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An action spectrum apparatus Brooks, Donald Elliott 1967

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AN ACTION SPECTRUM APPARATUS by DONALD ELLIOTT BROOKS B.Sc. (Hons.), University of British Columbia, 196k A THESIS SUBMITTED IN PARTIAL FULF31MENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of PHYSICS We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1967 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada - i - ABSTRACT An instrument is described which is capable of measuring the action spectrum of the removal of CO inhibition of respiration by light. In the method employed here, a cell suspension in a CO-Og atmos phere is alternately exposed to two wavelengths of light. Their photo chemical effects are balanced using an 0^ electrode as the null detector. The light intensities at the balance points from a series of wavelength pairs are used to determine the ratios of the extinction coefficients of the CO - oxidase complex, at the various wavelengths, to the extinction coefficient at a standard wavelength. An action spectrum for Bakers yeast is shown. - i i - ACKNOWLEDGMENTS I am deeply g r a t e f u l to Dr. C.P.S. Taylor f o r the guidance, support and continuous encouragement provided me throughout the course of t h i s work. His presence was always stimulating; I consider myself fortunate to have worked i n such close contact with him. I would also l i k e to thank Mr. Doug Stonebridge f o r h i s invaluable assistance i n the construction of the apparatus. F i n a n c i a l support from the National Research Council of Canada between 196*4- and 1966 i s g r a t e f u l l y acknowledged. - i i i - TABLE OF CONTENTS Page INTRODUCTION I Chapter I THE THEORY AND APPLICATION OF THE ACTION SPECTRUM METHOD 1-1. T h e o r e t i c a l Background 1.1 Problems to which Action Spectrum Methods are Suited 2 1.2 The CO-Oxidase Action Spectrum 2 1-2. Generalization of the K i n e t i c s 6 1-3. Necessary Conditions f o r Determining an Oxidase Action Spectrum • 6 I- ^ . General Outline of the Method 6 Chapter I I OPTICAL SYSTEM I I - l . The Monochromator and i t s Modifications 8 I I - 2. Cone Channel Condenser 2.1 Design Considerations 10 2.2 Construction of the Cone Channel Condenser ....... 13 Chapter I I I MEASUREMENT OF LIGHT INTENSITY I I I - l . General Requirements f o r Detector ................ 15 III-2. Comparison of Types of Detectors 2.1 Terms of Comparison 15 2.2 Types of Detectors Under Consideration 2.21 Thermal Detectors 17 2.22 Photoemissive Detectors 19 2.23 Photoconductive C e l l s 20 - i v - Page I I I - 3. Light I n t e n s i t y Measurements i n the Apparatus 3*1 Sampling the Beam f o r In t e n s i t y Measurements • 22 3.2 Detection of the Lead Sulphide C e l l Output 23 Chapter IV RESPIRATION RATE SENSOR IV- 1. P r i n c i p l e of the Respiration Rate Sensor 26 IV-2. Electrode Chamber Design 27 IV-3. Electrode Setting 27 IV- 4. Current Measurements 28 Chapter V CALIBRATION, OPERATION AND PERFORMANCE OF APPARATUS V- l . C a l i b r a t i o n 1.1 Electrode 29 1.2 Lead Sulphide Detector 1.21 I n t e n s i t y Response 30 1.22 Temperature Dependence of the Lead Sulphide Detector 30 1.23 Absolute C a l i b r a t i o n of the Detection System • 31 1.2^ Determination of Light I n t e n s i t i e s Incident on the C e l l Suspension 31 V-2. Determination of the A c t i o n Spectrum 2.1 Preparation of the B i o l o g i c a l Sample 32 2.2 Operating Procedure 33 2.3 C a l c u l a t i o n of Relative E x t i n c t i o n C o e f f i c i e n t s ... 35 V-3. Performance 3.1 Action Spectrum of Yeast • Jo 3.2 Accuracy of the Action Spectrum Determination ..... 36 - V - Page Chapter VI SUGGESTED IMPROVEMENTS VI-1. Reduction of Error in Present Apparatus kO VX-2. Alterations to Increase Range and Sensitivity of Apparatus kO BIBLIOGRAPHY kk - v i - LIST OF FIGURES Fol lowing page: 1-A. Block Diagram of A p p a r a t u s . . . . . . . 7 1-B. Op t i c a l Path Inside Monochromator 7 2, Ray Trac ing Diagram f o r Condenser Design 12 3. Semi-log P lo t of PbS Output v s . Temperature 21 k, PbS Detector Mounting and P o l a r i z i n g C i r c u i t 22 5. C i r c u i t Diagram of Phase Sens i t i ve Detector 2k 6. E lec t rode C h a m b e r . . . . . . . . 27 7. St/Sb C a l i b r a t i o n Curve . . 35 8. Ac t i on Spectrum of CO I n h i b i t i o n of Resp i r a t ion i n Bakers y e a s t . . . . 36 9. Thermistor Con t ro l l ed C i r c u i t f o r PbS P o l a r i z a t i o n . kl LIST OF TABLES Following page! Table I Comparison of P.S.D. and Thermopile Outputs 31 - 1 - Introduction An, action spectrum i s a representation of some b i o l o g i c a l a c t i v i t y which v a r i e s as a f u n c t i o n of the wavelength of l i g h t i ncident on the organism under observation. I f some measure of the a c t i v i t y being monitored i s p l o t t e d as a fun c t i o n of the wavelength of l i g h t incident on the sample, the r e s u l t i n g graph (the action spectrum) w i l l characterize the absorbing species c o n t r o l l i n g the action (1,2). The action that the present apparatus i s designed to characterize i s the l i g h t s e n s i t i v e i n h i b i t i o n of r e s p i r a t i o n by carbon monoxide. Warburg and h i s coworkers developed the c l a s s i c a l manometric technique f o r t h i s measurement (3). A more s e n s i t i v e apparatus designed to make the same measurement, using polarography, was l a t e r developed by Castor and Chance (4). This t h e s i s describes i n d e t a i l an ac t i o n spectrum appara tus based on t h e i r instrument. Chapter I outlines the t h e o r e t i c a l background of the method, discusses the conditions necessary f o r an act i o n spectrum measurement of t h i s type, and describes i n broad ou t l i n e the measuring procedure. Chapters I I , H I , and IV describe i n d e t a i l the various parts of the apparatus, the design considerations which l e d to t h e i r use, and t h e i r a p p l i c a t i o n i n the measuring system. The c a l i b r a t i o n , operation, and performance of the instrument i n measuring an oxidase action spectrum i s discussed i n Chapter V. Chapter VI o f f e r s some suggestions f o r improvements i n , and extensions t o , the apparatus as i t now e x i s t s . - 2 - CHAPTER I The Theory and A p p l i c a t i o n of the Action Spectrum Method I - l . T h e o r e t i c a l Background 1.1 Problems to which Action Spectrum Methods are Suited In p r i n c i p l e , an action spectrum may be taken of any b i o  l o g i c a l a c t i v i t y which i s l i n k e d to the absorption of s p e c i f i c wavelengths of l i g h t . The method has two advantages over absorption spectrophotometry: ( i ) a spectrum can be obtained when there are too few molecules to provide measurable absorption; ( i i ) the spectrum characterizes the molecule or molecules which are b i o l o g i c a l l y f u n c t i o n a l i n the process being observed. Thus, under the conditions to be described, r e l a t i v e e x t i n c t i o n c o e f f i c i e n t s may be determined f o r compounds which cannot be extracted i n an examinable form, or whose absorption bands may be masked by the absorption bands of other c e l l constituents. P r i m a r i l y , however, the method i s used to deter mine the ac t i v e molecule or molecules i n b i o l o g i c a l a c t i o n affected by l i g h t . 1.2 The CO-Oxidase Action Spectrum Biophysical and biochemical evidence gives some of the chemistry and mechanism of r e s p i r a t i o n and i t s l i g h t s e n s i t i v e i n h i b i t i o n by CO. (3) Based on such evidence, a set of chemical equations may be set up representing the combination of the enzymic i r o n i n the c e l l u l a r e l e c t r o n transport system with e i t h e r oxygen or carbon monoxide. In the equations and k i n e t i c analysis that follow, k1 . _ are r e a c t i o n v e l o c i t y - 3 - constants which are independent of l i g h t i n t e n s i t y , and i s the r eac t i on v e l o c i t y constant of the l i g h t induced breakdown, of the Fe^CO"*" complex at a wavelength A • The ana lys i s inves t iga tes the steady-state behaviour of the system when i r r a d i a t e d by monochromatic l i g h t of any chosen wave l e n g t h , and shows how one can ca l cu l a te the r e l a t i v e ex t i nc t i on of the Fe"-CO complex at each wavelength. The r e l a t i v e e x t i n c t i o n c o e f f i c i e n t i s the r a t i o €^ j££ , where s represents a chosen standard wavelength. The s to i ch iomet r i c equations a re : Fe" + O z Fe"-O z — - P r o d u c t s d - D (1-2) L e t : (e-p-q) = [Fe"J p = [Fe M-0 2] x = [0 2] q = [Fe«-CO] y= [coj Then, i t fo l lows from the law of Mass A c t i o n , t h a t : p = k,*ce-p-<i) - t k z + k s ) p d-3) The r e s p i r a t i o n r a t e , -x, i s g iven by: -x = p + k s p 1. Fe"-CO i s the abbrev iat ion f o r the complex formed by the carbon monoxide and the reduced heme i r o n of the enzyme. -1* - In a steady s t a t e : %-° p ss O which implies — X = k 5 |p and therefore the r e s p i r a t i o n rate i s d i r e c t l y proportional t o p. So, s o l v i n g f o r p i n ( I - l ) and (1-2): P = e 1 + K- + k « y X K 3 X where: K,»= k^+k 5 * K3 = k4 + k; 3 k, k P X K 3 X Let p = p_ be the concentration of Fe'^Og when no CO i s present (y = 0). Then e - 1 + |<m , and