High-Fidelity Raman Imaging Spectrometry: A Rapid Method Using an Acousto-optic Tunable Filter

rapid communication

High-Fidelity Raman Imaging Spectrometry: A Rapid Method Using an Acousto-optic Tunable Filter

PATRICK J. TREADO, IRA W. LEVIN, and E. NElL LEWIS* Laboratory of Chemical Physics, National Institute of Diabetes, Digestive and Kidney Diseases National Institutes of Health, Bethesda, Maryland 20892

In this communication, we describe a technique for obtaining high-fidelity Raman images and Raman spectra. The instrumentation provides the ability to rapidly collect large-format images with the number of image pixels limited only by the number of detector elements in the silicon charge-coupled device (CCD). Wavelength selection is achieved with an acousto-optic tunable filter (AOTF), which maintains image fidelity while providing spectral selectivity. Under computer control the AOTF is capable of ~s tuning speeds within the operating range of the filter (400-1900 nm). The AOTF is integrated with the CCD and holographic Raman filters to comprise an entirely solid-state Raman imager containing no moving parts. In operation, the AOTF is placed in front of the CCD and tuned over the desired spectral interval. The two-dimensional CCD detector is employed as a true imaging camera, providing a full multichannel advantage over competitive Raman imaging techniques. Images and spectra are presented of a mixture of dipalmitoylphosphatidylcholine (DPPC) and L-asparagine, which serves as a model system for the study of both lipid/peptide and lipid/protein interactions in intact biological materials. The Raman images are collected in only several seconds and indicate the efficacy of this rapid technique for discriminating between multiple components in complex matrices. Ad- ditionally, high-quality Raman spectra of the spatially resolved micro- scopic regions are easily obtained. Index Headings: Imaging; Microscopy; Raman spectroscopy; Acousto- optics; Charge-coupled devices; Lasers; Holographic filters; Amino ac- ids; Phospholipids.

INTRODUCTION

Within the past several years a variety of instrumental developments have expanded the utility of Raman spectroscopy and Raman imaging for materials analysis. These new technologies make the design and construction of compact, rugged Raman instruments capable of rapid operation a practical reality for process monitoring and clinical imaging applications. One of the primary developments in instrumentation has been the use of silicon charge-coupled devices (CCD) for collection of visible excitation Raman spectra. 1 CCDs have high quantum efficiency, low readout noise, and extremely low detector dark current. When appropriately cooled, these two-di-

Received 20 April 1992. * Author to whom correspondence should be sent. Address for corre-

spondence: National Institutes of Health, Building 2, Room 114, Be- thesda, MD 20892.

mensional solid-state arrays are capable of long integration times and are well suited for detection of low- light-level signals. Silicon CCD detectors are particularly attractive for Raman spectroscopy when combined with red wavelength laser excitation, 2,3 due to their extended red response, which offsets the decrease in Raman scattering cross section. Longer wavelength excitation has the additional advantage of producing significantly less sample fluorescence and laser-induced photothermal degradation, as well as the capability of spectroscopically sampling deeper within thick materials.

Eliminating unwanted thermal and photochemical perturbations is critical in imaging experiments, particularly for biological materials? Faithful image repro- duction is achieved only if the structural integrity of a sample is retained throughout spectral image acquisition. The conventional Raman microprobe) while suitable for Raman microspectroscopy, provides limited imaging capability. Image generation requires the systematic scanning of the sample, typically achieved either by translating the sample through a tightly focused, stationary laser beam or, alternatively, by raster scanning the beam itself. The signal-to-noise ratios obtainable with conventional microprobes require signal averaging at each spatial position, making the generation of high-quality images inherently slow. As a result, only crude spatial maps are usually obtained. In addition, the spatial resolution of laser illumination scanning techniques is defined by the focused laser spot size. For high-spatial-resolution imaging, the small spot sizes employed may generate extremely high local laser power densities at the sample. These power densities may induce gross thermal decom- position of a sample--an important consideration in the study of fragile biological samples. Total incident power densities must be reduced to below damage threshold levels but with a commensurate increase in image acquisition time.

