Volume 51, Number 12, 1997 APPLIED SPECTROSCOPY 18450003-7028 / 97 / 5112-1845$2.00 / 0q 1997 Society for Applied Spectroscopy

Rapid Micro-Raman Imaging Using Fiber-Bundle ImageCompression

JIAYING MA and DOR BEN-AMOTZ*Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393

A new technique for rapid Raman imaging and chemical analysisof micro-composites and biomaterials, with potential applications inreal-time robotic vision, automated manufacturing, and medical im-aging, is described and demonstrated. The key feature of this newinstrument is a ® ber-optic bundle used to compress two-dimensionalimages onto a one-dimensional ® ber stack, which serves as the en-trance slit of an imaging optical spectrograph. Thus a completeRaman spectrum is simultaneously collected from every point with-in a sample in a single scan of a charge-coupled -device (CCD) de-tector. The method is demonstrated by using Raman imaging of amicroscopic mixed-salt sample. Its ef® ciency relative to alternativeRaman imaging methods is quantitatively evaluated, and potentialapplications in other spectral imaging measuremen ts are discussed.

Index Headings: Raman spectroscopy; Spectral imaging; Chemicalimaging; Fiber optics; Chemical sensor.

INTRODUCTION

Conventional chemical analysis requires macroscopicquantities of material as well as suf® cient time for off-site laboratory analyses. This traditional approach doesnot rise to the challenge of emerging materials science,manufacturing, and biomedical applications, which re-quire real-time microchemical analysis of composites andbiomaterials. In this work a new technique based on mi-crospectroscopic optical imaging is proposed and dem-onstrated as a tool for use in such advanced chemicalanalysis applications. This new method approaches theoptimal theoretically attainable ef® ciency and speed,opening up the possibility of performing real-time Ra-man, ¯ uorescence, and/or optical absorption imaging ona microscopic scale.

This technique builds on recent advances in laser lightsources, optical ® lters, and array detectors that have givenrise to a new breed of chemical imaging instruments.These instruments may be viewed as hybrids of micro-scopes and optical spectrometers [Raman, infrared (IR),or ultraviolet-visible (UV-vis)] that produce a three-di-mensional (3D) data cube (x-y-l ) containing an opticalspectrum ( l ) at each spatial location (x-y) in the mate-rial.1 Raman chemical imaging is particularly promisingin this regard as it combines high spatial resolution (de-termined by the excitation laser wavelength) and the highchemical information content (in the form of molecularvibrational ® ngerprints). Here we present a new approachto Raman imaging in which an entire 3D data cube iscollected simultaneously without the need to scanthrough either the spectral or spatial dimensions.

Several scanning methods have previously been usedto generate Raman images, including line illumination

Received 13 February 1997; accepted 6 June 1997.* Author to whom correspondence should be sent.

with mechanical sample scanning and global illuminationwith tunable optical ® lter scanning. These approacheshave been compared in detail in several recent studies.1± 3

The global illumination method bene® ts from the powerdistribution advantage,2 and thus reduced sample heating,relative to line or point illumination. Recently developedglobal illumination imaging instruments employ fastacousto-optic tunable ® lters (AOTFs) with 50-cm2 1 res-olution4± 8 or, better yet, liquid crystal tunable ® lters(LCTFs) with 7-cm2 1 resolution, as demonstrated byMorris, Treado, and co-workers.9,10 These provide highimage and spectral ® delity, although only moderate depthresolution. On the other hand, point illumination offersthe higher depth resolution of confocal imaging, at theexpense of very slow data collection rates.11

All the above scanning methods are too slow to allowthe collection of a complete Raman spectral image inanything approaching real time. Our new technique, onthe other hand, makes it possible to collect Raman imagesin a single charge-coupled-device (CCD) scan with a100-s integration time (which could be improved to 1 sby using a higher power excitation laser operating at lev-els near the optical damage limits of the sample).