s u b s t i t u t i n g back gives: Km y _ K, r i - * l (i_5) e x I f x and y remain constant, the l e f t hand side of (1-5) i s a constant. Changing the wavelength or i n t e n s i t y of l i g h t i n c i d e n t on the reacting species w i l l a f f e c t only p, and K , through kA . So, at wavelengths A. and X , i f the r e s p i r a t i o n rates, and thus the values of p, are equal: Ku _ K M which implies k_ =. k_ • These r e a c t i o n v e l o c i t y constants are r e l a t e d to the e x t i n c t i o n c o e f f i c i e n t s of the FeM-C0 com plex, as shown below. Consider a l a y e r of suspended absorbing molecules of t h i c k  ness d containing a r e a c t i n g species at a concentration c. Let a mono chromatic beam of wavelength A and quantum i n t e n s i t y J ^ i l l u m i n a t e an area A of the s o l u t i o n , and l e t the e x t i n c t i o n c o e f f i c i e n t s of the - 5 - reacting species be j S • Assume that d and c are such that a negligible number of quanta are absorbed, so that a l l absorbing centers are exposed to equal intensities. Then, the number of mole quanta absorbed per second is given by J3 J x C A d . The number of moles per second reacting as a result of the light energy absorbed will be: where d)> is the photochemical yield, defined as the ratio of the number of molecules reacting, to the number of quanta absorbed. Thus and the reaction velocity constant, defined by k. = ~ — is given as: i C Converting the quantum intensity to energy units, and writing € = Z - 3 0 3 as the extinction coefficient based on the common logarithms: k x _ 4>e»Wx A (i-6) Z.303 N . h c ' where: = intensity in energy units N 0 = Avogadro*s number h = Planck's constant C* = speed of light Warburg and others (3»5) have shown that for a number of heme - CO complexes in different molecules, c)> is independent of wave length. Thus, under the conditions for which (1-6) applies: _____ = ____________ (i-7) - 6 - 1-2. Generalization of the K i n e t i c s The chemical equations 1-1 and 1-2 seem to be a reasonable representation of the l i g h t s e n s i t i v e i n h i b i t i o n of r e s p i r a t i o n by CO. E x p l i c i t knowledge of the r e a c t i o n constants and concentrations would be necessary i f the e x t i n c t i o n c o e f f i c i e n t s of the enzyme - CO complex were to be calculated absolutely. However, the r e l a t i v e values £ ^ / € s s o , do not depend on the k i n e t i c analysis f o r t h e i r v a l i d i t y . I f changing from quantum i n t e n s i t i e s to J ^ leaves the system chemically and k i n e t i c a l l y stant between the two i n t e n s i t i e s . No f u r t h e r d e t a i l s of the b i o l o g i c a l a c t i o n need be known. 1-3. Necessary Conditions f o r Determining an Oxidase Ac t i o n Spectrum The use of the a c t i o n spectrum method i n oxidase i d e n t i f i  c a t i o n depends on the f o l l o w i n g : ( i ) the i r o n containing enzyme reacts with oxygen ( i i ) the i r o n containing enzyme also binds carbon monoxide ( i i i ) the oxygen concentration controls the r e s p i r a t i o n rate ( i v ) the oxygen and carbon monoxide compete f o r the binding s i t e on the enzyme. 1-4. General Outline of the Method In the apparatus to be described, the measurement of the photochemical e f f e c t of a wavelength A was made by a l t e r n a t e l y i l l u m i n a t  i n g the r e a c t i o n s o l u t i o n with a narrow bandwidth monochromatic beam and unchanged, one need l o g i c a l l y assume only that the product con-- 7 - a comparison beam of f i x e d spec t r a l composit ion and va r i ab l e i n t e n s i t y . The i n t e n s i t y of the comparison beam was adjusted u n t i l the r e s p i r a t i o n rate sensor ind i ca ted no change when the beams were a l t e rna ted . This procedure was ca r r i ed out at a l l wavelengths f o r which the r e l a t i v e ex t i n c t i o n c o e f f i c i e n t s were measured. The b lock diagram of the measurement apparatus i s shown i n F igure 1. The e x t i n c t i o n c o e f f i c i e n t s , r e l a t i v e to that at A = 55 n nm, were ca l cu la ted from the express ion : g » C A WS»O 550 € s s o " W A CSSO A ( I " 8 ) which fo l lows d i r e c t l y from equat ion 1-7. In (1-8): C A = i n t e n s i t y of comparison l i g h t which gives same photochemical e f f e c t as A C 5 S 0 = i n t e n s i t y of comparison l i g h t which gives same photochemical e f f e c t as 550 nm. -k -c -8 1/ V. 0 > H~ O • <0 Q o O QJ Q o v. 0 o vj 0 .0 o • 0 V. <: u o' c o 5 3 d 8: 0 °7 Entrance S l i t Entrance, bsorn whife tijht from tungsten lamp Concave mirror FIGURE 1-8 Optical Pcxfh Inside- Monochr.oma+br - 8 - CHAPTER II Optical System I I - l . The Monochromator and its Modifications Any apparatus designed to take an action spectrum requires a source of monochromatic light of variable wavelength, and an optical system to deliver the light to the biological sample. In the present apparatus, the monochromatic light is supplied by a Bausch and Lomb Grating Monochromator employing a diffraction grating 50 mm x 50 mm ruled at 600 grooves/mm. The instrument has a linear dispersion of 6.6 nm/mm at the exit s l i t . Quartz condensers image the light from the source on the en trance s l i t which lies in the focal plane of a front surfaced concave mirror of focal length 250 mm. The collimated beam from this mirror is reflected to the grating, dispersed back to the concave mirror, condensed, and imaged at the exit s l i t . Wave length selection at the exit s l i t is made by rotating the grating about a vertical axis, thus scanning the dispersed beam across the exit s l i t . Wavelengths from 200 nm to 1,400 nm in the fi r s t order are available, although above 400 nm the second order begins to overlap, impairing the purity of the beam. The width of the s l i t deter mines the bandwidth of the monochromatic light passed. Optimum purity and intensity for a given s l i t width are obtained when the exit and entrance slits are set equal. Since the apparatus is designed for i n i t i a l use between 350 nm and 1,000 nm, a glass envelope bulb is used. The bulb is rated at 8 v - 50 w, is of Japanese make (commercial name: "Astron" or MAce M), and is housed in a water cooled jacket. It is run from a 10 v - 100 w Sorensen - 9 - DC power supply s t a b i l i z e d t o ± 0 . 2 5 $ . The filament i s surrounded by a glass bulb covered everywhere i n s i d e with a r e f l e c t i v e coating except where the beam emerges. The bulb thus acts as a s p h e r i c a l mirror to the part of the radiant energy that would normally be l o s t from the system. The added r e f l e c t e d l i g h t g r e a t l y increases the i n t e n s i t y a v a i l a b l e at the quartz condensing lenses of the monochromator, although the c o l l i m a t i o n caused by the mirror causes some defocussing at the g r a t i n g . The a c t i o n spectrum method used here requires that a beam of f i x e d s p e c t r a l content be alternated on the sample with the monochromatic l i g h t of v a r i a b l e wavelength. The f i x e d comparison beam must be of v a r i a b l e i n t e n s i t y but must not contain an appreciable amount of i n f r a - r e d r a d i a t i o n . Unless such r a d i a t i o n i s f i l t e r e d out, both thermal damage to the b i o l o g i c a l sample, and thermal i n s t a b i l i t i e s i n the electrode current may occur. The comparison beam l i g h t i s introduced by p l a c i n g a plane f r o n t surfaced mirror i n the white beam between the concave mirror and the d i f f r a c t i o n g r a t i n g . This beam i s r e f l e c t e d to the f l o o r of the monochromator box, where another plane mirror r e f l e c t s the l i g h t back to the f i r s t mirror, then to the con cave mirror, which focusses i t on the e x i t s l i t . The arrangement i s such that , as nearly as p o s s i b l e , the second plane mirror and the d i f f r a c t i o n grating are i n o p t i c a l l y equivalent p o s i t i o n s . A s e r i e s of p a r a l l e l louvers mounted above the second plane mirror acts as the i n t e n s i t y c o n t r o l . The louvers are ganged together, and t h e i r angular p o s i t i o n with respect to the beam determines how much of the l i g h t i s r e f l e c t e d to the e x i t s l i t . The louver angle i s c o n t r o l l e d by a micrometer head, mounted i n the side of the monochromator box. I t screws against a spring loaded arm to which the hinged louvers are attached. There i s a p r o v i s i o n f o r mounting f i l t e r s - 10 - above the set of louvers to l i m i t the s p e c t r a l content of the comparison l i g h t , but i t was found that l o c a t i n g the f i l t e r s i n t h i s p o s i t i o n i n t r o  duces a great deal of scattered l i g h t i n t o the system. Instead, the f i l t e r s are placed outside the monochromator box, between the quartz condensing lenses and the entrance s l i t . The change between the monochromatic and comparison beams i s made by mechanically i n s e r t i n g the f i r s t plane mirror i n t o the white beam from the concave mirror. The second mirror, i n t e n s i t y c o n t r o l l i n g louvers, and f i l t e r holder, are a l l out of the normal o p t i c a l path and remain st a t i o n a r y throughout the change. The f i r s t mirror runs on a track, the p o