requires a Silicon Intensified Target (SIT) vidicon camera tube operating in the pulsed mode; this detector system is not described elsewhere in this symposium. Described in this paper are three aspects of our work dealing with the verification of the performance of the system that may aid other users of the OMA: 1) the measurement of the deterioration of the image caused by pulsing the SIT vidicon and how to eliminate the deterioration, 2) the measurement of pincushion distortion, and 3) the use of a triangular mask in a spinning rotor to simulate an optical density wedge. Background Information H i s t o r i c a l . The u l t r a c e n t r i f u g e was developed i n the l a t e 1920 s by Svedberg and co-workers f o r the purpose o f generating high c e n t r i f u g a l f i e l d s t o study the sedimentation behavior o f macromolecules i n s o l u t i o n , thereby p r o v i d i n g i n f o r m a t i o n as to t h e i r s i z e and shape (3,4_). The f i r s t commercial instrument was constructed by a company that l a t e r became the Spinco D i v i s i o n o f Beckman Instruments, Inc. (Palo A l t o , CA). In s p i t e of the development of other commercial u l t r a c e n t r i f u g e s , most of the i n struments c u r r e n t l y i n use a r e v e r s i o n s of the o r i g i n a l d e s i g n updated w i t h improved components as they became a v a i l a b l e . 1

Rotor and C e l l s . I n the u l t r a c e n t r i f u g e a r o t o r spins about a v e r t i c a l a x i s a t speeds ranging from about 600 to 68,000 rpm. A v a r i e t y of r o t o r s can be used, h o l d i n g 2, 4 or 6 c e l l s . The c e l l s , 1 i n . i n diameter, a r e assembled from a p a i r o f t r a n s p a r ent windows, quartz o r sapphire, i n holders placed on both s i d e s This chapter not subject to US copyright. Published 1979 American Chemical Society Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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of a hollowed c e n t e r p i e c e . The p a r t s are assembled i n a c y l i n d r i c a l b a r r e l with a bottom l i p , and then a screwring on top i s tightened to prevent leakage. A f t e r the s o l u t i o n to be studied i s introduced with a s y r i n g e through a small hole i n the s i d e of the c e l l , a s e a l i n g plug i s screwed i n t o the b a r r e l . In F i g u r e 1 i s shown the top view of a f o u r - c e l l r o t o r con t a i n i n g a counterbalance and three kinds of c e l l s . The counter balance i s constructed with two D-shaped holes which provide r e f erence edges f o r the image corresponding to d i s t a n c e s of 5.7 and 7.3 cm from the center of the r o t o r . A l a r g e v a r i e t y of c e l l centerpieces are a v a i l a b l e f o r d i f f e r e n t purposes. U s u a l l y the w a l l s are constructed so as to l i e along r a d i i from the r o t o r center. (For other shapes there are convective disturbances i n the s o l u t i o n caused by d e n s i t y i n v e r s i o n s a r i s i n g from sedimen t a t i o n of s o l u t e molecules against or away from the w a l l s . ) Two s i n g l e - s e c t o r c e l l s are shown with d i f f e r e n t angular openings, and the t h i r d c e l l contains two s e c t o r s which can be f i l l e d w i t h two d i f f e r e n t s o l u t i o n s . U s u a l l y one i s the s o l u t i o n c o n t a i n i n g the s o l u t e of i n t e r e s t and the other i s the s o l v e n t . Absorption O p t i c a l System. During an u l t r a c e n t r i f u g e e x p e r i ment the s o l u t e , i f denser than the s o l v e n t , sediments toward the bottom of the c e l l , thereby generating a c o n c e n t r a t i o n g r a d i e n t . (There are many types of experiments; the two most common types w i l l be described l a t e r . ) The instrument i s constructed with two separate o p t i c a l systems that record the c o n c e n t r a t i o n gradient during an experiment. One of these, the absorption o p t i c a l s y s tem, i s e s p e c i a l l y u s e f u l i n biochemistry f o r the study of pro t e i n s and n u c l e i c a c i d s which have chromophores which absorb v i s i b l e or uv l i g h t . Since the detector system can only measure the l i g h t i n t e n s i t y , the c o n c e n t r a t i o n c i s determined from the Beer-Lambert law, A = l o g Io/I = E e l , where A i s the absorbance, I i s the l i g h t i n t e n s i t y a f t e r passage through the s o l u t i o n of l e n g t h 1, Io i s the i n t e n s i t y through s o l v e n t , and Ε i s the e x t i n c t i o n c o e f f i c i e n t f o r that p a r t i c u l a r wavelength of l i g h t . In the a b s o r p t i o n o p t i c a l system the c e l l i s i l l u m i n a t e d by monochromatic l i g h t obtained from a lamp with a f i l t e r or w i t h a monochromator. ( A d d i t i o n a l d e t a i l s of t h i s part of the system w i l l not be given here.) The remainder of the system i s shown i n F i g u r e 2. The l i g h t passes through the c e l l and then through a narrow s l i t (not shown) p a r a l l e l to a r a d i u s through the center of the r o t o r . The l i g h t continues through a condensing lens which converges the l i g h t to a narrower beam f o r passage through the camera l e n s . The camera lens-condensing lens combination focuses an image of the c e l l onto a s u i t a b l e d e t e c t o r , which provides the r a d i a l d i s t r i b u t i o n of l i g h t i n t e n s i t y i n the c e l l . The r a d i a l absorbance p r o f i l e i s obtained from a combination of the i n t e n s i ty p r o f i l e s from a s o l u t i o n and a solvent c e l l .

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Figure 1.

Ultracentrifuge Light Analysis

Schematic drawing of a four-cell rotor, top view

Figure 2. Scale drawing of camera lens system as used with OMA. (The vertical direction is enlarged about 8 times for chrity.) The system has been turned through 90°; the actual light path is vertical. L, is the condensing lens, f = 69.25 cm at 260 nm. L is the camera lens, f = 21 cm. U = 8.25 cm, V, = 9.37 cm, U = 54.7 cm, V = 29.2 cm. The thickness of the cell in the direction of the optical path is usually 1.2 cm. 2

2

t

2


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F i l m and P h o t o m u l t i p l i e r Detector Systems. In the o r i g i n a l u l t r a c e n t r i f u g e b u i l t by Svedberg and co-workers 03>A) and a l s o i n the commercial instrument, the l i g h t i n t e n s i t y p a t t e r n was r e corded on f i l m , r e q u i r i n g a densitometer to o b t a i n the a b s o r p t i o n profile. Schachman and co-workers (5,6) constructed a mechanic a l l y scanned p h o t o m u l t i p l i e r system i n the e a r l y 1960 s. In a d d i t i o n to a commercial v e r s i o n now a v a i l a b l e ( 7 ) , a number of other systems w i t h computer c o n t r o l l e d gathering of data have been constructed (8, 9_, 10, 11, 12). f