The key feature of this instrument is its ability to si-multaneously measure an array of optical spectra from agrid of points in a microscopically heterogeneous mate-rial. In this sense it is related to other multi-point probinginstruments, such as those developed by Vess and An-gel,12 in which an array of optical ® bers delivers differentRaman signals to a single CCD detector used to simul-taneously measure multiple spectra. Similarly, our newinstrument design utilizes a ® ber-optic bundle to com-press two dimensions of spatial information into a one-dimensional (1D) linear array, which serves as the en-trance slit of the spectrograph used to simultaneously dis-perse and detect multiple Raman spectra. In particular, anoptical image of the light emanating from the sample iscollected by using a bundle of close-packed ® bers ar-ranged in a circular array at the collection end and alinear stack at the detection end. The net result of this® ber-bundle image compression (FIC) strategy is that allthree axes of the 3D data cube (or cylinder) are simul-taneously measured in a single scan of a 2D array detec-tor (CCD) mounted to the exit port of the spectrograph.

A signal-to-noise (S/N) analysis is used to quantitatethe advantages of FIC strategy relative to previous mi-crospectrograph designs. The actual performance of anFIC instrument is demonstrated by measuring the Ramanimage of a microscopic mixed-salt sample. The capabil-ities and limitations of this new technology are discussed,along with its potential implementation in other spectro-

1846 Volume 51, Number 12, 1997

FIG. 1. A white-light image of the arrangement of ® bers in the collec-tion end of the 61-® ber bundle; numbers indicate the arrangement ofthe ® bers in the linear detection end of the bundle.

FIG. 2. The Raman scattering signal measured by the CCD detector isshown as a map of the detector output (left) and individual spectraobtained from different ® bers (right). Note the two distinct spectra ob-served at different locations. The KNO3 spectrum has a prominent peakat 1055 cm2 1, while the most intense peak of K2SO4 occurs at 975 cm2 1.Numbers on the left and right vertical axes indicate the pixel positionand ® ber number at each vertical location on the CCD. The horizontalaxis on the left represents the CCD pixel position and that on the rightthe corresponding Raman frequency shift.

scopic imaging applications [such as UV, vis, near-IR(NIR), IR absorption, ¯ uorescence, or re¯ ectance].

EXPERIMENTAL

In order to demonstrate the new FIC Raman imagingtechnique, we have used a ® ber bundle containing 61silica ® bers (with 100-m m core and 130-m m core pluscladding diameter). Figure 1 shows a white-light imageof the arrangement of these ® bers at the collection endof the bundle. The numbers on each ® ber correspond tothe position of the ® bers in the linear stack (detection)end.

The sample used to test the FIC instrument prototypeconsists of two salt fragments (KNO2 and K2SO4, eachabout 50± 100 m m in size) placed on a piece of sapphire(single-crystal c-axis cut) positioned under a microscope(Olympus BH-2 with 20- 3 11-mm-long working dis-tance objective). A holographic beamsplitter is used todirect a 40-mW helium± neon Raman excitation laser to-wards the sample. This portion of the instrument is sim-ilar to that used in previous micro-Raman studies,13 ex-cept that the sample is positioned about 1 mm below themicroscope focal plane, in order to produce a reasonablyuniform irradiation of an area of 100-m m diameter. TheRaman-scattered signal emerging from the microscope isimaged onto the collection end of the ® ber bundle byusing a 50-mm-focal-length camera lens (with an overallmagni® cation of about 53 ). Thus the Raman-scatteredphotons emanating from different regions of the sampleare directed into different ® bers (with each ® ber repre-senting an area of about 20-m m diameter in the sample).

The detection (linear stack) end of the ® ber bundle isbutted up against the entrance slit of a custom-built lens-coupled imaging spectrograph (with a focal length of250, 500-nm blazed 600-grooves/mm grating, 0.1-AÊ

wavelength resolution, and 10-m m spatial resolution). Ra-man scattering was recorded by a CCD detector (aPrinceton Instruments Corp. LN/1024 EUV CCD withST-135 controller) and processed by KestrelSpec soft-ware (Rhea Corp.).

RESULTS AND DISCUSSION

Figure 2 shows an image of the resulting CCD signalobtained after 100 s of integration. The spectra shown onthe left are obtained from selected individual vertical pix-els containing signi® cant Raman signal. Notice that twoclearly distinct spectra are obtained from the two differ-ent salts fragments. Note also that each ® ber is about fourpixels wide, and so about two times higher signal-to-noise ratio could be obtained by integrating the signalfrom all four pixels spanning each ® ber (rather than justone pixel).