s i t i o n on which i s c o n t r o l l e d by a l e v e r arm external to the box. Mech a n i c a l l y l i n k e d to t h i s arm are two f i l t e r holders, one of which introduces an i n f r a - r e d f i l t e r i n t o the o p t i c a l path when the comparison l i g h t i s i l l u m i n a t i n g the sample, but i s not i n the path when the monochromatic l i g h t i s being used. The second holder i s i n s e r t e d i n t o the o p t i c a l path only when the sample i s monochromatically i l l u m i n a t e d . This second holder i s used p r i m a r i l y f o r broad band f i l t e r s which eliminate interference from the second and t h i r d orders i n the monochromatic beam. II-2. Cone Channel Condenser 2.1 Design Considerations In order to produce a l i g h t i n t e n s i t y at the b i o l o g i c a l sample high enough to get measurable e f f e c t s even with the most i n s e n s i t i v e enzymes, the e x i t beam from the monochromator must be concentrated i n some way. Also, since the lamp filament i s focussed at the e x i t s l i t , the i n t e n s i t y w i l l vary across the s l i t and some scrambling of the beam w i l l be necessary t o get an even, d i s t r i b u t i o n throughout the drop, p a r t i c u l a r l y - 11 - i n the comparison beam. This i s important, since the d i s t r i b u t i o n of comparison and monochromatic beams must be proportional throughout the drop to avoid t r a n s i e n t e f f e c t s when the beams are interchanged. Further, the more even the i l l u m i n a t i o n on the i n d i v i d u a l c e l l s , the more c l o s e l y the assumptions w i l l apply under which (1-6) was derived. A quartz achro matic lens system, as well as being expensive, does not provide such a d i f f u s e beam, even when f i l t e r e d . A b e t t e r system i s provided by a cone channel condenser, a m o d i f i c a t i o n of the device described by Williamson (6) . The condenser used i s a four sided pyramid of square cross-section open at both ends. The inner surfaces are f r o n t surfaced mirrors. The base opening i s 11 mm square and s i t s on the e x i t s l i t of the monochromator. The top i s 1.6 mm square and f i t s f l u s h up against the cover s l i p forming the bottom of the electrode chamber. The condenser i s designed to produce an even i n t e n s i t y d i s t r i b u t i o n over a complete hemispherical s o l i d angle of 2TT at the e x i t of the cone, as well as increase the i n t e n s i t y at the e x i t over that at the base. The cone i s designed so that the most widely divergent ray of the entrance beam to the cone s u f f e r s multiple i n t e r n a l r e f l e c t i o n s u n t i l i t emerges at 90° to the o p t i c axis of the condenser. Less divergent rays entering s u f f e r fewer r e f l e c t i o n s and emerge at smaller angles to the a x i s . The length of the cone that w i l l give such r e f l e c t i o n s i s c r i t i c a l . I t i s determined by analyzing a ray t r a c i n g diagram showing the o p t i c a l path of the extreme ray from the monochromator entering the condenser. The diagram i s constructed as shown, below i n Figure 2 . The cone i s drawn on the o p t i c a x i s , and the extreme ray from the monochro mator i s drawn, at an angle V to the axis, entering the mouth of the cone. - 12 - Instead of t r a c i n g the path of the ray through i t s r e f l e c t i o n s , every time the ray reaches a r e f l e c t i n g surface the cone i s drawn as being r e f l e c t e d about that surface. Thus, the extreme ray w i l l appear as a s t r a i g h t l i n e on the diagram, while the cone ends and t h e i r r e f l e c t i o n s w i l l form two concentric polygons. As long as the cone angle i s small, the inner polygon may be replaced by a c i r c l e as shown i n the diagram, and the outer c i r c l e may be excluded during the a n a l y s i s . I f the extreme ray cuts t h i s inner c i r c l e , the ray w i l l emerge from the end of the cone at the angle the ray makes with the c i r c l e at that point. For the ray to emerge at 90° to the cone, i t must l i e tangent to the c i r c l e , as shown. The length of the cone, x, i s the distance between the mouth, of width 2s, and the c i r c l e . The cone angle, , i s that angle at the centre of the c i r c l e formed by the radius continuous with the cone edge. Figure 2 Ray Tracing Diagram f o r Condenser Design - 1 3 - The length x = x (c,s,V) i s found from: ( i ) cofan B - x + r = _r =*> r = c x S C S-C ( i i ) A = S co+an V ( i i i ) r = (A + X + r ) s i n \ / = » r ~ sin V(s c o f a n V + x) < - s m V E l i m i n a t i n g r and A from these three equations, and s o l v i n g f o r x gives: X = (s-e) cos V  c/s - sin V In the Bausch and Lomb monochromator, the angle V i s deter mined by the aperture stop, which i n t h i s instrument i s the grating area, and the f o c a l length of the condensing concave mirror. Thus: ton V = 25 mm 2 5 0 m m V = 5 °43 / The e x i t s l i t length = 2 s = 1 1 mm and the cone end width 2 c was taken as 1 . 8 mm. These f i g u r e s g i v e : x = 1 0 2 mm. 2 . 2 Construction of the Cone Channel Condenser The cone was constructed from glass s t r i p s of appropriate shape, ground f l a t on one edge (obtained from Emerald Glass Co., Toronto, Ont.). They were placed two at a time i n a j i g which supported them at the ends, and held them at r i g h t angles with one ground edge butted up against the surface of the other piece. The glass was held i n t h i s p o s i t i o n with s e t t i n g screws which determine the angle, and the s i z e of the two end openings. The j i g was then, hung i n an evacuated chamber and aluminum evapora ted onto the inner surfaces of the two pieces. A l l s e t t i n g and holding of the glass had to be mechanical up to t h i s point, since epoxy or other glues evaporated too r a p i d l y when the pressure was reduced i n the chamber, • - u - and the vacuum obtainable was i n s u f f i c i e n t to allow good aluminum evaporation. Once aluminized, the two pieces were glued with epoxy on the back of the j o i n t . Two such assemblies were then placed v e r t i c a l l y i n another j i g whose base and top were of proper s i z e to set the base pieces i n an 11 mm square and the top of the cone i n a 1.6 mm square. These two units were also epoxied on the back of the j o i n t to form the cone. Some adjustment to the length could have been made by coating the aluminized surface with p a r a f f i n , grinding the glass down, then, removing the p a r a f f i n by s e t t i n g the cone i n hot water. Unfortunately, an e r r o r i n the c a l c u l a t i o n of the extreme ray angle from the monochromator i n the o r i g i n a l cone design r e s u l t e d i n the cone being b u i l t 111 mm long. This longer length caused some loss i n i n t e n s i t y at the sample. However, even with the low power l i g h t source used, ample i n t e n s i t y was a v a i l a b l e at the end of the cone when s l i p widths of the order of 0.5 mm were used. - 15 - CHAPTER I I I Measurement of Light I n t e n s i t y I I I - l . General Design Requirements f o r the Detector The determination of an action spectrum requires an accurate, reproducible measure of l i g h t i n t e n s i t y . The l i g h t detector i n the appara tus must have a high s i g n a l to noise r a t i o , i t must be able to e f f i c i e n t l y detect r a d i a t i o n of wavelengths from 200 nm t o 1,000 nm, and i t should preferably be of small enough s i z e to be introduced e a s i l y i n t o the o p t i c a l system employed i n the apparatus. The r e l a t i v e cost of the detection system i s a lso an important consideration. III- 2 . Comparison of Types of Detectors 2.1 Terms of Comparison A quantitative comparison of the various types of l i g h t detectors a v a i l a b l e has been made by Jones (7). He considers them i n terms of t h e i r d e t e c t i v i t y . The d e t e c t i v i t y i s the r a t i o of the gain of the detector, that i s the number of v o l t s d e l i v e r e d by the detector f o r every watt of i n c i d e n t radiant power, to the r.m.s. noise voltage introduced i n t o the output by the detector i t s e l f . Since the response of the device w i l l i n general vary with the frequency at which the l i g h t beam i s modulated, the d e t e c t i v i t y , D ( f ) , w i l l be a function of the modulation frequency f . Thus: r.m.s. noise, where R ( f ) = r e s p o n s i v i t y , the r a t i o of the detector output i n v o l t s to - 16 - the rad iant power i npu t , i n watts . I f the noise vo l tage i s decomposed i n to spec t r a l components at frequency f , W ( f ) , such that the mean square noise vol tage n 2 i s g iven by : n*~- ^ W(f)df (111-2) then the d e t e c t i v i t y over a u n i t bandwidth of noise centered on a frequency f i s de f ined by: D,(f) =_R!