Analytical Chemistry with Spatial Resolution: Obtaining Spectral

12 Analytical Chemistry with Spatial Resolution: Obtaining Spectral Images with Multichannel Detectors JAMES B. CALLIS

Downloaded by COLUMBIA UNIV on January 21, 2018 | http://pubs.acs.org Publication Date: November 16, 1983 | doi: 10.1021/bk-1983-0236.ch012

Department of Chemistry, University of Washington, Seattle, WA 98195 ADAM P. BRUCKNER Aerospace and Energetics Research Program, University of Washington, Seattle, WA 98195 The concept of the "chemical composition/distribution matrix" i s introduced i n order to f a c i l i t a t e the interpretation of a chemical analysis when information about the spatial distribution of the analytes i s included. Methods for displaying the chemical composition matrix as a "chemical image" are discussed. Generally, the chemical content matrix i s not obtained directly, but must be deduced from spectroscopic measurements at each point i n space. This data set i s most conveniently thought of as a "spectroscopic image." Methods for obtaining the spectroscopic image by scanning are considered. I t i s shown that schemes which employ imaging detectors have large time and/or signal-to-noise ratio advantages over methods using single-channel detectors. As an example of the use of an imaging detector to obtain the spectral image, we present the "Multichannel Imaging Spectrophotometer" (MIS). I t is shown that a "multichannel disadvantage" limits the a b i l i t y of imaging devices to detect analytes embedded i n scattering media. Possible remedies for this problem are devised and data presented to support them. As the proceedings o f t h i s symposium a t t e s t , imaging d e t e c t o r s have an important r o l e t o p l a y i n a n a l y t i c a l o p t i c a l spectroscopy. They have been p a r t i c u l a r l y e f f e c t i v e i n s o l v i n g problems o f multicomponent a n a l y s i s , where i t i s necessary t o make s p e c t r o s c o p i c measurements a t m u l t i p l e wavelengths. Also, the multichannel advantage o f an o p t i c a l a r r a y i s o f t e n valuable when a chromatographic technique f o r s e p a r a t i o n i s combined with a s p e c t r o s c o p i c technique f o r f i n g e r p r i n t i n g ( 1 , 2 ) . The recent commercial introduction of liquid chromatography detectors c o n s i s t i n g o f r a p i d scanning UV-VIS spectrometers employing diode arrays demonstrates the growing a p p r e c i a t i o n o f the power o f these techniques ( 3 ^ 5 ) . 0097 6156/83/0236-0233S06.00/0 © 1983 A m e r i c a n C h e m i c a l S o c i e t y

Talmi; Multichannel Image Detectors Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


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MULTICHANNEL IMAGE DETECTORS