An improvement on point-scanning Raman microscopy that has been devised involves defocusing the excitation source in one dimension and systematically translating the sample through the laser slit illumination. The Raman line image is collected with a CCD detector placed at the focal plane of a spectrograph, e Although the line-scan Raman imaging approach is more efficient

Volume 46, Number 8, 1992 0003-7028/92/4608-121152.00/0 APPLIED SPECTROSCOPY 1211 © 1992 Society for Applied Spectroscopy

than point-scanning Raman microprobes, by the use of the two-dimensional capability of the multichannel detector, high local laser power densities may still be retained in the focused illumination dimension. Line-scan Raman imaging, like the scanning Raman microprobe, relies on translation stage technology to move the sample at small spatial increments through the line-focused laser beam. As a result, the maximum attainable resolution in the translated dimension is a convolution of the laser linewidth and the image step size. Positioning errors of the moving translation stage will tend to degrade the image quality and reduce the spatial resolving power. In general, any spectroscopic imaging strategy employing moving mechanical parts is subject to similar problems. Ideally, Raman imaging using widefield laser illumination and a CCD detector as an imaging camera represents optimal conditions. This approach provides a multichannel advantage over competitive techniques since the image fidelity is limited only by the number of detector elements in the CCD. In addition, the widefield optical configuration allows incident laser power densities to be moderated while still producing good-quality images and spectra.

Widefield illumination has been employed with Hada- mard transform Raman microscopy, 7,8 a technique which integrates multichannel CCD detection with spatial multiplexing for simultaneously collecting the three dimensions of information in Raman spectroscopic microscopy. The use of noninvasive widefield laser illumination in Hadamard imaging has been described as a spatial distribution multiplex advantage2 Hadamard microscopy, capable of imaging even relatively weak scatterers in only a few minutes, has defined the state of the art for vibrational spectroscopic microscopy, but it is not without limitations. In Hadamard imaging, the CCD detector is not employed to directly view the magnified image of the Raman scattered sample. Rather, the x and y spatial dimensions of the image are collected independently with a Hadamard mask and the CCD's vertical detector elements, respectively. While the y spatial dimension is reproduced at high quality directly with the CCD camera, problems associated with Hadamard multiplexing can corrupt the quality of the x spatial dimension image. In fact, mask defects, as well as mask alignment and positioning errors, can arise, TM ultimately compromising the quality of the images collected. Numerical methods have been devised to correct for Hadamard imaging defects, 8 but these techniques add several minutes of com- putation time. In addition, the raw images are spatially encoded and must be transformed before they can be viewed.

Another recent technological development is the application of holographic filter technology to Raman spectroscopy 11-~3 and Raman imaging. 8,~4 Holographic Raman filters are spectroscopic devices for Rayleigh line rejection and are designed for a variety of laser wavelengths. In general, the filters provide between 5 and 6 orders of laser rejection while uniformly transmitting 75- 80% of the Raman emission within 75 cm -1 of the ex- citing line. One, or even two, of these filters can be placed in front of a CCD detector to obtain very efficient Ray- leigh line rejection while maintaining image fidelity. Ho- lographic beamsplitters employed for laser epi-illumi-

1212 Volume 46, Number 8, 1992

nation and suitable for Raman spectroscopic imaging have also been described24 These spectral filters are almost ideal for Raman microscopy, as they reflect 90% of the excitation laser wavelength while transmitting almost 80% of the emitted radiation beyond a 400-cm -1 displacement from the Rayleigh line. The advantages of holographic filters, which include high throughput, uni- form transmission, and imaging quality, make them suitable choices for Raman spectroscopy and almost ideal for Raman imaging.

In this communication, we describe a completely solid- state approach for Raman spectroscopic imaging that relies on an image-quality-maintaining spectroscopic device. The method applies a relatively new commercial technology, digitally controlled acousto-optic tunable filters (AOTFs), to rapidly obtain high-fidelity Raman spectroscopic images. The technique uses a visible wavelength AOTF in conjunction with holographic laser filters, f/1 image collection optics, and a low-light-level CCD. The design and theory of the AOTF have been described previously for near-infrared vibrational ab- sorption microscopy. 15 The AOTF contains an optically transparent birefringent crystal, which can be viewed through directly and, therefore, placed between the emis- sive Raman scatterer and a Si focal-plane array detector. The result is a no-moving-parts spectroscopic imager capable of producing high-quality Raman spectra and multispectral Raman images in only several seconds.