£L_ [w(fjj v* (in-3) Fur the r , a time constant , t , which i s a measure of the response time of the de tec to r , may i n genera l be def ined by : r = P. ( fry,)* 4fD,(f)df (nuo where frv> i s the modulat ion frequency which maximizes D, (f). In many types of de t e c to r s , the r e spons i v i t y spectrum i s charac ter ized by the time constant i n the form: R(f) - [l + 0.7Tfr p)*]"* (in_5) where R r y > < * x i s the p a r t i c u l a r de t e c to r ' s maximum re spons i v i t y , and i s i t s time constant . Detectors are c l a s s i f i e d more genera l l y by Jones in to Class I or II. Since D ( f ) i s found to depend upon both z: and the sens i t i v e area A i n e i t h e r o f two ways, t h i s f a c t i s made the bas i s of the c l a s s i f i  c a t i o n : C lass I D « fe, [jj ^ - 17 - Class I I D = k» "E where k^ and k- are constants. In order to more e a s i l y compare detectors within a given c l a s s , t h e i r d e t e c t i v i t i e s are compared to the d e t e c t i v i t y of the h y p o t h e t i c a l l y " i d e a l " detector of the c l a s s . The value of the f r a c t i o n thus formed i s termed the f i g u r e of merit of the detector. The i d e a l Class I detector i s taken to be a p e r f e c t l y black thermal detector whose output i s l i m i t e d by only photon noise. I f the detector and the noise i t produces both have the same bandwidth i t s d e t e c t i v i t y i s given by (111-6) I = 3.62 x l O ' 0 ^ ' / * thus the Class I f i g u r e of merit, M^, i s given by M.= _D = 2-7-6 Or_V| , /* D e t e c t i v i t i e s i n Class I I are r e l a t e d to the t h e o r e t i c a l upper l i m i t f o r thermocouples and bolometers, estimated to be D H = 0 . 3 3 * 1 0 " j _ _ _ ' (III-8) AVa- Therefore the f i g u r e of merit, M_, i s given by M z = 3 K DA'X (331-8) X 2.2 Types of Detectors Under Consideration The types of detectors considered f o r use i n the action spectrum apparatus were thermal detectors, photoemissive detectors, and photoconductive c e l l s . 2.21 Thermal Detectors Thermal detectors operate by converting r a d i a t i o n power - 18 - i n t o a voltage by means of a temperature change i n the absorbing element. An important parameter i n t h e i r performance i s the p h y s i c a l time constant, "Cp = C / K , where C = thermal capacity per u n i t s e n s i t i v e area K = conductivity per u n i t area. These thermal detectors are of three types: ( i ) Thermocouple s: The t h e o r e t i c a l f i g u r e of merit of a Johnson noise l i m i t e d thermocouple i s given by fsy] _ 3*10 £ o  X~(kT*C)(L,*+L3rt ( I I I ~ 9 ) where: € = e m i s s i v i t y of r e c e i v e r S = thermoelectric power of p a i r of metals k = Boltzman*s constant T = absolute temperature C = thermal capacity of r e c e i v e r per u n i t area L i > a =^J>/~r f o r thermocouple wires 1 and 2 , where ) \ * = thermal conductivity, = e l e c t r i c a l r e s i s t i v i t y . The highest f i g u r e s of merit thus f a r obtained f o r thermo couples are about 1.0, while the Eppley thermopile i s quoted i n Jones (?) as having a f i g u r e of merit of approximately 0 . 0 2 5 , and a time constant of the order of 2 seconds. ( i i ) Radiation Bolometers: These are e s s e n t i a l l y resistance thermometers. Radiation heats a ribbon through which a steady current flows, changing i t s resistance. The highest f i g u r e of - 1 9 - merit obtained at room temperature f o r these detectors i s about 0 . 5 . ( i i i ) Golay Pneumatic Heat Detector: The Golay detector i s a g a s - f i l l e d chamber with an i n f r a - r e d transmitting f r o n t window, A low heat capacity metallized membrane absorbs the inc i d e n t r a d i a t i o n and increases i n temperature, exchanging heat t o the gas. The gas pressure increases and deforms a f l e x i b l e membrane which causes a r e f l e c t e d beam to d e f l e c t as a measure of the r a d i a t i o n absorbed. The Golay deviates from an i d e a l Class I detector only i n that there i s some absorption i n the entrance window, the heat absorbing membrane i s not per f e c t l y black, and there i s some conduction from t h i s membrane to the walls of the chamber. The combination gives a f i g u r e of merit of M 1 = 0 , 2 7 5 . 2 . 2 2 Photoemissive Detectors Photoemissive detectors employ the ph o t o e l e c t r i c e f f e c t t o detect r a d i a t i o n i n t e n s i t i e s . The secondary electrons emitted are accelera ted through a p o t e n t i a l d i f f e r e n c e and measured as a cathode current. This current i s e i t h e r measured immediately, as i n a vacuum phototube, or a f t e r i n t e r n a l a m p l i f i c a t i o n by a cascade process e i t h e r i n a gas (gas phototube) or between members of a se r i e s of dynodes i n a high f i e l d gradient (photo- m u l t i p l i e r ) . By f a r the most e f f i c i e n t of these three i s the photomultiplier, since i t supplies e s s e n t i a l l y noisefree a m p l i f i c a t i o n with gains of the order of 1 0 ^ . The f i g u r e of merit of a photomultiplier operating i n the v i s i b l e region, f o r instance, (the photoemissive surface type i s designated - 2 0 - S-Jj- f o r t h i s region of s p e c t r a l response) has a f i g u r e of merit, = _ i f 0 , 0 0 0 . An S-5 photomultiplier, operating i n the u l t r a v i o l e t and v i s i b l e has M^ = 1 2 , 7 0 0 . The f i g u r e s of merit of gas phototubes and non-amplifying phototubes are several orders of magnitude lower, a l l having M^ 1 0 0 . Photomultipliers do, however, have several disadvantages. The a c c e l e r a t i n g p o t e n t i a l s i n the tubes are of the order of k i l o v o l t s , and must be h i g h l y s t a b i l i z e d . Thus, extremely w e l l regulated high voltage power supplies are needed to run the photomultipliers, and t h e i r cost i s high. The tubes often e x h i b i t some gain i n s t a b i l i t y over a period of time. While the s e n s i t i v e areas may be small, the o v e r - a l l tube s i z e i s quite l a r g e , and the measuring system as a whole i s rather bulky and awkward to mount, p a r t i c u l a r l y in. the o p t i c a l system used i n the a c t i o n spectrum apparatus. More important, no one photomultiplier gives u s e f u l detection over the wavelength range required, so d i f f e r e n t tubes would have to be employed over d i f f e r e n t s p e c t r a l regions. Thus, photomultipliers would not seem to f u l f i l adequately the requirements of the apparatus. 2 . 2 3 Photoconductive C e l l s Photoconductive c e l l s are s o l i d state devices which produce u s e f u l d e t e c t i v i t i e s in. the u l t r a v i o l e t , v i s i b l e , and i n f r a - r e d regions of the spectrum. I t i s believed that the l i g h t quanta in c i d e n t on the detector r a i s e electrons from the ground band or impurity centers i n t o the conduction band of the s o l i d , thus decreasing the resistance of the detector. The increase i n current may be monitored as a measure of the r a d i a t i o n i n t e n s i t y . The Kodak Ektron Detectors, made of e i t h e r lead sulphide or lead selenide deposited on g l a s s , are t y p i c a l photoconducting c e l l s . They w i l l u s e f u l l y detect r a d i a t i o n between 2 0 0 nm and 5 » 0 0 0 nm with time - 21 - constants between 2 and 1,000 microseconds. Their d e t e c t i v i t i e s , normalized to u n i t area and bandwidth as given, i n the t e c h n i c a l pamphlet "Kodak Ektron Detectors f o r the Infrared" (i960) are between 10"^ and lO"^. This data gives, f o r the d e t e c t i v i t y of highest r e s p o n s i v i t y , a d e t e c t i v i t y of 3.5 x 10"^ at 600 nm. Taking the value of the time constant to be the upper advertised l i m i t of 1 x 10 J sec. which applies f o r the detectors of highest d e t e c t i v i t y , and an area of 0.04 cm , the f i g u r e of merit calcu l a t e d from (III-7) i s M^^6. This value i s lower than those f o r photo tubes and photomultipliers, but at l e a s t s i x times higher than those f o r thermal detectors. One disadvantage of the lead sulphide detector i s a s i g n i  f i c a n t temperature dependence of both the s i g n a l and the s i g n a l to noise r a t i o . The Kodak Ektron Detector handbook provides the semi-lot p l o t of s i g n a l versus temperature shown in. Figure J, C a l c u l a t i o n of the slope of the graph gives: . ^ _ 2 = -2.3 *\0 2 ±Z% deg-' -43.S1|.0 4C The temperature dependence of the output of the lead sulphide c e l l , and hence of any s i g n a l S proportional to i t , i s then given by: A(l<>3 S) = (-2.3*\o~2 tz7o) AT (111-10) The advantages of the s o l i d state device, however, outweigh i t s disadvantages. Lead sulphide c e l l s may be constructed with v i r t u a l l y any shape and si z e of s e n s i t i v e area; t y p i c a l l y of the order of 1 to 20 mm square. The power necessary to operate the detector i s supplied by an inexpensive 22.5 v o l t battery, allowing complete e l e c t r i c a l i s o l a t i o n , from the r e s t of the e l e c t r o n i c s i n the apparatus. Further, the entire system - 22 - i s p h y s i c a l l y compact and easy to mount. Thus, on the basis of i t s f i g u r e of merit, broad wavelength s e n s i t i v i t y , small s i z e and low cost, i t was decided t o use a lead sulphide c e l l as the r a d i a t i o n i n t e n s i t y detector i n the a c t i o n spectrum apparatus. IH-3» Light I n t e n s i t y Measurements i n the Apparatus 3.1 Sampling the Beam f o r Intensity Measurements In. operation, two PbS c e l l s are wired i n s e r i e s with a 22.5 v battery. One of the c e l l s i s exposed t o the r a d i a t i o n t o be measured, and the other i s masked to act as a load r e s i s t o r across which the voltage changes, proportional to