V i d i c o n Detector Systems. I t was obvious to many workers that the use o f a t e l e v i s i o n camera tube as the l i g h t d e t e c t o r would o f f e r a number of advantages over the p h o t o m u l t i p l i e r tube. The viewing o f the image i n r e a l time would a i d i n a l i g n i n g the o p t i c a l system and a l s o i n d e c i s i o n making d u r i n g an experiment. L l o y d and Esnouf (13) constructed a vidicon-based system of t h e i r own design f o r the u l t r a c e n t r i f u g e , but i t had a number o f drawbacks. Any refinements t o t h e i r system have not appeared i n publication. When the commercial OMA (Model 1205, P r i n c e t o n Applied Research C o r p o r a t i o n , P r i n c e t o n , NJ) became a v a i l a b l e , we recogn i z e d i t s p o t e n t i a l as a replacement f o r the p h o t o m u l t i p l i e r det e c t o r . The v i d i c o n d e t e c t o r s u r f a c e was d i v i d e d i n t o 500 chann e l s , the image could be seen on a cathode r a y tube (CRT) monitor i n r e a l time, the i n t e n s i t y p r o f i l e was a v a i l a b l e i n d i g i t a l form, the p r o f i l e could be time-averaged f o r any d e s i r e d number of video scans, and the f i n a l p r o f i l e was stored i n i n t e r n a l memory f o r t r a n s f e r to an e x t e r n a l output d e v i c e . Not only had a c o n s i d e r a b l e amount o f work gone i n t o i t s development and the v e r i f i c a t i o n of performance, but i t s p o t e n t i a l f o r use f o r a v a r i e t y of p h y s i c a l techniques would ensure the c o n s t r u c t i o n of enough u n i t s t o support f u r t h e r development of the system. Moreover the need f o r l o w - l i g h t - l e v e l d e t e c t o r s f o r other purposes would lead t o f u r ther improvements i n d e t e c t o r d e v i c e s . Our f i r s t work with the OMA demonstrated t h a t , with a modif i e d o p t i c a l system e a s i l y c o n s t r u c t e d , absorbance data could be obtained that were as good as those from e x i s t i n g p h o t o m u l t i p l i e r systems ( 1_). However, there were drawbacks. Even though the Model 1205F u v - s e n s i t i v e v i d i c o n d e t e c t o r head was used, our medium pressure Hg a r c lamp w i t h i n t e r f e r e n c e f i l t e r s d i d not prov i d e s u f f i c i e n t uv l i g h t i n the 250-280 nm range t o make up f o r the 1% duty c y c l e o f a c e l l i n a spinning r o t o r . I t was u n l i k e l y that any simple monochromator system could provide s u f f i c i e n t l i g h t . Moreover i t was necessary t o o b t a i n the i n t e n s i t y p r o f i l e from a s o l u t i o n and solvent c e l l spun s u c c e s s i v e l y , a time-consuming process. Before an OMA-based system could be considered as a p o t e n t i a l replacement f o r the p h o t o m u l t i p l i e r scanner, i t was necessary t o extend the wavelength c a p a b i l i t y t o the 250-280 nm range and t o f i n d a method of examining c e l l s i n d i v i d u a l l y i n a m u l t i c e l l


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r o t o r . One way of s o l v i n g these problems was to use an i n t e n s e pulsed l i g h t source, such as a Xe a r c or a l a s e r , but such systems were expensive and d i d not have the r e p r o d u c i b i l i t y of pulse amp l i t u d e s u i t a b l e f o r time-averaging. The manufacturer of the OMA a l r e a d y had a v a i l a b l e a d e t e c t o r head w i t h an SIT v i d i c o n (Model 1205D), which i s an image i n t e n s i f i e r stage coupled d i r e c t l y to a s i l i c o n v i d i c o n tube. With app r o p r i a t e a c c e l e r a t i n g v o l t a g e s a p p l i e d to the photocathode, a gain of s i g n a l s t r e n g t h up to 1500 times could be achieved. Moreover i t had been demonstrated that the i n t e n s i f i e r stage could be gated on and o f f by the a p p l i c a t i o n of f l a t - t o p p e d pulses from a s u i t a b l e pulsed power supply. The only drawback to the SIT v i d i c o n was that l i g h t of wavelength lower than 350 nm was not transmitted through the f i b e r o p t i c s f a c e p l a t e . They overcame t h i s problem by p l a c i n g a very t h i n f l u o r e s c e n t f i l m against the f a c e p l a t e to convert the uv l i g h t to higher wavelengths f o r passage through the f a c e p l a t e . The f i l m d i d not s i g n i f i c a n t l y a f f e c t o p e r a t i o n f o r l i g h t of wavelengths above 350 nm, but i t provided s u f f i c i e n t conversion e f f i c i e n c y , about 10%, to make the SIT v i d i c o n c o n s i d e r a b l y more s e n s i t i v e than the uv v i d i c o n . Once p r e l i m i n a r y experiments with a pulsed SIT v i d i c o n s y s tem had demonstrated that i t performed s a t i s f a c t o r i l y i n the d e s i r e d uv r e g i o n and that i t could be s u c c e s s f u l l y gated to examine i n d i v i d u a l c e l l s i n a m u l t i c e l l r o t o r , we constructed an i n t e r f a c e - c o n t r o l l e r f o r automatic o p e r a t i o n of the OMA w i t h s i n g l e - or double-sector c e l l s i n m u l t i c e l l r o t o r s ( 2 ) . The c e l l l o c a t i o n i n the r o t o r and the s t a r t and stop of the gate pulse i s obtained from a phase-lock loop c i r c u i t . A mark on the r o t o r i n t e r r u p t s a l i g h t beam r e f l e c t e d from the r o t o r to a p h o t o c e l l , p r o v i d i n g a r e f e r e n c e p u l s e f o r each r o t a t i o n . The time between each p u l s e i s d i v i d e d i n t o 3600 p a r t s i r r e s p e c t i v e of the r o t o r speed. Appropriate switches are used to s e l e c t the c e l l h o l e i n the r o t o r and the pulse width, i n terms of the angle, f o r s i n g l e or double-sector c e l l s . A d d i t i o n a l d e t a i l s concerning the cont r o l s f o r automatic data gathering and the performance of the system are p u b l i s h e d elsewhere (2). When 8 - b i t microprocessor-based computers became a v a i l a b l e , we decided that such a computer, even though slower than m i n i computers, would be adequate to operate the OMA and our i n t e r face c o n t r o l l e r w i t h the u l t r a c e n t r i f u g e . We now have i n the system an A l t a i r 8800 computer w i t h 28K of memory (MITS, Albuquerque, NM), a hard-wired a r i t h m e t i c board and two m i n i f l o p p y d i s k d r i v e s (North S t a r , Berkeley, CA), a 700 ASR Terminal (Texas Instruments, D a l l a s , TX), and a 7202A Graphic P l o t t e r (HewlettPackard, Palo A l t o , CA) . Appropriate software, w r i t t e n i n B a s i c , has been developed to c o l l e c t i n t e n s i t y data from the OMA autom a t i c a l l y and a l s o to t r e a t and p l o t the data at the end of the experiment. D e t a i l s of the system and software w i l l be published elsewhere. We a l s o have an improved i l l u m i n a t i o n system with a 200 W Hg-Xe arc lamp and a Model H-20 monochromator w i t h a h o l o -