The u n i v e r s i t y o f Washington group has concentrated on the use o f imaging d e t e c t o r s i n f l u o r e s c e n c e spectrophotometry ( 6 ) . Our instrument, the Video Fluorometer ( 7 ) , uses a unique o p t i c a l concept which allows us t o record i n d i g i t a l form a s e r i e s o f f l u o r e s c e n c e s p e c t r a as a sequenced s e t o f up t o 256 e x c i t a t i o n wavelengths i n a time as short as 16.7 msec without mechanical scanning. The e f f e c t o f t h i s novel scheme i s t o produce an image which c o n t a i n s both e x c i t a t i o n and emission s p e c t r a . To capture t h i s image q u a n t i t a t i v e l y , we use an SIT v i d i c o n as a s e n s i t i v e multichannel imaging d e v i c e i n t e r f a c e d t o a PDP 11/04 computer through a h i g h speed frame b u f f e r memory. The performance o f the Video Fluorometer has been thoroughly t e s t e d (8,9) and a s e r i e s of algorithms has been designed t o a l l o w q u a n t i t a t i v e (10,11) and q u a l i t a t i v e (12) a n a l y s i s o f complex mixtures. Recently, we have i n t e r f a c e d the Video Fluorometer t o a l i q u i d chromatograph so t h a t i n d i v i d u a l components o f a complex mixture c o u l d be separated and t h e i r f l u o r e s c e n c e scanned i n r e a l time as they emerged from the column (13). We are c u r r e n t l y c o n s t r u c t i n g a second g e n e r a t i o n o f video fluorometers, r e p l a c i n g the SIT camera with an even more s e n s i t i v e i n t e n s i f i e d diode array, and i n c o r p o r a t i n g a r a p i d l y scanned n i t r o g e n l a s e r pumped dye l a s e r as e x c i t a t i o n source. T h i s system i s b e i n g i n t e r f a c e d t o a c a p i l l a r y gas chromatograph v i a a molecular beam so t h a t u l t r a - h i g h r e s o l u t i o n spectroscopy can be performed " o n - t h e - f l y " (14). While a p p l y i n g o p t i c a l imaging d e t e c t o r s t o a n a l y t i c a l spectroscopy, we g r a d u a l l y began t o a p p r e c i a t e t h e i r p o t e n t i a l f o r e l u c i d a t i o n o f those problems o f multicomponent analysis where i t was d e s i r e d not o n l y t o i d e n t i f y and q u a n t i t a t e each component but a l s o t o s p e c i f y i t s l o c a t i o n i n space as w e l l , i.e. when a n a l y t i c a l chemistry was t o be performed with s p a t i a l resolution. I t i s our b e l i e f t h a t a n a l y t i c a l chemists w i l l become i n c r e a s i n g l y concerned with t h i s problem i n the next decade (15/16) and t h a t o p t i c a l imaging d e t e c t o r s w i l l p l a y a major r o l e i n i t s s o l u t i o n . Conceptual Framework The d e s i r e d endpoint o f the task o f a n a l y t i c a l chemistry with s p a t i a l r e s o l u t i o n i s a l i s t o f the a n a l y t e s present, t h e i r amounts, and the s p a t i a l l o c a t i o n s o f each. The b e s t means f o r organizing such a list i s i n the form of a "chemical composition/distribution tensor," Ç , each element o f which i s a v e c t o r c o f dimension 3+R, whose f i r s t t h r e e elements r e f e r t o the s p e c i f i c l o c a t i o n i n space, and the remaining R elements g i v e the amounts o f each o f the R d i s t i n c t chemical components recognized, i . e .

i,j,k,m,n

)

[1]

Here, the i n d i c e s i , j , k r e f e r t o the t h r e e s p a t i a l c o o r d i n a t e s .


12.