E X P E R I M E N T A L

Figure 1 is a schematic of the acousto-optic filtered Raman imaging spectrometer, which consists of image collection and transfer optics, a tellurium dioxide (TeO2) AOTF (Brimrose TEAF-.6-1.2L), and a slow-scan, liquid-nitrogen-cooled, silicon CCD (Spex Spectrum One). The AOTF is an electronically tunable spectral bandpass filter which can be constructed for operation in the ul- traviolet, visible, or infrared regions. The device employed in this study has a clear aperture of 7 × 7 mm with two piezoelectric transducers bonded to one side for im- proved resolution. Application of an rf signal to the transducer produces acoustic waves in the birefringent crystal, which in turn generates a periodic modulation of the index of refraction by the elasto-optic or "Raman-Nath" effect. TM By digital control of the rf frequency applied to the transducer, an electronically addressable spectral bandpass filter is constructed. We employ the AOTF as an image-quality notch filter, similar in operation to a transmission diffraction grating, to effectively replace the dispersive spectrograph commonly employed for Raman spectroscopy. The filter retains image fidelity while simultaneously providing a spectral bandpass (FWHM) of approximately 2 nm (50 cm -1) at 630 nm. In addition, the AOTF is tunable to 0.1 nm (2.5 cm -1) and has a wavenumber repeatability of 0.05 nm (1.2 cm-1).

Raman excitation at 514.5 nm is provided by an Ar + laser (Coherent Innova-100) delivering I W of laser power to the sample. Illumination is performed at an oblique angle of 45 ° relative to the sample, which is placed on a standard microscope slide. The laser is defocused to a 1.5-mm-diameter spot size prior to impinging on the sample, to reduce the local power density. The illumi-

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V - - Sample AOTF F2

Fla. 1. Schematic representation of the acousto-optic filtered Raman imaging spectrometer. The optical train shows the path of the incident laser beam and Raman-scattered light. The lipid/amino acid sample is mounted on a microscope slide positioned 45 ° relative to the incident laser; 90 ° Raman scattering is collected and spectrally filtered with the AOTF. Holographic Raman filters are placed after the AOTF to eliminate intense Rayleigh scatter before the image is focused onto a liquid-nitrogen-cooled CCD.

nation spot is defocused until the magnified and projected laser image approximately matches the CCD detector active area. The imaging detector is a low-noise CCD providing 16 bits of dynamic range and configured as a rectangular array of 576 × 384 pixels, where each pixel is 20 #m square.

In operation, the laser is transmitted through a di- electric notch filter (Omega Optical 514.5 RN 10), F1 in Fig. 1, to attenuate extraneous laser plasma emission lines. The laser is defocused with lens L1 (50 mm f.1.) before impinging on the sample. Lens L2 (25 mm f.l., ]'/1) collects and collimates the radiation scattered at 90 ° to the incident beam. The collimated light is stopped down with aperture A1 and projected through the AOTF, where the scattered light is spectrally filtered. A beam- stop and aperture A2 are used to spatially select the (+) order diffracted beam while blocking both the ( - ) order diffracted beam [orthogonal in polarization to the (+) order] as well as the zero-order undiffracted beam, which contains all other wavelengths including the intense Ray- leigh scattered radiation. Two 514.5-nm holographic Ra- man edge filters (Physical Optics RHE 514.5), F2 in Fig. 1, are used to attenuate the Rayleigh line before the Raman image is focused by the image formation lens L3 (50-mm-f.1. camera lens) onto the CCD detector. The holographic Raman filters employed here provide 5 orders of rejection of the laser line at 514.5 nm while transmitting 80% of the radiation beyond 519.8 nm (200 cm-1). The AOTF, operable between 400 and 1900 nm, selects a series of wavelengths by applying approximately 2 W of rf power to the crystal in the frequency range of 40- 170 MHz. At each discrete wavelength a CCD frame is collected, digitized, and stored. The size, wavelength range, wavelength increment, and exposure time for each image frame may be modified under computer control. Unlike dispersive multichannel Raman spectrographs, there is effectively no trade-off with acousto-optic filtered spectrometry between spectral resolution and wavelength coverage. Higher spectral resolution is provided by increasing the physical interaction length of the crystal, which reduces the overall throughput of the device and is analogous to closing down the slits of a dispersive spectrograph. In general, the AOTF provides tuning coverage of approximately 11,000 cm -1, which would be adequate to perform full Stokes/anti-Stokes Raman spectroscopy for almost any type of material. This feature is in contrast to the setup of a spectrograph that

would employ a higher-groove-density diffraction grating to increase resolution at a cost of reduced spectral coverage.