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graphic g r a t i n g (Jobin-Yvon, Metuchen, NJ) (14). Performance of the System The manufacturer p r o v i d e s s p e c i f i c a t i o n s f o r the performance of the OMA, i n c l u d i n g l i n e a r i t y of counts as a f u n c t i o n of i n t e n s i t y and the geometric d i s t o r t i o n and channel-to-channel c r o s s t a l k of the v i d i c o n . However, the user needs methods f o r v e r i f y i n g the performance of h i s OMA o p e r a t i n g w i t h h i s system. T h i s informat i o n i s needed both f o r d e s i g n i n g c o r r e c t i o n schemes i f d i s t o r t i o n i s found and f o r determining whether f u r t h e r improvements i n the system are needed. I d e a l l y the methods should be simple so that the t e s t i n g can be performed on a r o u t i n e b a s i s . Obtaining the g r e a t e s t p o s s i b l e accuracy i n molecular weight determinations and p o l y d i s p e r s i t y analyses r e q u i r e s the g r e a t e s t p o s s i b l e accuracy i n the absorbance p r o f i l e . As a s t a r t i n g t a r g e t we would be s a t i s f i e d w i t h an accuracy of 0.001 absorbance u n i t s i n the range from 0 to 1 absorbance u n i t s . Further e f f o r t to achieve greater accuracy would be warranted only when the t a r g e t l e v e l was reached. During the development of our OMA-based d e t e c t o r system, we performed a number of experiments checking the performance by measuring the absorbances of uniform s o l u t i o n s spun i n the u l t r a c e n t r i f u g e . I t was observed that the f l a t absorbance p r o f i l e s f o r the hundreds of p o i n t s f o r each c e l l v a r i e d only by about 0.002 absorbance u n i t s and a p l o t of the average absorbance f o r each c e l l v s . the c o n c e n t r a t i o n was l i n e a r from 0 to 1 absorbance u n i t s and even higher (]L,2). However, such a demonstration with uniform s o l u t i o n s does not prove that the c o r r e c t absorbance p r o f i l e i s obtained from a c e l l w i t h a c o n c e n t r a t i o n g r a d i e n t . Not only can e r r o r s i n absorbance measurements a r i s e from n o n - l i n e a r i t y i n the d e t e c t o r c i r c u i t r y , but d i s t o r t i o n i n the l i n e a r i t y of the p o s i t i o n of channel d e t e c t o r elements can lead to a corresponding d i s t o r t i o n of the measured i n t e n s i t y p r o f i l e . The geometric d i s t o r t i o n of the SIT v i d i c o n , as s t a t e d i n the s p e c i f i c a t i o n sheet s u p p l i e d by the OMA manufacturer, i s t y p i c a l l y 2 channels between channels 100 and 400 f o r a 2.5 mm high image centered on the tube. T h i s d i s t o r t i o n i s s u f f i c i e n t to r e q u i r e c o r r e c t i o n of data obtained f o r experiments w i t h steep concentrat i o n gradients i n our system. During the development of our OMA-based d e t e c t o r system we performed a number of t e s t s aimed a t v e r i f y i n g the c o r r e c t beh a v i o r of v a r i o u s components. With the completion of the e n t i r e system, i t was important to evaluate i t s performance by the examination of images obtained from o b j e c t s w i t h known dimensions and s o l u t i o n s of known sedimentation behavior. Before beginning these s t u d i e s , the o p t i c a l system was a l i g n e d according to the u s u a l procedures (15) and the d e t e c t o r head e l e c t r o n i c s were adjusted f o l l o w i n g the procedures given i n the Operating and S e r v i c e Manual supplied by the manufacturer.


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For the SIT v i d i c o n the l i n e - s c a n amplitude i s reduced to g i v e a 5 by 12.5 mm scanning p a t t e r n and adjusted so as to center the image on the tube f a c e . Adjustment of the symmetry c o n t r o l i s e s p e c i a l l y important. Misadjustment w i l l cause a d i s t o r t i o n i n the i n t e n s i t y p r o f i l e of a knife-edge image; a t the bottom i t w i l l appear rounded or i t w i l l f a l l below the zero i n t e n s i t y l e v e l . The image p a t t e r n presented to the face of the SIT v i d i c o n i s about 12 mm i n the h o r i z o n t a l d i r e c t i o n , corresponding to the r a d i a l dimension o f the c e l l , and about 1 mm i n the v e r t i c a l d i r e c t i o n , r e p r e s e n t i n g a b l u r r e d image of the defocused s t a t i o n a r y s l i t above the r o t o r . For each r e v o l u t i o n o f the r o t o r the v i d i con i s pulsed f o r both the r e f e r e n c e counterbalance h o l e and f o r the d e s i r e d c e l l s e c t o r (Figure 1 ) , r e s u l t i n g i n an image w i t h a long r e c t a n g l e (from the c e l l ) w i t h a s m a l l r e c t a n g l e on both ends (from the two counterbalance h o l e s ) . Ruled L i n e Target. For the measurement of p i n c u s h i o n d i s t o r t i o n , we decided t o examine a r u l e d l i n e t a r g e t . I t turned out that the i n t e n s i t y p r o f i l e s obtained f o r these measurements were a l s o u s e f u l f o r e v a l u a t i o n o f the d i s t o r t i o n caused by the p u l s i n g of the SIT v i d i c o n and a l s o f o r measurement of channel-to-channel crosstalk. The manufacturer of the u l t r a c e n t r i f u g e provides a r u l e d d i s k that when placed i n a s u i t a b l e holder i n a s t a t i o n a r y r o t o r a t the plane corresponding to half-way through the c e l l , serves both as a t a r g e t f o r f o c u s i n g the camera l e n s and as a s e r i e s of l i n e s of known spacing f o r determination of the o p t i c a l m a g n i f i c a t i o n f a c tor. The d i s k , constructed of g l a s s w i t h blackened, etched l i n e s 1 mm a p a r t , was not s u i t a b l e f o r our purposes, s i n c e i t could not transmit uv l i g h t and a l s o because there were small blemishes a t the edges o f the l i n e s . With the a i d o f Micrometrology ( D a l l a s , TX) we constructed a quartz d i s k w i t h a r u l e d p a t t e r n . A 10-times enlarged p a t t e r n was cut w i t h a blade i n t o R u b y l i t h u s i n g a coordinatograph with an accuracy b e t t e r than 0.001 i n . A photograph, reduced e x a c t l y 10 times, was obtained, and the image was t r a n s f e r r e d from the photograph t o a quartz d i s k u s i n g p h o t o r e s i s t technology. The f i n a l p a t t e r n c o n s i s t e d of 35 transparent l i n e s , 0.080 mm wide and spaced e x a c t l y 0.500 mm apart on an opaque background. The edges were s h a r p l y d e f i n e d , w i t h o n l y an o c c a s i o n a l blemish. Behavior o f Pulsed SIT V i d i c o n . Before proceding w i t h the experiments i n v o l v i n g the pulsed SIT v i d i c o n , i t i s necessary to d i s c u s s the e f f e c t of p u l s i n g the tube on the image. The s i g n a l gain o f the SIT v i d i c o n i s determined by the photocathode v o l t a g e , which can be r e g u l a t e d w i t h an adjustment potentiometer. When an image i s examined c o n t i n u o u s l y , the c o r r e c t v o l t a g e f o r the f o cusing g r i d of the i n t e n s i f i e r stage i s s u p p l i e d by the c i r c u i t , w i t h no adjustment r e q u i r e d when the g a i n i s changed. Operated i n the pulsed mode, the i n t e n s i f i e r stage r e q u i r e s an optimal pulse