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BRUCKNER

Analytical

Chemistry

with Spatial

Resolution

and the R i n d i c e s m, n ... enumerate the R distinct components. The chemical composition t e n s o r can be w r i t t e n c o n v e n i e n t l y as a 3+R by L matrix, C, which which c o n s i s t s o f L ordered rows which are the v e c t o r s c, and the s c a l a r L i s the t o t a l number o f s p a t i a l elements. I n t e r p r e t a t i o n o f the chemical composition tensor as a t a b l e q u i c k l y becomes d i f f i c u l t f o r complex o b j e c t s with m u l t i p l e a n a l y t e s . A much b e t t e r r e p r e s e n t a t i o n i s i n the form o f a "chemical image" d i s p l a y e d on a computer g r a p h i c s terminal. For a two-dimensional o b j e c t t h i s i s r e a d i l y done because there is a one-to-one correspondence between the p o s i t i o n s on the o b j e c t and l o c a t i o n s on the x,y d i s p l a y . Each chemical e n t i t y can be r e a d i l y i d e n t i f i e d i f i t i s encoded as a p a r t i c u l a r c o l o r with the i n t e n s i t y b e i n g p r o p o r t i o n a l t o l o c a l concentration. For three-dimensional o b j e c t s , the t a s k i s more difficult. One simple method i s t o f o l l o w the example o f computed tomography and t o d i s p l a y the o b j e c t as a s e r i e s o f two-dimensional slices. In cases where the d i s t r i b u t i o n s o f s p e c i f i c e n t i t i e s are o r g a n i z a b l e as d i s c r e t e subobjects, e.g. i n c l u s i o n s i n a rock, s u r f a c e s e n c l o s i n g each o b j e c t may be defined, the three-dimensional scene projected on a two dimensional plane, the v i s i b l e s u r f a c e s f a l s e - c o l o r encoded, and hidden o b j e c t s removed. In the case where o b j e c t s are embedded w i t h i n other o b j e c t s , one can d e f i n e " c l i p p i n g " planes or use semitransparent outer s u r f a c e s . Some b e a u t i f u l examples o f the use o f these techniques f o r p o r t r a y i n g the s t r u c t u r e s and i n t e r a c t i o n s o f biomolecules have been given by Langridge and coworkers (17). u n f o r t u n a t e l y , the chemical composition t e n s o r i s not a d i r e c t l y measureable q u a n t i t y . Instead, i t must be deduced from i n d i r e c t data. In the f i r s t p l a c e , s p e c t r o s c o p i c measurements w i l l u s u a l l y be used t o i d e n t i f y and q u a n t i f y the chemical substances present. There may o r may not be a one-to-one r e l a t i o n s h i p between the i n t e n s i t y a t one p a r t i c u l a r wavelength and the l o c a l concencentration o f a p a r t i c u l a r chemical e n t i t y . In the second p l a c e , i f the a n a l y s i s i s t o be three-dimensional and non-destructive, the chosen range of electromagnetic r a d i a t i o n must be t r a n s m i t t e d through the o b j e c t f o r m u l t i p l e orientations of the object. The reconstruction of three-dimensional i n f o r m a t i o n from a s e r i e s o f two-dimensional p r o j e c t i o n s i s not a t a l l t r i v i a l , but we s h a l l leave t h i s aspect of t h i s problem t o a l a t e r date. Of major concern i n t h i s work i s the i r r e f u t a b l e f a c t t h a t when h i g h r e s o l u t i o n chemical images are being sought, where a number o f a n a l y t e s are t o be i d e n t i f i e d and q u a n t i f i e d , huge amounts o f data w i l l be involved. For example a single two-dimensional o b j e c t r e s o l v e d t o 256 p o i n t s i n both s p a t i a l domains with absorbance measured a t 256 wavelengths t o a p r e c i s i o n o f one p a r t i n 256 w i l l r e q u i r e 16.78 Mbytes f o r storage. While such data storage and p r o c e s s i n g requirements w i l l s t r a i n the c a p a c i t i e s o f present-day computers, recent advantages i n o p t i c a l storage techniques (such as the read/write M