Raman images are collected of aggregate films containing dipalmitoylphosphatidylcholine (DPPC) (Avanti Polar Lipids) and L-asparagine (Sigma Chemical Co.) deposited on a microscope slide. These two materials are chosen as model compounds for lipid/peptide interactions to stress the relevance of the method for the study of biological materials. The aggregates are formed by placing individual crystals on a slide, co-dissolving the samples with 5 uL of CHC13 to promote fusion of the two materials, and then removing solvent traces. Raman images of the fused aggregates are collected at 2880 cm -1, the asymmetric methylene stretching modes for the acyl chains of DPPC, and at 3390 cm -1, the Fermi resonance NH2 stretching mode component for L-asparagine. All Raman images presented in this work are obtained by binning 9 CCD elements (3 × 3) over 384 x 384 total CCD pixels, resulting in images containing 128 × 128 pixels. For comparison with the Raman images, the biological samples are viewed under a high-quality, brightfield-illuminated, transmission microscope (Olympus BH- 2) with the use of a 4 × plan achromat objective (N.A. 0.10) and a 2.5× projection eyepiece. The white light transmission image is detected with a video CCD camera (Javelin) and digitized with a frame grabber (Univision Scorpion 16G) at 8-bit dynamic range (256 gray levels).

Data collection is performed with an 80386-based computer with software written primarily in the C program- ming language. The software provided the ability to collect and to store data from the CCD as a series of images comprised of floating point numbers, in addition to con- trolling the wavelength setting of the AOTF. While the slow-scan CCD provides 16 bit integer numbers, the data are stored as floating point numbers in order to minimize round-off errors during subsequent mathematical ma- nipulations. For example, background correction by image ratioing, frame averaging, and truncation to 8 bits for image display are all potential sources of round-off errors that degrade image quality. These primary spectral image processing tasks are performed with software developed to manipulate floating point images before mapping the images to 8 bits for manipulation with a commercial software package. Final image contrast en- hancement and display is performed with a Windows 3.0 image-processing package (BioScan Optimas 3.0). For

APPLIED SPECTROSCOPY 1213

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2700 2800 2900 3000 3100 3200 3300 3400 3500

Wavenumber (cm 1)

FIG. 2. Raman spectra of DPPC (A) and L-asparagine (B) collected between 2700 and 3500 cm 1 at 5-cm -1 increments. The spectra represent a 3 × 3 pixel region of the CCD. Incident laser power is 1 W of defocused radiation, and the collection time is 10 s per image frame. These two spectra are selected from two different regions of the sample and are representative of the 16,384 total Raman spectra collected over the entire image field of view.

publication, images are photographed from the computer VGA monitor.

RESULTS AND DISCUSSION

Figure 2A and 2B show spectra of a mixture of DPPC and L-asparagine aggregates collected with the imaging spectrometer. Figure 2A is a spectrum taken from a region of the sample containing only lipid molecules; Fig. 2B reflects a sample region which contains only the amino acid. These spectra are obtained by tuning the AOTF between 2700 and 3500 cm -~ at 5-cm -1 increments. Im- ages having 128 × 128 pixels are collected at each spectral interval. The resulting three-dimensional data set contains 176 stacked images or, alternatively, 16,384 spatially resolved Raman spectra, each containing 176 data points. A full discussion of the principles governing acousto-optic multispectral image collection and han- dling has been described previously: 5 By extraction of spectral vectors of intensity from each image at a fixed image x y coordinate, the spectra in Figs. 2A and 2B are obtained. The integration time for each frame of 10 s, as well as the total spectral image data acquisition time, could have been reduced significantly by binning more CCD pixels, but at a cost to image fidelity and spatial resolution. Alternatively, instead of the use of a slow- scan CCD for collecting survey microspectra, a fast sin- gle-point detector could be employed for rapid collection of entire spectra in only several seconds, not the several minutes required in this study. While the CCD exposure time in the current experimental system is controlled mechanically, the shutter can be removed entirely and replaced by the intrinsic switching capabilities of the AOTF. In operation, the AOTF would be switched on to coincide with the beginning of the CCD integration, and turned off before the CCD is read out.