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v o l t a g e which v a r i e s with the photocathode v o l t a g e . As the v o l tage of the pulse i s changed i n e i t h e r d i r e c t i o n from i t s optimal v a l u e , the sharpness of the image d e t e r i o r a t e s and the m a g n i f i c a t i o n of the image changes. During the r i s e and f a l l periods of the pulse d u r a t i o n , the i n t e n s i f i e r voltage i s incorrect, r e s u l t i n g i n a deterioration i n the f i d e l i t y of the f i n a l image, s i n c e i t w i l l c o n t a i n a s e r i e s of images which p r o g r e s s i v e l y change i n sharpness and s i z e . The magnitude and the c h a r a c t e r i s t i c s of the d e t e r i o r a t i o n i n the image, accumulated d u r i n g the p u l s e , depend upon the d u r a t i o n of the r i s e and f a l l periods r e l a t i v e to the t o t a l pulse time. Presumably the d e t e r i o r a t i o n i n the image can be reduced to a n e g l i g i b l e l e v e l by u s i n g a pulse generator with r i s e and f a l l times very short compared to the t o t a l pulse time. Procedure f o r Examination of Ruled Disk. Since the l i n e s on the r u l e d d i s k are s t r a i g h t and not c o n c e n t r i c a r c s about the center of the r o t o r , the sharpest image of the l i n e s i s obtained w i t h a s t a t i o n a r y r o t o r . The d i s k i n i t s holder was placed i n the r o t o r , which was coupled to the d r i v e . The c o r r e c t o r i e n t a t i o n of the l i n e s , p e r p e n d i c u l a r to a r a d i u s of the r o t o r , was accomplished by r o t a t i n g the d i s k holder to give the sharpest l i n e edges as examined on the monitor i n r e a l time (see F i g u r e 3 ) . The camera lens was focused to produce the sharpest l i n e images. Determination of Optimum Pulse Voltage f o r the SIT V i d i c o n . A number of procedures f o r the determination of the optimal pulse v o l t a g e were evaluated. Comparison of a s e r i e s of accumulated p a t t e r n s obtained at d i f f e r e n t v o l t a g e s proved to be no more s e n s i t i v e than examination of the image i n r e a l time as the v o l tage was changed. Since the m a g n i f i c a t i o n of the image changes w i t h v o l t a g e , the g r e a t e s t s e n s i t i v i t y was achieved by 1) f i n d i n g the l i n e near the center of the p a t t e r n from the r u l e d d i s k which did not move as the v o l t a g e was changed and 2) moving the v i d i c o n r e l a t i v e to the image u n t i l t h i s l i n e was centered between two channels. The center l i n e image, magnified h o r i z o n t a l l y and v e r t i c a l l y , was examined as the v o l t a g e was changed; at the o p t i mal v o l t a g e , the v e r t i c a l s i z e was maximum and the minima between the l i n e images were the lowest and the f l a t t e s t . For the p a r t i c u l a r gain s e t t i n g (about 700) that we used, the pulse v o l t a g e which gave the sharpest l i n e images f o r the r u l e d d i s k was about 700 V, as measured with a Model 465 o s c i l l o s c o p e (Tektronix, Inc., Beaverton, OR) u s i n g a 100X probe. The v o l t a g e was about the same whether u s i n g the Model 1211 ( P r i n c e t o n A p p l i e d Research Corp., P r i n c e t o n , NJ) or the Model 340 (Velonex, Santa C l a r a , CA) high v o l t a g e p u l s e generator. D e t e r i o r a t i o n of Image Caused by P u l s i n g the SIT V i d i c o n . When we began measuring the pincushion d i s t o r t i o n with the r u l e d d i s k , we had assumed that a pulse width of 10 ysec obtained from


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Figure 3. Intensity patterns from ruled disk obtained with varying pulse widths. Ordinate, relative intensity from 0 to 1; abscissa, channel number from 0 to 500. (a) unpulsed; (b) 10 psec; (c) 5 psec; (d) 2 psec; (e) 1 fisec; (f) 1 psec but changed from 700 to 800 V.


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the Model 1211 pulse generator would be s u f f i c i e n t to render negl i g i b l e the c o n t r i b u t i o n (1%) of the defocused image accumulated during the 50 nsec r i s e and f a l l periods of the p u l s e . When i t was found that the l i n e images from the pulsed SIT v i d i c o n were not as sharp as those from the v i d i c o n operated i n the continuous mode, a more d e t a i l e d study of the e f f e c t of pulse width on image q u a l i t y was undertaken. For these s t u d i e s the r u l e d d i s k was placed i n the r o t o r and adjusted as d e s c r i b e d e a r l i e r . The lamp p o s i t i o n was adjusted to give the most uniform i l l u m i n a t i o n p o s s i b l e at a wavelength of 265 nm. The pulse frequency, 578 Hz, was e q u i v a l e n t to a r o t o r speed of 33,467 rpm. For each pulse width, a s u f f i c i e n t number of scans were accumulated to g i v e between 80,000 and 90,000 counts maximum. For the s h o r t e r p u l s e widths, the entrance s l i t of the monochromator was widened to i n c r e a s e l i g h t throughput. The accumulated p a t t e r n s f o r the r u l e d d i s k were c o r r e c t e d f o r nonuniform i l l u m i n a t i o n and nonuniform response of the v i d i c o n with a n o r m a l i z i n g p a t t e r n obtained when the r o t o r was turned to an open h o l e . The number of scans f o r the l a t t e r p a t t e r n was r e duced to give about the same maximum number of counts. The i n t e n s i t y of each channel of the r u l e d d i s k p a t t e r n was d i v i d e d by the i n t e n s i t y of the same channel of the open p a t t e r n obtained at the same pulse width. The r e s u l t i n g r e l a t i v e i n t e n s i t y p a t t e r n was normalized by m u l t i p l y i n g by a constant so as to give a value of 1 f o r the channel with the maximum r e l a t i v e i n t e n s i t y . The r e l a t i v e i n t e n s i t y p a t t e r n s obtained from t h i s study are shown i n F i g u r e 3. Compared to the unpulsed p a t t e r n ( a ) , the l i n e images from the 10 ysec pulsed p a t t e r n (b) are l e s s sharp. The minima i n the center are elevated and the e l e v a t i o n i n c r e a s e s p r o g r e s s i v e l y on both s i d e s away from the center. Enlarged port i o n s of the s i d e s and center of the p a t t e r n s are shown i n F i g u r e 4a and b. These p a t t e r n s are c o r r e c t l y shown as bar graphs to emphasize that each channel segment gives an average of the l i g h t s t r i k i n g i t . The l i n e s f o r the unpulsed SIT v i d i c o n are symmetr i c a l across the p a t t e r n . For the pulsed SIT v i d i c o n the l i n e s i n the center are symmetrical, but the l i n e s away from the center become p r o g r e s s i v e l y rounded at the bottom on the s i d e f a c i n g away from the c e n t e r . Since a 1% c o n t r i b u t i o n of changing defocused images (based upon the r e l a t i v e times of the r i s e and f a l l periods of the pulse to the t o t a l duration) would produce only a small e f f e c t on the f i n a l p a t t e r n , one can only conclude that i n s i d e the i n t e n s i f i e r stage there e x i s t s a defocused s t a t e that i s c o n s i d e r a b l y longer than the 100 nsec combined r i s e and f a l l times measured o u t s i d e the tube. Moreover the p r o g r e s s i v e d i s t o r t i o n away from the center of the p a t t e r n i s what one would expect from a s u b s t a n t i a l c o n t r i b u t i o n of images of greater m a g n i f i c a t i o n caused by reduced v o l t a g e s during the r i s e and f a l l of the v o l t a g e i n s i d e the i n t e n s i f i e r stage. More i n f o r m a t i o n as to the a c t u a l d u r a t i o n of the defocused