the l i g h t i n t e n s i t y , appear. A masked PbS detector i s used as the load r e s i s t o r because i t s resistance w i l l be affected by humidity, temperature, applied voltage and other external conditions i n the same way as that of the PbS c e l l a c t u a l l y detecting the r a d i a t i o n . Thus the voltage changes a c t u a l l y measured should be proportional to the true values of i n t e n s i t i e s i ncident on the exposed c e l l . Unfortunately, t h i s matching only holds i f the two detectors are equally illuminated, since the temperature dependence of the resistance i n the photoconducting state d i f f e r s from that i n the dark, g i v i n g r i s e to the expression (111-10). The temperature dependence would be much greater i f an ordinary r e s i s t o r were used as the load r e s i s t o r . The PbS c e l l employed i n the apparatus has a s e n s i t i v e area 2 mm x 2 mm covered by a quartz cover s l i p . I t i s mounted at the end of the e x i t s l i t of the monochromator, adjacent t o the base of the cone channel condenser as shown in. Figure 4. The l i g h t beam s u f f i c i e n t l y f i l l s the s l i t t h a t an appreciable amount i s a v a i l a b l e f o r the i n t e n s i t y measurement outside the cone. By sampling the beam outside the cone, none of the Qone channs.1 cono/eo-sefy Mercury rher mo meter L _ ads J-ro m d&t actors to PLS p o l a r i z i n g cct. Monochromator . _//'/, length = 2O^m/ \ L/ghf 6earn 'Back to book PbS defectors Front ^surFaced rn/rror r e f l e c t s part of beo-m onto s&ns'/fii/e. o-r-ea. of Pb S c/e fe. c for 30 i/ PbS c e l l exposed to be.CLrn A A A - -AAA 330K 69K Masked P6S ceil used as /OOLJ re.s/sfor Output FIGURE 4- PbS Defector: Mounting and Pofori zing C/rcuif - 23 - i n t e n s i t y incident on the sample i s l o s t . The masked PbS c e l l which acts as the load r e s i s t o r i s mounted back to back with the measuring detector, and i s therefore i n the same p h y s i c a l environment. Both c e l l s are i n thermal contact with the bulb of a mercury thermometer, the readings from which were used to correct a l l measurements t o 2k° C. The remaining c i r c u i t r y necessary to operate the PbS c e l l s as a detector i s mounted in. an e l e c t r i c a l l y shielded box on top of the monochromator adjacent t o the s l i t . E f f e c t i v e s h i e l d i n g of the detector and i t s leads was found to be very important f o r stable s i g n a l measurements. 3.2 Detection of the Lead Sulphide C e l l Output To determine the r e l a t i v e e x t i n c t i o n c o e f f i c i e n t s by the a c t i o n spectrum method, one must measure sets of l i g h t i n t e n s i t i e s through out the spectrum. Since the i n t e n s i t i e s always enter the c a l c u l a t i o n as the r a t i o of one i n t e n s i t y to another, the e l e c t r i c a l output of the PbS detector may be used in. place of actual l i g h t i n t e n s i t i e s providing t h i s output i s a l i n e a r function, of i n t e n s i t y over the range of wavelengths and i n t e n s i t i e s used. Because such a r e l a t i o n s h i p was found to hold f o r the PbS detector used i n the apparatus (see Section V - 1.21), only a stable measurement of the PbS s i g n a l i s required. The method of measurement i s as f o l l o w s . The monochromator l i g h t source i s chopped at 235 Hz with a s l o t t e d wheel d r i v e n by a synchro nous motor. Since the inherent frequency v a r i a t i o n i n the mains d r i v i n g the motor i s small, no s t a b i l i z a t i o n , i s necessary. The a m p l i f i e r i n the d e t e c t i o n network i s only broadly tuned to the chopping frequency and i s therefore i n s e n s i t i v e to small changes i n i t . The AC s i g n a l from the PbS c e l l i nserted i n t o the modulated - zh - beam i s AC coupled to the phase s e n s i t i v e detector (hereafter r e f e r r e d to as the PSD) whose c i r c u i t i s shown i n Figure 5» The i n i t i a l a m p l i f i c a t i o n i s c a r r i e d out by an active f i l t e r c o n s i s t i n g of a P h i l b r i c k K2-W packaged a m p l i f i e r with a Twin-T r e j e c t i o n f i l t e r i n the feedback loop. The notch f i l t e r i s designed with a low Q (Q = 6.2) to allow e f f e c t i v e l y constant a m p l i f i c a t i o n at 235 Hz since i t was found the chopper wheel had a 3 Hz beat superimposed on i t s frequency output. The cause of the beat i s obscure and i s more e a s i l y allowed f o r e l e c t r i c a l l y than corrected mechanically. The reference voltage c a r r i e s the phase information that determines how much of the s i g n a l amplitude appears as DC at the output of the PSD. When the s i g n a l and reference are p r e c i s e l y i n phase, a complete h a l f wave i s detected, while i f the two d i f f e r by a phase angle )f , the f r a c t i o n of the s i g n a l amplitude detected i s proportional to COS X'• The reference voltage originates from a b a r r i e r l a y e r c e l l onto which the beam from an automobile lamp f a l l s . This beam i s also chopped by the s l o t t e d wheel. The phase of the reference s i g n a l , r e l a t i v e t o that from the PbS detector, i s set by adjusting the azimuthal p o s i t i o n of the b a r r i e r l a y e r c e l l at the rim of the chopper wheel. The output from the PSD i s f i l t e r e d through a 0.05 sec. time constant f i l t e r between the high and low terminals of a Keithley Model 150-A Microvoltmeter-Ammeter operated d i f f e r e n t i a l l y . This f i l t e r stage cuts out the 60 Hz voltage between the c i r c u i t and chassis grounds of the microvoltmeter which are a design l i m i t a t i o n of the instrument. The DC measurement i s made potentiometrically, using the Keith l e y plus an Esterline-Angus Recording Milliammeter as the n u l l detector. The bucking voltage i s obtained from an external c i r c u i t . The potentiometric measurement •f-30oy ALL CAP. VALUES IN MICROFARADS ELECTRODE OFF J PSD SWITCH POSIT/ONS fZO/c FIGURE 5 C i r c u i t Diagram of Pha.se. Sensitive. De-fe.ctor' - 25 - has the advantages that expanded scales on both the microvoltmeter and the recorder may be used for more accurate measurements and""that the measurement does not depend on the accuracy of the meter's calibration. The ultimate noise bandwidth of the PSD is then just the bandpass of the Esterline-Angus chart recorder, of the order of 0.5 Hz. - 26 - CHAPTER IV Resp i ra t ion Rate Sensor IV-1. P r i n c i p l e of the Resp i ra t ion Rate Sensor The apparatus of Castor and Chance (4), on which the present instrument i s based, was b u i l t to monitor the r e s p i r a t i o n rate of m ic ro  organisms i n the presence of CO. Since t h e i r r e s p i r a t i o n i s known to be i n h i b i t e d by CO, and s ince the degree of i n h i b i t i o n decreases with increased i n t e n s i t y o f i l l u m i n a t i o n , i t i s poss ib l e to take an a c t i on spectrum of the r e l i e f o f i n h i b i t i o n i f one can. obta in a convenient measure of the r e s p i r a  t i o n r a t e . A measure o f t h i s ra te may be obtained us ing a po l a r i z ed plat inum e l ec t rode , which operates as f o l l ows . When Pt o r Hg i s po l a r i zed - 0,6 v to - 0,9 T wi th respect to a Ag + AgCl reference e l ec t rode , 0^ i n a so lu t i on i n contact with the cathode i s e l e c t r o l y t i c a l l y reduced, causing a current to f low i n the externa l c i r c u i t . The r eac t i on i s genera l l y s p e c i f i c f o r 0^ i n t h i s type of system s ince other ions which are e a s i l y reduced e l e c t r i c a l l y i n t h i s vo l tage range, such as Ag , Cu , or Pb , are not normally used i n b i o l o g i c a l media. Fur ther , the r eac t i on i s pH independent, s ince H + reduct ion does not occur at po l a r i z a t i ons under 1 v o l t (8). Since the 0^ concentrat ion at the Pt surface i s zero , the ra te of r educ t ion , and therefore the e l e c t r i c a l cu r ren t , i s dependent on the rate at which 0 2 can d i f f u s e to the e l e c t rode . Th is d i f f u s i o n rate i s d i r e c t l y p ropor t i ona l to the oxygen concentrat ion i n the s o l u t i o n . When an e lect rode i s p laced i n a drop, roughly of the same - 2? - diameter as the electrode, containing l i v i n g c e l l s , the opposing e f f e c t s of r e s p i r a t o r y uptake by the c e l l s and d i f f u s i o n of the oxygen i n from the drop surface cause a steady state oxygen gradient to be set up* Changes i n r e s p i r a t i o n cause changes i n the steady s t a t e , which are measured as current changes i n the electrode current, IV - 2 . Electrode Chamber Design The electrode chamber assembly i s as shown i n Figure 6 , The needle of a hypodermic syringe was used both as a means of introducing the c e l l suspension to the t i p of the Pt electrode, and as a base f o r the reference electrode, A 4 to 5 ram hollow s i l v e r needle was crimped to i t s t i p and e l e c t r o p l a t e d with AgCl to act as t h i s reference. Other methods of mounting the s i l v e r were t r i e d , but i t was found any arrangement using solder slowly poisoned the c e l l s , and caused the time over which they remained active to decrease to well under an hour. When the apparatus i s i n operation, the