The image shown in Fig. 3A is a brightfield transmission image of the lipid/amino acid mixture. The two components of the aggregate mixture are both white in appearance and are virtually indistinguishable under a conventional light microscope. Figures 3B and 3C are

Raman images of the same material shown in Fig. 3A based upon the Raman-shifted frequencies of 2880 and 3390 cm -1, which are associated with the CH2 and NH2 stretching modes for the lipid and amino acid, respectively. The Raman images are corrected for fluorescent background and inhomogeneous sample illumination by dividing the images in the collected data set by an image at 2700 cm -1, a baseline region devoid of Raman emission. Total magnification provided by the optics used in this work is estimated at 13. The reference bar in image 3C indicates 0.1 mm, which corresponds to 8 um per image pixel. In Fig. 3B, the imaging spectrometer is tuned to 2880 cm -1, the CH2 asymmetric stretching mode of lipid acyl chains. The bright portion of the image represents predominantly the lipid component of the sample, while the faint region in the upper half of the image corresponds to amino acid Raman scattering. The spectrum of Fig. 2A indicates that, while the L-asparagine CH2 stretching mode region maximum occurs at 2940 cm -~, there is still significant Raman intensity at 2880 cm -~, accounting for the additional image intensity. De- spite the presence of amino acid Raman scatterers in the predominantly lipid CH2 stretching mode region, it is possible to discriminate between the two components. The distinction between the lipid and amino acid be- comes more apparent, however, in panel 3C, where the spectrometer has been tuned to the NH2 stretching mode feature at 3390 cm -1. Since the lipid component of the sample contains no NH2 functional groups, there is no intensity contribution from the lipid at this frequency; hence the lipid constituent is not imaged. The amino acid component is, however, visualized and appears as the brighter region in the upper part of the panel. The differences in the images 3B and 3C provide a clear dem- onstration of the chemical inhomogeneities in this par- ticular sample and provide additional information not available in the white light image in Fig. 3A.

Figure 4 shows two Raman spectra of polycrystalline DPPC. The spectrum in Fig. 4A is obtained with the imaging spectrometer after application of a Fourier deconvolution, while the spectrum in Fig. 4B is collected at 5 cm -1 resolution with the use of a scanning double monochromator system and a photomultiplier tube. Al- though the 50-cm -1 bandpass of the AOTF is relatively broad, tuning at 5-cm -~ increments and applying standard Fourier self-deconvolution techniques ~7 enhance additional detail obscured in the raw data set. The spectrum clearly shows features due to both the symmetric (2850 cm -1) and asymmetric (2880 cm -1) CH2 stretching modes of the lipid hydrocarbon chain, as well as the chain terminal (2936 cm -1) and headgroup (2968 cm -~) CH3 stretching modes. A visual comparison between the AOTF spectra and the data obtained from the double monochromator shows the similarity in the spectra.

In the simplest application, the spectral discrimination afforded by this technique relative to white light imaging provides enhanced image contrast between sample regions differing in molecular composition and distribution and exhibiting quite distinct Raman spectra. This is the man- ner in which the data collected for this paper have been presented. More ambitious approaches would employ the Raman imaging technique to extend many of the biomo- lecular problems for which traditional, nonimaging, Ra-


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2700 2750 2800 2850 2900 2950 3000 3050 3100

W a v e n u m b e r ( c m -~)

FIG. 4. CH stretching mode region (2700-3100 cm -1) Raman spectra of DPPC collected with the acousto-optie filtered imaging spectrometer (A) and a dispersive scanning monochromator at 5-cm-' resolution (B). Fourier self-deconvolution is applied to spectrum A. The two spectra show similar features due to methyl and methylene vibrations arising from both the hydrocarbon chain and headgroup portions of the lipid.