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time p e r i o d can be obtained by examination of p a t t e r n s obtained for s h o r t e r p u l s e s . At 5 ysec (Figure 3c) the p r o g r e s s i v e d e t e r i o r a t i o n of l i n e s away from the center i n c r e a s e s . At 2 ysec (d) the envelope of maxima and manima narrows more r a p i d l y away from the center, then remains f l a t . At 1 ysec (e) the p a t t e r n changes i t s appearance, with increased d e t e r i o r a t i o n away from the c e n t e r . At 1 ysec, i n c r e a s i n g the p u l s e v o l t a g e from 700 to 800 V improves the sharpness of the l i n e s i n the c e n t e r , but the l i n e s away from the center d e t e r i o r a t e p r o g r e s s i v e l y then appear to improve s l i g h t l y at the edges of the p a t t e r n ( f ) . The envelope minima and maxima appear to converge, meet (or c r o s s o v e r , perhaps), then diverge again i n a manner suggestive of a phase i n v e r s i o n , and the spacing of the l i n e images i s discontinuous at the crossover r e gions. Moreover the p o s i t i o n s of the crossovers changed with p u l s e magnitide and width. There are at l e a s t two kinds of changes i n the appearance of the p a t t e r n s as the pulse width i s decreased, which suggests that more than one process i s i n v o l v e d as the v o l t a g e s of the photocathode r i s e and f a l l at the beginning and end of the p u l s e . We did not i n v e s t i g a t e the defocused c o n d i t i o n any f a r t h e r , as there were two ways of e l i m i n a t i n g the defocused p o r t i o n of the image. With the u l t r a c e n t r i f u g e the SIT v i d i c o n can be gated outs i d e the c e l l opening, thereby l i m i t i n g the defocused s t a t e to p e r i o d s of darkness while p r o v i d i n g c o r r e c t f o c u s i n g f o r the c e l l opening. However, p r o p e r l y focused images of the r u l e d d i s c are s t i l l needed to provide the m a g n i f i c a t i o n f a c t o r and a measure of the pincushion d i s t o r t i o n f o r u l t r a c e n t r i f u g e p a t t e r n s . For both pulse generators that we have examined, the shape of the p u l s e edges, as shown on the o s c i l l o s c o p e screen, remains the same as the p u l s e width i s p r o g r e s s i v e l y i n c r e a s e d . One can hyp o t h e s i z e that a c o r r e c t p a t t e r n f o r the focused s t a t e might be obtained from two pulses of d i f f e r e n t l e n g t h , but both s u f f i c i e n t l y long to allow the i n t e n s i f i e r stage to reach the c o n d i t i o n s f o r c o r r e c t focus. I t i s l i k e l y that the c o n t r i b u t i o n of the r i s e and f a l l periods to the f i n a l image would be the same f o r both p u l s e s . By s u b t r a c t i n g the image of the s h o r t e r pulse from that of the longer p u l s e , one would o b t a i n a c o r r e c t l y focused d i f f e r e n c e image corresponding to the c e n t r a l p o r t i o n of the longer p u l s e . T h i s hypothesis was t e s t e d by combining the images obtained for d i f f e r e n t widths shown above to o b t a i n d i f f e r e n c e images, even though the procedure f o r data gathering was not f u l l y s a t i s f a c t o r y . Only the r e l a t i v e i n t e n s i t y p a t t e r n s , not the i n d i v i d u a l d i s k and a i r p a t t e r n s , had been recorded on the d i s k e t t e s . Moreover s i n c e the r o t o r had been moved to record d i s k and a i r patterns f o r each p u l s e width, there was no assurance that the d i s k had always been returned to i t s o r i g i n a l p o s i t i o n , even though a r e f e r e n c e mark on the r o t o r and a s t a t i o n a r y p o i n t e r had been used as alignment a i d s . F i n a l l y a v a r i e t y of s l i t widths had been used f o r the monochromator.


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To o b t a i n a d i f f e r e n c e p a t t e r n from the normalized p a t t e r n s , i t was necessary to reduce the magnitude of the p a t t e r n f o r the s h o r t e r pulse to a s i z e corresponding to the r e l a t i v e l e n g t h of the p u l s e s ; t h i s was accomplished by m u l t i p l y i n g the i n t e n s i t i e s f o r the s h o r t e r pulse by the r a t i o of the pulse widths. Difference p a t t e r n s f o r 10-5, 10-3, 5-3 and 3-2 ysec (not shown) were of a q u a l i t y s i m i l a r to the p a t t e r n obtained from the continuous SIT v i d i c o n , with minima near the zero l e v e l . However, the 2-1 ysec d i f f e r e n c e p a t t e r n was u n s a t i s f a c t o r y , with the l i n e maxima prog r e s s i v e l y decreasing i n i n t e n s i t y away from the c e n t e r . In Table I are l i s t e d the m a g n i f i c a t i o n f a c t o r s obtained f o r the d i f f e r e n c e p a t t e r n s and the 1 ysec p a t t e r n (1-0 ysec) from the l e a s t squares s t r a i g h t l i n e through the centers of the 12 l i n e s i n the center of the p a t t e r n . The i n c r e a s e i n m a g n i f i c a t i o n f o r the 2-1 and 1 ysec p a t t e r n s suggests that during the time p e r i o d s encompassing the f i r s t and l a s t microsecond of the p u l s e , the SIT v i d i c o n i s i n a defocused s t a t e . The c o n t r i b u t i o n from these periods i s s u f f i c i e n t to warp p a t t e r n s obtained from pulses 3-10 ysec long and even longer. In s p i t e of the d e f i c i e n c i e s i n the data gatheri n g , t h i s study demonstrates that the defocused p o r t i o n of a patt e r n can be removed by the use of a p p r o p r i a t e d i f f e r e n c e p a t t e r n s . TABLE I M a g n i f i c a t i o n of D i f f e r e n c e P a t t e r n s Difference Pattern

10-5 10-3 5-3 3-2 2-1 1-0

ysec ysec ysec ysec ysec ysec

Magnification Factor Channels/mm 26.69 26.68 26.56 26.65 27.19 28.39

Since the study d e s c r i b e d above was performed with a borrowed Model 1211 pulse generator, we deemed i t a d v i s a b l e to examine patterns obtained w i t h our Model 340 Velonex pulse generator to see i f i t e x h i b i t e d the same behavior. The experimental procedures were changed to avoid the problems d e s c r i b e d above and two new parameters were i n v e s t i g a t e d . The wavelength was changed to 405 nm. The l i n e images should be sharper, s i n c e more of the l i g h t i s transmitted d i r e c t l y , thereby a v o i d i n g the randomization of the entrance angle caused by wavelength conversion w i t h i n the s c i n t i l l a t i o n f i l m . Even though r e l a t i v e i n t e n s i t y p a t t e r n s had been obtained e a r l i e r f o r 5 v o l t a g e s encompassing 650-750 V, i t was p o s s i b l e that d i f f e r e n c e p a t t e r n s would provide more i n f o r m a t i o n as to the e f f e c t of v o l t a g e on image q u a l i t y . Therefore d i f f e r ence p a t t e r n s were obtained f o r 725 V, the v o l t a g e that gave the