drop of c e l l suspen sion i s formed between the ground t i p of the Pt electrode, the reference electrode, and the cover s l i p forming the bottom of the chamber, IV-3. Electrode S e t t i n g I t was found e m p i r i c a l l y that the s t a b i l i t y of the current depended both on the rate of 0^ uptake by the c e l l s , and on the distance between the electrode and cover s l i p . Following Castor and Chance, i t was found the best s e t t i n g f o r the electrode separation was made by f i r s t measuring the current with the chamber f i l l e d with phosphate buffer, g i v i n g a current of the order of 6 x 10~ amps. The electrode was then screwed Q down u n t i l the current i n a drop of b u f f e r was about 2 ,5 x 10"* amps. Nut to adjust electrode, lei/el ^Copper iA//r& use.lcJe.ct -to Pt r/<-<_. le c frico/Vy rounded 2. ctiom. lucite rod-^ Ele c tribal conn, to ret. electrode clamped here •Syr/nge. holding cell Suspension sS•siting scr&uj to adjust chamber location -AgCI plated Aj tip o-cts as reference electrode ^'/:aj Aluminum collar 'SUpporf/ng ctxxrnber Cone channeJ Condenser FIGURE S Electrode Ct>cx.m6er - 28 - This s e t t i ng was re ta ined when the drop of c e l l suspension was formed at the end o f the e l e c t rode . The c e l l concentra t ion used was that concentrat ion of r e s  p i r i n g c e l l s which would use up a l l the oxygen i n the c e l l suspension sample i n one minute. Th is t ime , c a l l e d the r e s p i r a t i o n t ime, was d e t e r  mined by us ing a separate Pt e lec t rode system set i n a r o t a t i ng sample ho lde r . The c e l l suspension was d i l u t e d with phosphate b u f f e r u n t i l a r e s p i r a t i o n time of the order of one minute was obta ined. At t h i s concen t r a t i o n , the c e l l s remained ac t i ve i n the ac t i on spectrum apparatus f o r over two hours i n most cases . IV-4> Current Measurements The current measurements were made by bucking out the e lect rode current with a v a r i ab l e opposing cu r ren t . The bucking current was con t ro l l ed by a ten tu rn he l i po t whose f u l l sca le d e f l e c t i o n corresponded to between 100 na and 0.1 na on fou r succeeding decade ranges. The d e v i a  t i ons from n u l l as the steady s ta te changed were observed on the Ke i th ley 150-A Microvoltmeter-ammeter. When the ammeter i s connected to the Ester l ine-Angus recorder , the smal lest d i v i s i o n on the chart represents -12 2 x 10 amps. The b ias of the e lectrodes i s set by another simple dry c e l l c i r c u i t con t ro l l ed by a potent iometer . A l l t h i s c i r c u i t r y i s housed i n the cabinet with the phase s ens i t i v e detec tor and i t s assoc ia ted amp l i  f i e r s and t ransformers . The cable to the e lec t rode i s sh i e l ded , and the s h i e l d i s grounded through the cabinet plug to the c i r c u i t ground of the phase sens i t i v e de tec to r . I t i s i s o l a t e d , however, from the monochromator box, which i s grounded through the sh i e l d on the cable from the PbS de tec tor to the phase sens i t i v e de tec tor cab inet . - 29 - CHAPTER V C a l i b r a t i o n , Operation, and Performance of Apparatus V - l . C a l i b r a t i o n 1.1 Electrode The Pt electrode was used only as the i n d i c a t o r of the balance between the photochemical e f f e c t s of the monochromatic and compari son l i g h t beams. Thus, no c a l i b r a t i o n of the electrode current as a f u n c t i o n of oxygen concentration was necessary f o r the operation of the apparatus. However, some parameters associated with the electrode's opera t i o n had to be determined. The p o l a r i z a t i o n voltage was determined by s e t t i n g the Pt and Ag/AgCl electrodes i n a 1 N NaCl s o l u t i o n i n equilibrium with atmos pheric oxygen. The Pt was made the cathode, the p o l a r i z i n g voltage was v a r i e d , and the current flowing i n the external c i r c u i t p l o t t e d as a f u n c t i o n of voltage. The r e s u l t i n g curve was sigmoid shaped under - 0.9 v, followed by a steep r i s e at voltages ( ^ 1.0 v) high enough to reduce the H + ions i n s o l u t i o n . The p o l a r i z a t i o n voltage was taken, from the top of the S, to be - 0.7 v, well below the H^ producing p o t e n t i a l s . To check that the current was proportional to oxygen con centration, and not to some other substance i n a b u f f e r s o l u t i o n , the electrode current was measured as a function of oxygen concentration i n a _3 phosphate b u f f e r s o l u t i o n 10 J N i n NaCl. The oxygen concentration was c o n t r o l l e d by mixing varying q u a n t i t i e s of the a i r e q u i l i b r a t e d s o l u t i o n , i n which the oxygen concentration should have been 20.9$, with a s o l u t i o n through which nitrogen had been bubbled to remove a l l d i s s o l v e d oxygen. - 30 - The p l o t of current vs. d i l u t i o n was approximately a s t r a i g h t l i n e passing through the o r i g i n . 1.2 Lead Sulphide Detector 1.21 Intensity Response The i n t e n s i t y of the monochromatic l i g h t was v a r i e d by i n s e r t i n g a number of c l e a r glass s l i d e s i n t o the inc i d e n t beam i n f r o n t of the entrance s l i t of the monochromator. The l i g h t was allowed to f a l l i n t u r n on the lead sulphide c e l l , and on the s e n s i t i v e element of an Eppley Thermopile i n the same p o s i t i o n . The lead sulphide c e l l was used with i t s incident l i g h t chopped at 235 Hz and the output detected on both an o s c i l l o s c o p e and the phase s e n s i t i v e detector. The thermopile output f o r each i n t e n s i t y was taken as the average over ten measurements of the thermopile voltage change a f t e r a 30 second exposure to the unchopped l i g h t beam. These DC voltages were detected by the Keithl e y microvoltmeter and recorded on an Esterline-Angus recording milliammeter. In a l l cases, the reading e r r o r from the chart was of the same order of magnitude as the standard d e v i a t i o n i n the voltage values. I t was found, as in d i c a t e d i n the Kodak Ektron Detector handbook, that the AC voltage change across the load r e s i s t o r of the detec t i o n c i r c u i t was d i r e c t l y proportional to the l i g h t i n t e n s i t y . Further, the DC output of the PSD was also found to be d i r e c t l y proportional to the i n t e n s i t y . The voltages could thus be used i n the c a l c u l a t i o n of the r e l a t i v e e x t i n c t i o n c o e f f i c i e n t s . 1.22 Temperature Dependence of the Lead Sulphide Detector As discussed i n section _II-2.23> the output of the lead sulphide detector, and therefore the phase s e n s i t i v e detector, i s tempera ture dependent, the v a r i a t i o n of s i g n a l with temperature being given by - 31 - equation III-3D. This expression was checked against a se r i e s of measure ments on the PSD taken over the temperature range 24° C to 29° C. The in s i d e mean value of the temperature c o e f f i c i e n t , used as suggested by -2 -1 Wilson (9), was determined from these readings to be - 2.4 x 10 deg with a standard d e v i a t i o n of 0.2. 1.23 Absolute C a l i b r a t i o n of the Detection System While the output of the lead sulphide detector i s d i r e c t l y p r oportional to the i n t e n s i t y at a given wavelength, the p r o p o r t i o n a l i t y constant i s somewhat wavelength dependent. To determine t h i s dependence, and thus to allow the determination of the absolute l i g h t i n t e n s i t i e s causing a photochemical change, a f u r t h e r c a l i b r a t i o n was made. A serie s of i n t e n s i t y measurements were taken comparing the outputs of the thermopile and a lead sulphide c e l l . Readings were taken at 10 nm i n t e r v a l s between 3^5 nm and 1000 nm. Two layers of red cellophane were i n s e r t e d i n the beam f o r the readings between 750 nm and 1000 nm to remove the v i s i b l e second order. The lead sulphide output was f e d i n t o the phase s e n s i t i v e detector, and the DC output corrected f o r temperature deviations from 24° C. A l l voltages from the phase s e n s i t i v e detector were recorded at the 10 X gain s e t t i n g . The thermopile voltage changes were measured as described in. Section 1.21 above. Table I contains the r e s u l t s of these readings. 