FIG. 3. Brightfield (A) and Raman images (B, C) of a co-precipitated mixture of DPPC and L-asparagine. The images in B and C represent those recorded at 2880 cm-' (asymmetric CH2 stretching mode) and 3390 cm -1 (Fermi resonance NH2 stretching component) Raman dis- placements, respectively. B indicates the position and appearance of the lipid portion of the sample, while C reveals only the L-asparagine

m a n spect roscopy has been utilized. For example , we an t ic ipa te using R a m a n spect roscopy imaging to deter- mine the domain character is t ics re levant to cellular plas- m a m e m b r a n e s and intracel lular organelles. R a m a n spec t roscopy provides an ex t remely sensit ive and noninvasive probe for examining molecular reorganizat ions in biological m e m b r a n e s by moni tor ing the s t ruc tura l and dynamic proper t ies of bo th the lipid and pro te in f ract ions in ei ther in tac t or model membranes . In ad- di t ion to dist inguishing be tween the d is t r ibut ion of the various componen t s within the m e m b r a n e assembly, the associated R a m a n spect ra l fea tures for a specific domain can be re la ted to v ibra t ional modes describing the con- format iona l behavior of specific molecular moiet ies of individual bi layer const i tuents . For example , individual spect ra l da ta reflecting the lipid por t ion of the m e m b r a n e m a y be assigned to ei ther the hydrophi l ic headgroup or hydrophobic acyl chain regions of the bilayer. In par t icular, exper imenta l ly observed spectra l intensit ies, frequencies, and l inewidths respond different ly to a bi layer reorganizat ion induced by changes in sample t e m p e r a - ture or by the in t roduct ion of various classes of m e m - b rane pe r tu rban t s , such as sterols, anesthet ics , proteins , or drugs.

F U T U R E D I R E C T I O N S

This s tudy demons t r a t e s t ha t acousto-opt ic fil tered R a m a n imaging spec t rome t ry has potent ia l as a tool for the s tudy of a wide range of chemical , biochemical , and biophysical problems. In this p re l iminary work, a s imple l ip id /pept ide mix tu re is employed as a model for fu ture deta i led studies of biological membranes . T h e sample discussed here measures app rox ima te ly 1 m m 2 and is recorded a t low magnif icat ion with the use of two s imple lenses, ye t relat ively h igh-qual i ty R a m a n images are ob- ta ined a t a spat ia l resolut ion of only several microns.

component. The size bar in C corresponds to 100 gm, indicating 8 urn/ pixel in the 128 x 128 pixel images.

APPLIED SPECTROSCOPY 1215

Studies are current ly being unde r t aken in this l abora tory to in tegrate a research-qual i ty refract ive microscope with a high-resolut ion AOTF, holographic beamspl i t te rs , and the CCD to collect d i f f ract ion- l imited R a m a n images and R a m a n microspect ra . Current ly , images having only 128 × 128 pixels are collected because of the large amoun t s of disk space required to store the th ree-d imens iona l floating poin t da ta sets collected with this method. By employing optical read/wri te drives as s torage media , we an t ic ipa te being able to collect full CCD fo rma t (576 × 384) images in the near future. Where high image fidelity is p a r a m o u n t , the acousto-opt ic fi l tered m e t h o d would be compat ib le with commerc ia l ly available detectors having 4K x 4K elements , and would provide spectra l image qual i ty compet i t ive with t h a t for high-resolut ion pho tography . While the sys tem descr ibed in this com- munica t ion is employed for R a m a n spectroscopy, the basic pr inciples could be used for any emission spec- t roscopy, including fluorescence imaging. T h e rapid tuning capabi l i ty of the A O T F combined with fas t imaging de tec t ion allows adequa te t iming resolut ion to record dynamic events on the ms t ime scale. Of course, the rapid imaging an t ic ipa ted in fluorescence microscopy should be a t ta inable , to some degree, in R a m a n imaging as well. Wi th the p lanned i n s t r u m e n t enhancemen t s we hope to image mate r ia l s as rap id ly as 1 f rame/s and collect Ra- m a n "v ibra t iona l movies" of dynamic events. In the future , we will refine our capabil i t ies in apply ing R a m a n imaging spec t roscopy to the s tudy of biological and m a n y other kinds of mater ia ls . In par t icular , we envision being

able to spectra l ly image organelles, whole cells, and in tac t t i s s u e - - s y s t e m s which a t p resen t are not easily handled with a l te rna t ive imaging methods .

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High-Fidelity Raman Imaging Spectrometry: A Rapid Method Using an Acousto-optic Tunable Filter