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sharpest p a t t e r n f o r the Velonex p u i s e r , and a l s o at 700 and 750 V. A f t e r a d j u s t i n g the i l l u m i n a t i o n of the c e l l f o r 405 nm l i g h t the camera l e n s was moved to refocus the r u l e d d i s k . With the r u l e d d i s k i n the same p o s i t i o n , p a t t e r n s f o r 8.96 and 4.48 ysec p u l s e widths were obtained at the three v o l t a g e s mentioned above. Since the v o l t a g e of the Velonex pulse generator increased s l i g h t l y when the pulse width was reduced, a s l i g h t adjustment of the v o l t a g e c o n t r o l was r e q u i r e d to maintain the same v o l t a g e f o r the s h o r t e r p u l s e . Moreover the v o l t a g e across the pulse was not constant; i t was a r b i t r a r i l y decided to adjust the height of the s h o r t e r p u l s e , as observed on the o s c i l l o s c o p e screen, to match the f i r s t h a l f of the longer p u l s e . For each p a t t e r n s u f f i c i e n t scans were accumulated to b r i n g the maximum counts to between 90,000 and 100,000 counts, but the monochromator s l i t was not touched. A f t e r the s i x p a t t e r n s f o r the r u l e d d i s k were obtained, s i x p a t t e r n s were obtained f o r a i r with the same sequence of v o l t a g e s and p u l s e widths. For each image, the i n t e n s i t i e s were d i v i d e d by the number of scans. Then f o r each of the three v o l t a g e s the d i f f e r e n c e i n the images f o r the d i s k at the two pulse widths was d i v i d e d , channel by channel, by the d i f f e r e n c e i n the images f o r a i r . Then the r e l a t i v e d i f f e r e n c e p a t t e r n s were normalized as before to g i v e a maximum v a l u e of 1. The l i n e images f o r the p a t t e r n s obtained at the two pulse widths were d i s t o r t e d as was shown i n F i g u r e 3. However, the q u a l i t y of the l i n e images i n the normalized d i f f e r e n c e p a t t e r n f o r 725 V (Figure 4c) were as good as those obtained f o r the unpulsed SIT v i d i c o n . There i s only a small amount of increased curvature at the minima on the s i d e of the l i n e s away from the center. The d i f f e r e n c e p a t t e r n s f o r 700 and 750 V (not shown) were more rounded at the minima between the l i n e s , but c a r e f u l examination provided no improved c r i t e r i a f o r t h e i r use i n determining the optimum focus v o l t a g e . Even though the i n t e n s i t y p r o f i l e of a l i n e or knife-edge image i s d i s t o r t e d by p u l s e shape and pincushion d i s t o r t i o n , one would expect the p r o f i l e obtained from an image with n e a r l y u n i form i l l u m i n a t i o n to be c o n s i d e r a b l y l e s s a f f e c t e d . The normali z e d a i r p a t t e r n s obtained at 725 V f o r the two pulse widths (not shown) were the same, except f o r small d i f f e r e n c e s at the s i d e s of the p a t t e r n . A comparison of the d i f f e r e n c e p a t t e r n f o r the pulsed SIT v i d i c o n (Figure 4c) and the p a t t e r n f o r the unpulsed SIT v i d i c o n (a) r e v e a l s that the minima between the l i n e s are c l o s e r to the zero l e v e l f o r the former p a t t e r n . Since we had changed the wavel e n g t h from 265 to 405 nm to i n v e s t i g a t e the e f f e c t of the f l u o rescent f i l m on channel-to-channel c r o s s t a l k , i t seemed l i k e l y that the increased sharpness of the l i n e s f o r the d i f f e r e n c e p a t t e r n was due to t h i s change. To confirm t h i s c o n c l u s i o n and a l s o to v e r i f y that the improved methodology gives good d i f f e r e n c e patterns i n a r e p r o d u c i b l e f a s h i o n the 'ruled d i s k and a i r p a t t e r n s


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Figure 4. Magnified line images for ruled disk at sides and center of image, (a) unpulsed; (b) 10 yisec pulse; (c) difference pattern for 8.96-4.48 sec.


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were obtained f o r the two pulse widths at 725 V using l i g h t at 265 nm. The normalized d i f f e r e n c e p a t t e r n (not shown) c l o s e l y resembled the p a t t e r n f o r the unpulsed SIT v i d i c o n (Figure 4a) with r a i s e d minima, thereby v e r i f y i n g that the f l u o r e s c e n t f i l m s l i g h t l y increased the channel-to-channel c r o s s t a l k . Pincushion D i s t o r t i o n . The major aim of the study i n v o l v i n g the r u l e d d i s k was the measurement of the d i s t o r t i o n of the image introduced by the i n t e n s i f i e r stage of the SIT v i d i c o n , which i s known to be of the "pincushion type. Since the spacing of the l i n e s on the d i s k i s accurate to 1 ym, the d i s t o r t i o n i s e a s i l y measured, i n p r i n c i p l e , by p l o t t i n g the channel number of the peak p o s i t i o n of the l i n e image against the d i s t a n c e of that l i n e from the f i r s t l i n e . The measurements from the d i s k can be t r a n s l a t e d to d i s t a n c e s from the center of the r o t o r by comparison with the channel numbers obtained from the r e f e r e n c e edges of the counterbalance c e l l (Figure 1). Knowing the s i z e of the image on the f a c e p l a t e of the SIT v i d i c o n , one could a l s o measure the d i s t o r t i o n i n terms of the d i s t a n c e from the center of the tube. Based on e a r l y measurements with f i l m placed at the image plane, we know that the image has a m a g n i f i c a t i o n of about 0.5. Since experiments are performed at a v a r i e t y of wavelengths, r e q u i r i n g a change i n camera lens p o s i t i o n with a change i n m a g n i f i c a t i o n , we are concerned only with the f i n a l m a g n i f i c a t i o n of the e l e c t r o n i c image compared to the d i s tance i n the c e l l . The "Frame Scan" potentiometer i s f r e q u e n t l y adjusted to move the image of the r e f e r e n c e holes to p o s i t i o n s near the outer edges of the p a t t e r n ; a m a g n i f i c a t i o n f a c t o r i s then obtained with the r u l e d d i s k . Thus, f o r our purposes the measurement of d i s t o r t i o n r e l a t i v e to d i s t a n c e s on the f a c e p l a t e serves no purpose. The patterns obtained from the study of d i s t o r t i o n caused by p u l s i n g were examined f o r r a d i a l d i s t o r t i o n . Since the l i n e im ages are not n e c e s s a r i l y symmetrical and most of the l i g h t f o r each l i n e i s spread over 8 to 10 channels, a problem a r i s e s con c e r n i n g the d e f i n i t i o n of the l i n e center. Based upon an a p p r o x i mation to a gaussian shape, y = exp(-x^), we chose to f i t the l o g of the i n t e n s i t i e s f o r each group of p o i n t s to a cubic equation. The use of a c u b i c equation permitted a b e t t e r approximation to the asymmetric p r o f i l e . The p o s i t i o n of the l i n e was determined by c a l c u l a t i n g the χ p o s i t i o n where the s l o p e , determined from the f i r s t d e r i v a t i v e , was 0. (A d i f f e r e n t procedure, not t r i e d , would be to sum the i n t e n s i t i e s and to f i n d the f r a c t i o n a l channel num ber that would p l a c e h a l f the l i g h t i n t e n s i t y on both s i d e s of that number.) Examination of many p l o t s of image l i n e p o s i t i o n v s . a c t u a l l i n e p o s i t i o n revealed that the center 11 to 13 p o i n t s were n e a r l y l i n e a r , with the other p o i n t s , upon moving on e i t h e r s i d e away from the center, d e v i a t i n g p r o g r e s s i v e l y from a s t r a i g h t l i n e . To best demonstrate the d i s t o r t i o n , the p o s i t i o n s f o r the 12 l i n e s i n 11