1.24 Determination of Light I n t e n s i t i e s Incident on the C e l l Suspension In order to determine the l i g h t i n t e n s i t y incident on the sample, the r a t i o of the i n t e n s i t i e s at the top of the cone channel con denser, and at the lead sulphide detector beside the base of the cone had to be found. The r a t i o was expected to vary with wavelength, since i t i s TABLE I Comparison o f P .S»D. and Thermopile Outputs Note: ( i ) P o l a r i z a t i o n vo l tage of PbS p a i r = 15 v ( i i ) Thermopile i n t e n s i t y c a l i b r a t i o n = 0.059 uv/uw/cm P.S.D. Output i n mV DC Wavelength Corrected to 24° C and Thermopile Output 10 X Gain i n microvo l ts DC S l i t Width = 2.00 mm 365 21.11 t .19 0.80 2 -02 370 23.74 t .18 0.88 2 .02 380 29.22 ± .17 1.04 2 »02 390 34.64 ± .21 1.31 * .03 400 39.55 * .15 1.60 I .03 410 46.12 I .15 1.96 1* .03 420 53.19 - .15 2.32 ± .04 430 60.66 ± .15 2.60 ± .04 440 67.73 2 .15 2.90 ± .06 450 75.38 - .15 3.32 ± .06 S l i t Width = 1.515 mm 450 41.72 1* .17 1.95 - .06 460 48.07 ± .17 2.23 ± .06 470 53.97 J .17 2.50 t .06 480 57.39 - .19 2.65 - .06 490 61.43 ± .20 2.83 * .06 500 65.05 ± .21 3.02 t .06 510 70.00 t .21 3.28 ± .06 520 78.38 * .21 3.70 * .06 530 82.48 ± .21 3.85 * .06 540 84.38 ± .21 3.96 ± .06 550 86.83 * .21 4.06 ± .06 560 89.48 t .21 4.17 ± .06 570 92.23 - .21 4.31 ± .06 580 94.60 * .22 4.37 * .06 590 96.80 ± .22 4.54 ± .06 600 98.70 t .22 4.63 - .06 P.S.D. Output i n mV DC Wavelength Corrected to 24 C and 10 X Gain Thermopile Output i n microvo l ts DC S l i t Width = 1.00 mm 600 40.38 t .15 1.61 t .02 610 41.28 ± .15 1.61 ± .02 620 42.40 ± .16 I.65 ± .02 630 42.75 * .16" I.65 ± .02 640 48.42 t .17 1.83 - .02 650 50.67 ± .17 1.92 ± .02 660 49.82 - .17 1.86 t .03 670 49.22 t .17 1.79 - .03 680 48.87 ± .17 I.76 ± .03 690 48.64 ± .18 I.72 t .03 700 48.14 ± .18 1.69 ± .03 710 46.49 t .18 1.62 t .03 715 49.49 t .18 1.71 ± .03 720 54.24 t .18 1,89 ± .03 730 57.44 ± .18 I.96 ± .03 740 55.44 ± .18 1.89 i .03 750 53.74 ± .18 1.80 ± .03 S l i t Width = 1.00 mm Two l aye rs red cel lophane i n o p t i c a l path 750 28.48 ± .1 1.06 t .02 760 27.80 ± .1 1.01 ± .02 770 27.35 - .1 0.98 ± .02 780 26.8 ± .1 0.96 ± .02 790 26.4 ± .1 0.96 ± .02 800 25.94 - .1 0.89 * .02 810 25.64 ± .12 0.89 * .02 820 25.44 ± .12 0.86 ± .02 830 25.36 ± .12 O.83 ± .02 840 25.48 t .12 O.85 ± .02 850 25.83 t .12 O.87 ± .02 860 26.39 * .12 0.88 ± .02 870 27.40 ± .16 0.90 ± .02 880 28.27 * .17 0.90 ± .02 890 29.07 - .17 0.93 - .02 900 29.82 ± .17 0.93 * .02 910 30.57 * .17 0.96 ± .02 920 31.22 ± .17 1.00 i .02 930 31.5^ ± .16 0.98 t .02 940 31.90 ± .16 1.01 ± .02 950 32.17 2 ' l 6 1.01 ± .02 960 32.44 I .16 0.98 t .02 970 32.65 ± .16 1.01 + .02 980 32.73 t .16 1.03 t .03 990 32.73 ± .16 1.02 t .02 1000 32.66 t .16 0.99 * .02 - 32 - a measure both of the e f f e c t of the cone on the spec t r a l i n t e n s i t y , and the a b i l i t y of the g ra t ing to f i l l the s l i t un i formly over the range of wavelengths used# The s igna l s from l ead sulphide detectors mounted both permanently at the base of the cone, and d i r e c t l y on i t s end, were measured sequen t i a l l y on the phase sens i t i v e de tec to r . The detec tor on the end of the cone was f i x e d i n the p o s i t i o n which gave the maximum output, and was separated from the cone only by a g lass cover s l i p . Measurements were made i n chopped l i g h t from 300 nm to 1000 nm, (a red f i l t e r was p laced i n the beam from 700 nm to 1000 nm to remove the second order ove r l ap ) . The phase s ens i t i v e de tec tor readings were a l l cor rected to Zk° C and an amp l i f i e r ga in s e t t i ng of 100 X. The r e s u l t s , expressed as the r a t i o , St/Sb, of the corrected s i g n a l at the top of the cone, to that at the bottom, are d i s  p layed i n F igure 7» V-2. Determination o f the Ac t i on Spectrum 2,1 Preparat ion of the B i o l o g i c a l Sample The c e l l suspension used here to t e s t the apparatus was Bakers yeas t . I t was maintained f o r two weeks i n 0,15 M phosphate bu f f e r s o l u t i o n , 1$ to 2$ ethanol by volume, at pH 6.5 to 7.5. The suspension was cont inuously bubbled with f i l t e r e d a i r . S t e r i l e procedures were not fo l l owed , and the yeast was l e f t exposed to the a i r throughout the experimental p e r i o d . When requ i red f o r an experiment, a sample of the _3 suspension was made 10 N i n NaCl dur ing d i l u t i o n to a c e l l concentrat ion which gave a r e s p i r a t i o n time of 1 min . - 33 - 2.2 Operating Procedure The procedure f o r the operation of the act i o n spectrum apparatus i s as follo w s : ( i ) The e l e c t r o n i c s are allowed to warm up f o r at l e a s t an hour before any measurements are attempted. ( i i ) The p o s i t i o n of the electrode chamber r e l a t i v e to the cone channel condenser i s adjusted with s e t t i n g screws u n t i l the electrode i s centered d i r e c t l y over the center of the end of the cone. The correc t p o s i t i o n may be found by looking up through the cone from i n s i d e the monochromator with the a i d of a mirror. The Pt wire i n the end of the glass electrode i s seen as a small br i g h t dot i n the multiple r e f l e c t i o n s i n the cone's w a l l s . The center p o s i t i o n i s e a s i l y determined by watching the multiple patterns vary i n symmetry as the electrode i s s h i f t e d . ( i i i ) The phase s e n s i t i v e detector output i s maximized by: (a) Set t i n g the reference s i g n a l to a symmetrical square wave, as seen on an o s c i l l o s c o p e . (b) Adjusting the phase of the reference s i g n a l with respect to the PbS c e l l s i g n a l to give the maximum DC output. This i s conveniently done by changing the azimuthal p o s i t i o n of the b a r r i e r l a y e r c e l l from which the reference s i g n a l o r i g i n a t e s . ( i v ) The entrance and e x i t s l i t s are set f o r the desired bandpass. (v) The electrode chamber i s f i l l e d with a gas mixture, saturated with water vapour to prevent evaporation of the - 34 - drop, of 4 parts CO to 1 part Og. ( v i ) With the electrode spacing set as i n Section IV-3, the e x i t s l i t f i s h t a i l closed, and the chopper wheel turned o f f , a drop of c e l l suspension i s introduced between the Pt and reference electrodes. The drop, held by surface tension between the two electrodes and the cover s l i p , should be roughly of the same diameter as the t i p of the glass-sealed Pt electrode. The drop of c e l l s i s allowed to a t t a i n a steady state of r e s p i r a t i o n , as i n d i c a t e d by a reasonably stable electrode current. This takes 15 to 20 min., and the current i s u s u a l l y between 5 and 50 na f o r yeast. ( v i i ) The monochromator i s set to 550 nm and the e x i t s l i t f i s h t a i l opened. The electrode current should decrease because the l i g h t , by reducing the i n h i b i t i o n by CO, speeds up the r e s p i r a t i o n . For yeast suspensions, the decrease was always about 1 to 2 na. ( v i i i ) The comparison beam i s switched onto the sample, and i t s i n t e n s i t y a l t e r e d u n t i l the two beams produce the same steady state electrode current. ( i x ) When balance i s obtained the l i g h t chopper i s switched on, the i n t e n s i t i e s of the monochromatic and reference beams measured on the phase s e n s i t i v e detector, and the temperature at the lead sulphide detector recorded. (x) The wavelength i s v a r i e d and the measurements repeated as long as the c e l l s remain active enough to keep the electrode current stable, u s u a l l y about two hours or - 35 - more. I f i t i s necessary to change gain settings on the phase s e n s i t i v e detector during the course of the experi ment, the gain r a t i o should be determined, since i n the present apparatus the gain seems to depend somewhat on the external conditions. ( x i ) When the experiment i s completed, the chamber i s flushed several times with water, then l e f t f i l l e d with d i s t i l l e d water, to prevent the Ag/AgCl reference electrode from d e t e r i o r a t i n g . 2.3 C a l c u l a t i o n of Relative E x t i n c t i o n C o e f f i c i e n t s With the data obtained from the preceeding procedure, the r e l a t i v e e x t i n c t i o n c o e f f i c i e n t s of the terminal oxidase of the c e l l s under examination may be determined as follo w s : ( i ) The phase s e n s i t i v e detector readings from the monochromatic and comparison beams are corrected f o r temperature dependence to the standard value of Zh° C. ( i i ) The temperature corrected voltages corresponding to the monochromatic l i g h t i n t e n s i t i e s at the base of the cone are adjusted to give the i n t e n s i t i e s incident on the sample. Figure 7 gives the f a c t o r , St/Sb, by which the i n t e n s i t y at each wavelength must be m u l t i p l i e d to give the f i n a l value WA . In general, these f i g u r e s should i n t u r n be m u l t i p l i e d by the r a t i o of the thermopile readings to those of the lead sulphide detector, as determined i n Section V-1 .23. However, the r a t i o i s a slowly varying f u n c t i o n of wavelength, varying only because the d e t e c t i v i t y 300 3 5 0 400 4 5 0 500 5 5 0 600 650 X mu FIGURE T St/Sb C a l i b r a t i o n Curve. - 36 - of the lead sulphide c e l l increases i n the i n f r a - r e d . Over the wavelength i n t e r v a l used i n the present experiment, the r a t i o f l u c t u a t e s about the mean with a standard de v i a t i o n of l e s s than 1$, so no such f a c t o r i s used i n the c a l c u l a  t i o n . Also, i t i s not necessary to make e i t h e r an St/Sb, or a thermopile adjustment to the voltages associated with the comparison beam. I t i s of f i x e d wavelength, and there fore both numerical f a c t o r s cancel out i n the computation of the r e l a t i v e e x t i n c t i o n c o e f f i c i e n t s , ( i i i ) The r a t i o of the e x t i n c t i o n c o e f f i c i e n t s at some wavelength X , to that at 550 nm, i s calculated from: V- 3• Performance 3.1 Action Spectrum of Yeast The action spectrum of the terminal oxidase of Bakers yeast, as determined by the above method, i s i l l u s t r a t e d i n Figure 8. The l o c a t i o n of the peaks coincides with the peaks of the absorption spectrum of cytochrome a-a^, and thus i d e n t i f i e s cytochrome as the terminal oxidase. 