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video d i s k ) and inexpensive p i p e l i n e d a r r a y p r o c e s s o r s g i v e us optimism t h a t t h i s p a r t o f the problem i s t r a c t a b l e . There s t i l l remains the d i f f i c u l t y o f a c q u i r i n g a l l o f the d a t a i n a f i n i t e p e r i o d o f time. For example, c o n s i d e r the a c q u i s i t i o n o f d a t a for the 256x256 image mentioned above u s i n g a c o n v e n t i o n a l spectrometer which takes one second t o o b t a i n a 256-point spectrum from one s p a t i a l element on the o b j e c t : a t o t a l o f 18 hours would be needed! C l e a r l y , m u l t i c h a n n e l d e t e c t o r s would be of enormous b e n e f i t i n reducing the d a t a a c q u i s i t i o n time and/or improving the s i g n a l - t o - n o i s e r a t i o (SNR) f o r a g i v e n a c q u i s i t i o n time. We shall now consider several methods f o r acquiring s p e c t r o s c o p i c images u s i n g o p t i c a l imaging d e t e c t o r s . In the f i r s t method, shown s c h e m a t i c a l l y i n F i g u r e 1, a one-dimensional a r r a y d e t e c t o r i s used a t the output o f a spectrometer t o a c q u i r e a l l the d e s i r e d s p e c t r a l d a t a simultaneously a t one spatial l o c a t i o n a t a time. The s p a t i a l scanning may be achieved e i t h e r by displacement o f the o b j e c t through a f i x e d measuring spot as shown i n F i g u r e 1A o r by displacement o f the image o f the f u l l y illuminated object through a fixed aperture before the spectrometer as shown i n F i g u r e IB. E i t h e r o f these methods w i l l p o t e n t i a l l y y i e l d a time advantage o f Ν over a s i n g l e - c h a n n e l d e t e c t o r , where Ν i s the number o f s p e c t r a l elements a c q u i r e d . For convenience, we have shown l a s e r s as the l i g h t sources, but incoherent sources may be used as w e l l and indeed w i l l be needed f o r a b s o r p t i o n / r e f l e c t a n c e imaging. Even more e f f i c i e n t methods f o r c o l l e c t i n g the s p e c t r a l image are shown i n F i g u r e 2. In these schemes, a two-dimensional imaging d e t e c t o r i s used t o capture a s p a t i a l image o f the object. In the case where the spectral measurement is a b s o r p t i o n , r e f l e c t a n c e , o r e x c i t a t i o n , the o b j e c t i s i l l u m i n a t e d by a sequence o f uniform monochromatic wavelengths produced by a tunable l a s e r o r lamp/monochromator combination, as shown i n F i g u r e 2A. One s p a t i a l image i s a c q u i r e d a t each wavelength o f i n t e r e s t and s t o r e d i n d i g i t a l o r analog form. In the case where the s p e c t r a l measurement i s o p t i c a l emission o r Raman s c a t t e r i n g , a monochromator i s used as an imaging wavelength f i l t e r , and one s p a t i a l image a t each wavelength o f i n t e r e s t i s obtained, as shown i n F i g u r e 2B. T h i s scheme has been s u c c e s s f u l l y used t o produce Micro-Raman images o f h i g h c o n t r a s t and r e s o l u t i o n ( 1 8 ) . In the case o f f l u o r e s c e n c e imaging, the schemes o f F i g u r e s 2A and 2B can be combined t o y i e l d an instrument capable o f r e c o r d i n g both e x c i t a t i o n and emission i n f o r m a t i o n on an extended object. Obviously, the above s t r a t e g i e s employing a two-dimensional s p a t i a l d e t e c t o r have a much l a r g e r m u l t i c h a n n e l advantage than schemes using a one-dimensional spectral detector. Their p o t e n t i a l time advantage i s MXN, where M i s the number o f s p a t i a l l y r e s o l v e d elements i n the X d i r e c t i o n and Ν i s the number o f s p a t i a l l y r e s o l v e d elements i n the Y direction. E s s e n t i a l l y , designs based upon two-dimensional d e t e c t o r s can a c q u i r e a complete spectral image i n the same time as a


Analytical

CALLIS AND BRUCKNER

BEAM EXPANDING TELESCOPE


OBJECT

Chemistry

with Spatial

Resolution

APERTURE

·

LENS

Y-AXIS TRANSLATOR

X-AXIS TRANSLATOR

LASER MONOCHROMATOR

Figure I. Schemes for using a one-dimensional array detector to acquire spectral images. Key: top, scanning of object; and bottom, scanning of image.

BEAM EXPANDING TELESCOPE

IMAGING LENS IMAGING OBJECT MONOCHROMATOR

MONOCHROMATOR

OBJECT

^FILTER

LENS^

—LIGHT

SOURCE

Figure 2. Schemes for using a two-dimensional array detector to acquire spectral images. Key: top, acquiring an absorption, reflectance, or excitation image; and bottom, acquiring an emission or scattering image.


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c o n v e n t i o n a l non-imaging spectrometer o f a homogeneous m a t e r i a l .