334


the center were f i t with a l e a s t mean squares s t r a i g h t l i n e , then the d i f f e r e n c e between the measured l i n e p o s i t i o n s and the p o s i t i o n s c a l c u l a t e d by extending the s t r a i g h t l i n e was c a l c u l a t e d . Three such p l o t s f o r the unpulsed SIT v i d i c o n and the d i f f e r e n c e p l o t s obtained f o r 725 V w i t h i l l u m i n a t i o n at 265 and 405 nm are shown i n F i g u r e 5. The χ dimension has been converted to d i s tances i n the c e l l measured from the center of the r o t o r . The d e v i a t i o n approaches 0.02 cm at the e x t r e m i t i e s , which corresponds to 4 channel numbers. The p a t t e r n d e v i a t e s more on the r i g h t , i n d i c a t i n g that the frame scan i s not centered w i t h r e s p e c t to the f a c e p l a t e , an adjustment that could e a s i l y be made. I t should be emphasized that these p l o t s represent the com bined d i s t o r t i o n caused by the camera l e n s and the SIT v i d i c o n . The p l o t s could be used to c o r r e c t data obtained from u l t r a c e n t r i f u g e experiments. For some experiments only the 6.7 to 7.2 cm r e g i o n of the c e l l i s a c t u a l l y used. Thus a 10 ysec pulse p a t t e r n was obtained w i t h the v i d i c o n moved a d i s t a n c e corresponding to 125 channels, so as to p l a c e i t s center over the r e g i o n of i n t e r e s t . The p l o t of d e v i a t i o n v s . channel number, f o r t h i s image, was the same as that obtained f o r the centered image, i n d i c a t i n g that the d i s t o r t i o n arose e n t i r e l y from the SIT v i d i c o n . Channel-to-Channel C r o s s t a l k . In one of the brochures sup p l i e d by the manufacturer of the OMA the channel-to-channel c r o s s t a l k f o r the SIT v i d i c o n i s s t a t e d i n these terms: with a 10 ym l i n e centered on a channel, more than 60% of the s i g n a l amplitude i s centered i n that channel and more than 98% of the s i g n a l i s i n that channel and the two adjacent channels. Some of the p a t t e r n s obtained from the r u l e d d i s k s t u d i e s were examined to see i f they were s u i t a b l e f o r the measurement of c r o s s t a l k . The l i n e s on the r u l e d d i s k are 80 ym wide i n the r a d i a l d i r e c t i o n ; at the plane of the SIT v i d i c o n f a c e p l a t e , they are r e duced to h a l f that width, or about 40 ym. The width of an OMA channel element i s 25 ym. Thus one can make an estimate of c r o s s t a l k by c e n t e r i n g a l i n e between two channels. The r e l a t i v e i n t e n s i t i e s f o r such a l i n e are normalized so that the sum has a value of 2. The normalized r e l a t i v e i n t e n s i t y f o r one of the center channels i s Î Q + f-^; f o r the next three channels i t i s l ^2* ^2 + 3> d 3> r e s p e c t i v e l y , where f g r e f e r s to the f r a c t i o n of s i g n a l i n the center channel, and f]_, f 2 , and are the f r a c t i o n s f o r the s u c c e s s i v e channels on both s i d e s . The sum, f0 + 2 ( f + f +. f ) , should be 1. The c a l c u l a t e d f values f o r the unpulsed p a t t e r n a t 265 nm and the d i f f e r e n c e p a t t e r n at 405 nm are given i n Table I I . The c r o s s t a l k f o r the f i r s t p a t t e r n was h i g h e r ; i n f a c t an f ^ v a l u e should have been i n c l u d e d . The higher values are due to the e f f e c t of the f l u o r e s c e n t f i l m . We have no way of determining whether a 10 ym l i n e centered on a channel would give the same values with our system as the values given by the manufacturer f

+

f

x

a n

2

f

3



'ure 5.

Deviation plots for ruled line spacing. (·—·) Unpuhed, 265 nm; ( · - - · ) sec, 405 nm; ( · · · · · ) 8.9-4.48 sec, 265 nm.

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336

f o r t h e i r system. Since our o p t i c a l system i s more complicated, i t i s p o s s i b l e that our higher values arose from r e f l e c t i o n of l i g h t from the d i s k and/or l e n s s u r f a c e s . Nevertheless, we can check, when necessary, to see whether a c o r r e c t i o n f o r c r o s s t a l k w i t h our values would change s i g n i f i c a n t l y the measured i n t e n s i ties. TABLE I I Channel-to-Channel

f q Û

ft

n

Crosstalk

Unpulsed Image at 265 nm

Difference Pattern a t 405 nm

0.248 0.227 0.091 0.057

0.324 0.236 0.081 0.020

a

Average of 3 l i n e measurements Accuracy of Measurement of Absorbance G r a d i e n t s . As s t a t e d e a r l i e r , we s e t as a s t a r t i n g t a r g e t the a b i l i t y of our OMA s y s tem to measure c o n c e n t r a t i o n g r a d i e n t s corresponding to the absorbance range from 0 to 1 with an accuracy of 0.001 absorbance u n i t s . The assessment of the performance of the system r e q u i r e s the measurement o f the i n t e n s i t y p r o f i l e f o r an o p t i c a l d e n s i t y wedge w i t h a p r e c i s e l y known g r a d i e n t . The c l a s s i c a l way of a s s e s s i n g the behavior of an u l t r a c e n t r i f u g e o p t i c a l system has been to measure the sedimentation beh a v i o r o f s o l u t e s , u s u a l l y p r o t e i n s . In one type o f experiment, c a l l e d sedimentation v e l o c i t y , a uniform s o l u t i o n i s spun i n a r o t o r a t a h i g h speed, and the shape of the boundary i s examined as i t moves toward the bottom of the c e l l . The shape of boundary, w h i l e resembling an i n t e g r a t e d gaussian curve, i s d i s t o r t e d by a number of e f f e c t s d i f f i c u l t t o assess, i n c l u d i n g d i f f u s i o n , d e v i a t i o n from i d e a l behavior, inhomogeneity of c e n t r i f u g a l f i e l d , shape of c e l l , and even c o n v e c t i o n . The u s u a l method, then, i s to perform a sedimentation e q u i l i b r i u m experiment, i n which a s o l u t i o n c o n t a i n i n g a s o l u t e of known behavior i s spun a t a much lower speed u n t i l the concent r a t i o n gradient remains i n v a r i a n t w i t h time. The d i s t r i b u t i o n of s o l u t e f o r an i d e a l , two-component system should correspond to the equation, where c i s the c o n c e n t r a t i o n of s o l u t e w i t h dine d r

2

=

M(l - ν ρ ) ω

2

2RT

molecular weight M and p a r t i a l s p e c i f i c volume V, r i s the r a d i u s from the center of the r o t o r , ω i s the angular v e l o c i t y , R i s the


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gas constant, Τ Is the absolute temperature, and ρ i s the d e n s i t y of the s o l v e n t . The behavior of the o p t i c a l system i s assessed by p l o t t i n g In c (absorbance i n t h i s case) v s . r . The p l o t should be l i n e a r and the molecular weight c a l c u l a t e d from the known p a r t i a l s p e c i f i c volume and the other constants should agree c l o s e l y w i t h the known v a l u e , t y p i c a l l y w i t h i n 1 or 2%. The r e s u l t s from a recent experiment w i t h the o x i d i z e d d i s u l f i d e form of coenzyme A are shown i n F i g u r e 6. F i v e doubles e c t o r c e l l s , each c o n t a i n i n g s o l u t i o n and s o l v e n t , were placed i n a s i x - c e l l rotor,, which was allowed to s p i n overnight i n order to achieve e q u i l i b r i u m . The absorbance p r o f i l e s f o r l i g h t a t 265 nm were recorded, a d j u s t i n g the number of scans so as to g i v e about 300,000 counts f o r the lower c o n c e n t r a t i o n r e g i o n . The p l o t s f o r In A v s . Ar (Figure 6) are l i n e a r , w i t h the expected amount of s c a t t e r f o r the absorbance range 0.3-0.8. The agreement of the slopes (0.2323, 0.2368, 0.2325 and 0.2369 w i t h an average value of 0.2349__cm"^) i s s a t i s f a c t o r y . An exact value of V i s not known, but we have obtained a value of 0.556 ml/g from p r e l i m i n a r y sedimentation e q u i l i b r i u m measurements i n mixtures of H2O and D2O (16). A v a l u e of 0.563 ml/g was estimated_ from known atomic and molar volumes (17,18) . These v a l u e s f o r V gave molecular weights of 1480 and 1500, r e s p e c t i v e l y , i n good agreement w i t h the known molecular weight of 1532 f o r the a c i d form of o x i d i z e d coenzyme A. We have a l s o ob tained good molecular weight values f o r sperm whale myoglobin. Even though one can o b t a i n good agreement between the mea sured and c a l c u l a t e d l i g h t i n t e n s i t y p r o f i l e s f o r sedimentation e q u i l i b r i u m experiments, t h i s method has drawbacks f o r the r o u t i n e assessment of the performance .of the o p t i c a l system. There i s not commercially a v a i l a b l e a standard s o l u t e with guaranteed p u r i t y and c e r t i f i e d sedimentation behavior. Moreover each experiment r e q u i r e s a c o r r e c t i o n b a s e l i n e obtained from the c e l l s spun w i t h both s e c t o r s f i l l e d with s o l v e n t . Since a time p e r i o d of 8 hr or more i s r e q u i r e d to achieve e q u i l i b r i u m , i t becomes a f o r m i d i b l e task to i n v e s t i g a t e the e f f e c t of a number of parameters. We turned to the p o s s i b i l i t y of u s i n g an o p t i c a l d e n s i t y wedge to provide a l i g h t i n t e n s i t y g r a d i e n t . For sedimentation v e l o c i t y experiments, the wedge should have a range from 0 to about 1.5 absorbance u n i t s over a d i s t a n c e of about 1.3 cm, the normal span of an u l t r a c e n t r i f u g e c e l l . For sedimentation e q u i l i b r i u m experiments a s u i t a b l e wedge would range from 0 to 1.2 absorbance u n i t s over a d i s t a n c e of 3-4 mm. The wedge should transmit uv l i g h t . The wedge could be e i t h e r h e l d s t a t i o n a r y or spun i n an a p p r o p r i a t e h o l d e r i n a r o t o r . I t i s u n l i k e l y that such small wedges are commercially a v a i l a b l e . They could be spe c i a l l y c o n s t r u c t e d , but, b e f o r e use, t h e i r d e n s i t y p r o f i l e would have to be measured with a densitometer of proven accuracy u s i n g a very narrow s l i t . Such wedges would be d i f f i c u l t to make. We decided to t e s t the f e a s i b i l i t y of s p i n n i n g a geometrical