3.2 Accuracy of the Action Spectrum Determination The l i m i t of accuracy with which any cal c u l a t e d point of the a c t i o n spectrum may be determined i s the sum of the inherent errors and u n c e r t a i n t i e s i n each of the terms i n equation 1-8: ( i ) Uncertainties i n : (a) Both the random e r r o r , and the reading e r r o r from the chart introduced l e s s than 1$ erro r i n Cx Wssa 550 WA Cssa X (1-8) 6 550 and were neglected. €.550 2.0 f.d 1.6 U IZ 1,0 0.8 0.6 0.4 0.2 h 0.0 360 Mote- x => Data of Costo r and Chance • => Da fa. -From presenf work. Error bars denote t Cr x 1/10 400 440 480 520 A rnjj FIGURE 6 560 600 640 Action Spectrum of the CO Ihibifion of Respiration in Baker's Yeast - 37 - (b) The c a l c u l a t i o n of the temperature c o e f f i c i e n t and i t s a p p l i c a t i o n t o the PSD output introduces a 1$ uncertainty i n the monochromatic i n t e n s i t i e s , and a n e g l i g i b l e uncertainty i n the comparison i n t e n s i t i e s , as shown below: A temperature corrected voltage from the PSD i s determined from the expression log S f = log Si + o c A T where Sf = s i g n a l corrected to 24° C Si' = s i g n a l at a temperature d i f f e r i n g from 24° C by A T Thus, the actual e r r o r i n l o g S f , A (fog Sf) 3 w i l l be the sum of the a c t u a l errors i n l o g S / , and o c A T . The e r r o r i n l o g S i , Af log Si)} i s found by d i f f e r e n t i a t i o n to be A ( looS i ) s A S . Si where A S i i s the uncertainty i n S due to randomness and reading e r r o r s . Since 04 i s taken to be known to * 0.1° C, the uncertainty i n OL A T w i l l be: (. oz +• .1 \ o c A T ^ A T ; T h u s s A f l o g S A m A S i +r.oz + .i W A T Si ^ AT; The uncertainty i n the f i n a l value S^ ., w i l l then b e A S f =A ( foo J S f ) x ^ - 38 - or, expressed as a percentage iOQ'ASf = A(loy Sf) *iOO S f * \ A S i +('Ql WAT] x\0O So, f o r a t y p i c a l temperature c o r r e c t i o n of the s i g n a l corresponding to a monochromatic beam: A T = 2 C° S± = 0.1 mv These f i g u r e s give the uncertainty i n the corrected voltage as approximately ± 1$, and applying f i g u r e s f o r the comparison beam i n t e n s i t i e s gives a possible e r r o r much l e s s than 1%, (c) Due to a v a r i a t i o n In the gain l e v e l s of the PSD, the values of St/Sb are only reproducible to t 5$, introducing t h i s f u r t h e r e r r o r i n t o . This e r r o r i s not inherent to the method, however, and can probably be removed (see Chapter VT). (d) The balance point, at which the photochemical e f f e c t s of the monochromatic and comparison l i g h t s are the same i s found by equa l i z i n g the slopes of the recorded current output under the two i l l u m i n a  t i o n s . The estimation l i m i t s the determination of the balance point to within - 1$. Since such a balance i s required twice f o r any point on the action spectrum, a t 2^ e r r o r could be introduced i n t h i s way. 20 mv oC = 2.3 x 10' - 39 - (e) The error in the ratio s s 0 / h is negligible. Thus, the accuracy of the ratio G\ / £ s s o is found to be: •550 SO A J " ^/QSS is only sure, then, to within - 14$. - 40 - CHAPTER VI Suggested Improvements VI-1. Reduction of E r r o r i n Present Apparatus Considerable improvement i n the accuracy of the instrument, without d r a s t i c a l t e r a t i o n s , can be obtained i n the fol l o w i n g ways: ( i ) Removing the uncertainty i n the gain of the am p l i f i e r when the ranges are changed, or a l t e r i n g the gain r a t i o s so that a l l measurements could be taken on one s e t t i n g , should reduce the e r r o r i n St/Sb to 1 or 2$. ( i i ) Changing the system of introducing f i l t e r s when the beams are switched should remove the tra n s i e n t e f f e c t s on the drop, and eliminate the uncertainty i n the balance point determination. These improvements should reduce the l i m i t s of accuracy to t 6$ f o r the act i o n spectrum of any given b i o l o g i c a l sample. VI-2. A l t e r a t i o n s to Increase Range and S e n s i t i v i t y of Apparatus The following are a few suggestions r e l a t e d t o possible extensions of the apparatus, both i n accuracy and range. ( i ) A i r condition the room i n which the apparatus i s located to c o n t r o l temperature v a r i a t i o n s . I f these v a r i a t i o n s could be reduced to' £ 0,5° C or l e s s , then ( i i ) below would not be needed. ( i i ) Make the output of the lead sulphide c e l l independent of temperature by adding a temperature c o n t r o l l e d compen sating c i r c u i t element to the detector's voltage supply. - 41 - One such method would be to put a thermistor of appropriate s i z e i n se r i e s with the supply battery, as shown below. Thermistor Figure 9 Thermistor Controlled C i r c u i t f o r PbS P o l a r i z a t i o n I f the thermal c h a r a c t e r i s t i c s of the thermistor and detector are s i m i l a r over the normal range of room tempera tures, the measurements of l i g h t i n t e n s i t y should be i n  dependent of temperature. ( i i i ) Replace the present system f o r introducing the comparison l i g h t with a beam from a separate bulb outside the box. A l l f i l t e r s and the i n t e n s i t y c o n t r o l could go int o the comparison beam outside the monochromator. The beam could enter the monochromator with the beam axis p a r a l l e l t o , but ju s t below, the e x i t s l i t . The beams could then be interchanged by s l i d i n g i n a small mirror to d e f l e c t t h i s beam onto the s l i t and at the same time cut of f the monochromatic beam. The s l i t would thus be f i l l e d with l i g h t at a l l times. - 42 - Such a change would necessitate changing the method of chopping the l i g h t . A possible and b e t t e r method would be to have the detector mounted on one prong of a magneti c a l l y d r i v e n tuning fork (Bulova Watch Co.). Such devices are a v a i l a b l e with v i b r a t i o n amplitudes of s u f f i c i e n t magnitude to switch the s e n s i t i v e area i n and out of the l i g h t , i f masks are employed. The fork could be mounted beside the base of the cone, i n the same o p t i c a l l o c a t i o n as the present detection system. The l i g h t chopping wheel could then be discarded from the system e n t i r e l y . The reference s i g n a l f o r phase s e n s i t i v e detection could be taken from the other h a l f of the f o r k , using i t as a mechanical chopper i n a c i r c u i t . The above ou t l i n e d arrangement would have several advantages over the present system: (a) V i r t u a l l y no scattered l i g h t would be introduced i n t o the o p t i c a l path by r e f l e c t i o n from the f i l t e r s and i n t e n s i t y c o n t r o l l i n g louvers. While not s i g n i f i c a n t at the i n t e n s i t i e s and wave lengths used i n the i n i t i a l t e s t i n g of the apparatus, the scattered l i g h t i s of appreciable i n t e n s i t y , as observed by the unaided eye, and could become important at shorter wavelengths where the a v a i l a b l e i n t e n s i t y i s low. (b) Since the e x i t s l i t would be f i l l e d with l i g h t throughout the t r a n s i t i o n between beams, the drop - 43 - would be fully illuminated at a l l times, and no transient effects would appear. This would greatly increase the speed at which the action spectrum could be taken. (c) Elimination of the chopping wheel would allow uninterrupted illumination of the sample throughout the intensity measurements, (iv) The ultra-violet range of the instrument could be extended by using a hydrogen lamp as the source in the shorter wavelength region. A combined u.v.-visible source could be obtained by mounting the tungsten lamp in the usual way, and mounting a hydrogen lamp directed at 90° to be optical path of the incandescent light. A dichroic mirror placed at the intersection of the two light beams, at 45° to each of them, would reflect the u.v. light from the hydrogen lamp into the monochromator while allowing the visible light from the tungsten lamp to pass straight through unreflected. Such filters are available which will reflect greater than 90% of the u.v. while passing more than 90$ of the intensity of the visible light. - 44 - BIBLIOGRAPHY 1. Setlow, R.B. and P o l l a r d , E.C., "Molecular Biophysics" Addison- Wesley Publishing Co., Inc. (1962) pp. 267-305. 2. Horio, T. and Taylor, C.P.S.; J . B i o l . Chem. 240: 1772 (1965). 3. Warburg, 0., "Heavy Metal Prosthetic Groups and Enzyme Action* Oxford U n i v e r s i t y Press (1949). 4. Castor, L.N. and Chance, B.; J . B i o l . Chem. 217_: 453 (1955). 5. Bucher, T. and Kaspars, J . ; Biochim et Biophys. Acta, 1: 21 (1947). 6. Williamson, D.E.; J . Opt. Soc. Amer. 42: 712 (1952). 7. Jones, R.C., i n "Advances i n E l e c t r o n i c s " V o l . V, L. Morton, E d i t o r ; Academic Press Inc. (1953)* 8. Davies, P.W., i n " P h y s i c a l Techniques i n B i o l o g i c a l Research" V o l . IV, W. Nastuck, E d i t o r ; Academic Press Inc. (1962). 9. Wilson, E.B., J r . "An Introduction to S c i e n t i f i c Research" McGraw- H i l l Book Co. Ltd. (1952) pp. 232-276. 

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