takes t o scan the

spectrum


The M u l t i c h a n n e l Imaging Spectrometer In t h i s s e c t i o n , we w i l l d e s c r i b e an instrument, the "Multichannel Imaging Spectrometer" (MIS) which i s capable o f r e c o r d i n g the a b s o r p t i o n or r e f l e c t a n c e spectrum at up t o 64,000 p o i n t s on an o b j e c t i n a time as s h o r t as two minutes. As a paradigm f o r s p a t i a l l y r e s o l v e d a n a l y t i c a l chemistry we have used MIS t o analyze m a t e r i a l s on t h i n - l a y e r chromatographic p l a t e s both qualitatively and quantitatively (19). Several data a n a l y s i s s t r a t e g i e s have been devised t o e f f i c i e n t l y use the s p e c t r a l data from MIS t o r e s o l v e o v e r l a p p i n g spots. A schematic o f our s i n g l e beam imaging spectrophotometer i s given i n F i g u r e 3. A twenty-watt tungsten halogen lamp operated from a h i g h l y s t a b l e DC power supply served as the l i g h t source. Wavelength s e l e c t i o n was accomplished with a Bausch and Lomb 1/4 meter monochromator equipped with a 600 lines/mm g r a t i n g b l a z e d f o r 300 nm. S l i t s were s e t t o provide a s p e c t r a l bandpass o f 7 nm. Wavelength scanning was accomplished by means o f a v a r i a b l e speed motor . In order t o uniformly i l l u m i n a t e the t h i n - l a y e r p l a t e , an image o f the d i f f r a c t i o n g r a t i n g was p r o j e c t e d on t o the p l a t e by means o f a lens p l a c e d a t the e x i t s l i t ( f i e l d stop ). L i g h t t r a n s m i t t e d through the p l a t e was c o l l e c t e d by an f/0.78 CCTV l e n s , and focused onto the p h o t o s e n s i t i v e s u r f a c e o f the SIT vidicon camera (Quantex QX-10). Our digital television photometry system has been p r e v i o u s l y d e s c r i b e d ( 8 ) . The camera was operated t o c o l l e c t a sequence o f images synchronously with the scanning o f the wavelength d r i v e o f the monochromator. The software f o r t h i s o p e r a t i o n was developed by Hershberger, e t . a l . (13) f o r f l u o r e s c e n c e d e t e c t i o n o f l i q u i d chromatographic e f f l u e n t s but was r e a d i l y modified t o serve the present need. Real-time d i s p l a y s o f the monochromatic p l a t e image and the a b s o r p t i o n spectrum at two l o c a t i o n s on the p l a t e are a v a i l a b l e . Data c o n s i s t i n g o f a s e r i e s o f images taken over a s e l e c t e d wavelength r e g i o n are s t o r e d on d i s k f o r l a t e r post-run p r o c e s s i n g , e i t h e r by the PDP 11/04 o r by a remote VAX 11/780. In F i g u r e 4 we i l l u s t r a t e the c a p a b i l i t y o f MIS t o analyze s e v e r a l one dimensional chromatograms simultaneously. The images are from a Whatman LHP-K p l a t e which was spotted with v a r i o u s amounts o f H TPP porphine i n f i v e separate lanes (1.1, 2.2, 3.3, 4.4 and 5.5 #g r e s p e c t i v e l y , r i g h t t o l e f t i n 4A). In lane 3, a mixture o f H TPP along with ZnTPP and PdEP was spotted. The p l a t e was then developed i n a dichloromethane/cyclohexane (50:50) mixture f o r 3.5 min. F i g u r e 4A shows the raw t r a n s m i s s i o n image o f the sample p l a t e and a blank p l a t e , r e s p e c t i v e l y , at 420 nm. In 4B the two images o f 4A are combined i n t o an absorbance image and d i s p l a y e d as an i s o m e t r i c p r o j e c t i o n . T h i s l a t t e r d i s p l a y i s u s e f u l f o r v i s u a l i z i n g the q u a n t i t a t i v e r e l a t i o n s h i p s among the absorbances o f the spots. The upper t r a c e i n F i g u r e 4C i s the 2

2



12.

Analytical

CALLIS AND BRUCKNER

TUNGSTEN

Chemistry

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Resolution

LAMP

-LENS MONOCHROMATOR

4IRROR

< π — K ' TLC

2

TV

MONITOR

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11/04

PLATE

SHUTTER

SIT CAMERA

GRAPHICS TERMINAL

Figure 3. Block diagram of MIS.


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Analytical Chemistry with Spatial Resolution: Obtaining Spectral

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