Figure 6.

Sedimentation equilibrium of oxidized coenzyme A. Speed, 40,000 rpm; temperature, 20.0°; wavelength, 265 nm; solvent, 0.05M potassium phosphate, pH 6.8.

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shape to provide a l i g h t f l u x gradient i n the r a d i a l d i r e c t i o n . A s p e c i a l mask p a t t e r n was t r a n s f e r r e d to a quartz window of a c e n t r i f u g e c e l l u s i n g the same procedure described above f o r the r u l e d d i s k . On the mask were two, 1° s e c t o r openings, one of which tapered t o a p o i n t over the range corresponding t o 6.7 t o 7.10 cm. Centered between them were three r a d i a l r e f e r e n c e l i n e s . The edges of the two openings were measured with a Nikon Model 6c shadowgraph to an accuracy of 1.25 ym, and i t was found that the equations f o r the edges agreed extremely w e l l with those c a l c u l a t e d . The mask was spun i n a c e l l f i l l e d with water. On the f i r s t attempt the l i g h t i n t e n s i t y p r o f i l e agreed c l o s e l y with that c a l c u l a t e d from the geometry o f the two p r o f i l e s . However, with the e n t i r e system improved, recent measurements w i t h the s p i n n i n g mask showed a l a r g e r d e v i a t i o n between the two p r o f i l e s . No improvement was observed when the focus plane was changed from the one-half plane o f the c e l l to the mask plane, which was on the lower s u r f a c e of the window. Changing the mask window t o the top of the c e l l , w i t h the camera l e n s focused on the mask, o f f e r e d no improvement. We a r e b a f f l e d as to the cause of the discrepancy between the c a l c u l a t e d and measured i n t e n s i t y curves. The mathematics of the c a l c u l a t e d curve needs to be r e f i n e d t o i n c l u d e the width of the s l i t above the r o t o r , which we had assumed to be n e g l i g i b l e . Perhaps the e f f e c t o f changing the s l i t width and the focus plane i n the c e l l should be examined. Based on the good r e s u l t s obtained from sedimentation e q u i l i b r i u m experiments, we b e l i e v e that the SIT v i d i c o n , OMA e l e c t r o n i c s , and our software c o n t r o l l i n g the gathering of data are a l l behaving p r o p e r l y , but perhaps there are s t i l l problems to be s o l v e d . Conclusions In g e n e r a l , we are s a t i s f i e d w i t h the performance of our OMAbased l i g h t d e t e c t o r system f o r the a b s o r p t i o n o p t i c a l system i n the u l t r a c e n t r i f u g e . Many of the problems that we had to s o l v e were inherent i n the o p t i c a l system and the c e l l s , but were hidden to users w i t h p h o t o m u l t i p l i e r d e t e c t o r systems. Other problems a s s o c i a t e d w i t h the SIT v i d i c o n have been s a t i s f a c t o r i l y s o l v e d . We intend t o examine the problem of the mask i n the s p i n n i n g r o t o r . We a l s o intend to c o n s t r u c t a quartz d i s k w i t h l i n e s that are a r c s about the center o f the r o t o r . Placed i n a s p e c i a l h o l d er a t a plane e q u i v a l e n t to the mid-plane i n a normal c e l l , i t would provide r a d i a l m a g n i f i c a t i o n and c o r r e c t i o n i n f o r m a t i o n f o r any experiment. We are l o o k i n g , with a n t i c i p a t i o n , to the development of improved s o l i d - s t a t e d e t e c t o r s with increased s e n s i t i v i t y i n the uv r e g i o n and no d i s t o r t i o n .


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Acknowled gmen t T h i s work was supported by the M e d i c a l Research S e r v i c e o f the Veterans A d m i n i s t r a t i o n and USPHS NIH grant HL #14938.

Literature Cited 1. Richards, E.G., and Rockholt, D., Arch. Biochem. Biophys. (1973) 158, 864. 2. Rockholt, D.L., Royce, C.R., and Richards, E.G., Biophysical Chem. (1976) 5, 55. 3. Svedberg, T., and Rinde, H., J . Am. Chem. Soc. (1924) 46, 2577. 4. Svedberg, T., and Pederson, Κ.,"TheUltracentrifuge."The Clarendon Press, Oxford (1940). Reprinted, the Johnson Reprint Corp., New York (1959). 5. Hanlon, S., Lamers, K., Lauterbach, G., Johnson, R., and Schachman, H.K., Arch. Biochem. Biophys. (1962) 99, 157. 6. Lamers, Κ., Putney, F . , Steinberg, I.Α., and Schachman, H.K., Arch. Biochem. Biophys. (1963) 103, 379. 7. Chervenka, C.A., Fractions, Spinco Division of Beckman Instruments, Inc., Palo Alto, CA. (1971). 8. Crepeau, R.H., Edelstein, S.J., and Rehman, M.J., Analyt. Biochem. (1972) 50, 213. 9. Williams, Jr., R.C., Biophysical Chem. (1976) 5, 19. 10. Spragg, S.P., Burnett, W.A., Wilcox, J.K., and Roche, J., Biophysical Chem. (1976) 5, 43. 11. Cohen, R., Cluzel, J., Cohen, H., Male, P., Moigner, M., and Soulié, C., Biophysical Chem. (1976) 5, 77· 12. Wei, G.J., and Deal, Jr., W.C., Arch. Biochem. Biophys. (1977) 183, 605. 13. Lloyd, P.H., and Esnouf, M.P., Analyt. Biochem. (1974) 60, 25. 14. Rockholt, D.L., and Richards, E.G., Fed. Proc. (1976) 35, 1457. 15. Schachman, H.K., Gropper, L . , Hanlon, S., and Putney, F . , Arch. Biochem. Biophys. (1962) 99, 175. 16. Richards, E.G., and Rockholt, D.L., Fed. Proc. (1978) 37, 1710. 17. Cohn, E . J . , and Edsall, J.T., eds., "Proteins, Amino Acids and Peptides." Reinhold Publishing Corp., New York (1943). 18. McMeekin, T.L., Groves, M.L., and Hipp, N.J., J . Am. Chem. Soc. (1949) 71, 3298. RECEIVED

February 7, 1979.


Use of the OMA for Analyzing Light Intensity ... - ACS Publications

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