Another way of viewing the above data is representedin Fig. 3, which contains shaded regions indicating the® bers from which a Raman spectrum of each of the twosalts was obtained. These results clearly demonstrate thatboth the chemical identity and location of each micro-scopic region in the sample may be obtained from thesimultaneously collected microspectroscopic imagingdata.

The most signi® cant feature of the FIC instrument de-sign is that it allows the simultaneous measurement ofoptical spectra at an array of image resolution elements.This feature represents a signi® cant advance in ef® ciencyrelative to previous imaging techniques [which require

APPLIED SPECTROSCOPY 1847

FIG. 3. Shaded regions indicate the ® bers containing signi® cant Ramanscattering signal from KNO3 (\\\) and K2SO4 (///). Note that the bundlehas four broken ® bers (numbers 3, 6, 27, and 49).

scanning either l , or x-y, or Hadamard transform (HT)masks]. Thus an FIC instrument can be used to performmeasurements of small changes in spectral peak position,width, and/or depolarization with no loss of signal col-lection ef® ciency and speed. Such detailed spectroscopicmeasurements are required, for example, in order to de-tect microscopic stresses, fractures, contaminants, orchemical composition changes in semiconductors andother microcomposites,14 which would be dif® cult, if notimpossible, to perform with other spectroscopic imagingtechniques.

In addition, tunable ® lter scanning methods typicallyrequire systematic prescreening of the material of interestin order to select an optimal set of detection wavelengths.The FIC technique, on the other hand, does not requiresuch prescreening, since an entire spectrum is obtainedin every measurement. Thus the FIC technique, in addi-tion to being more ef® cient, may also be used in theanalysis of unknown samples and the detection of un-expected spectral changes that might be missed by dis-crete wavelength imaging.

In analyzing the relative ef® ciency of various imaginginstruments, only designs utilizing sheet illumination areconsidered, as point and line illumination strategies (alsotermed `̀ image reconstruction’ ’ ) are clearly less ef® cientbecause they do not bene® t from the power distributionadvantage.2 The two alternative sheet illumination strat-egies employed in previous designs are (1) tunable ® lterimaging (TFI), in which images are collected at a seriesof discreet wavelengths, and (2) Hadamard transform im-aging,15 in which a sequence of masked images is com-bined onto one CCD axis and wavelength is dispersed onthe other axis. Note that, unlike the new FIC strategy,both the TFI and HT designs require scanning of eitherthe wavelength or spatial masks in order to generate the3D data cube.

The signal-to-noise ratios of the TFI and HT methodhave been previously compared in detail by Puppels etal.2 The results of that analysis suggest that, under mostconditions of practical interest, the TFI method is supe-rior to the HT approach [except perhaps for applicationsin which a relatively small number of the total array de-tector pixels contain useful spectral information, the sig-

nal-to-background (S/B) ratio is very large, or the num-ber of independent spectral bands of interest is verylarge]. Since the HT and FIC methods have been previ-ously compared, we restrict the following discussion toa quantitative comparison of TFI and our new FIC im-aging strategy.

The analysis assumes that, for both the TFI and FICmeasurements, a total time of N seconds is available fordata collection, and that the Raman spectrum has a back-ground of B counts per second, a signal intensity of Scounts per second (above background), and a read noiseof R counts per pixel (or resolution element). Further-more, the spectrum is assumed to be limited by countingnoise (rather than ® xed-pattern or other non-Poissonsources of noise). Under these conditions, the root-mean-square noise in the spectra obtained by using the FICdesign may be expressed as s 5 Ï SN 1 2BN 1 2R2.Furthermore, assuming that the spectra are not read-noiselimited (which is again a reasonable assumption) then 2R2

! SN 1 2BN, and the predicted S/N ratios for the FICand TFI designs become

SN NS/N 5 5 S andFIC ! S 1 2BÏ NÏ S 1 2B

N/MS/N 5 STFI ! S 1 2B

where M is the number of independent wavelengths atwhich Raman imaging spectra must be taken in order tomap the chemical constituents of interest (and thus N/Mis the integration time available at each wavelength).Clearly the FIC design has a factor of Ï M advantage inS/N ratios relative to the TFI design. Stated another way,a TFI instrument would have to collect data for M timeslonger than an FIC instrument in order to obtain the sameS/N ratio. Note that in practice M may be as small as 2if only one spectral (Raman) peak and one backgroundregion are required, although in general 10 , M , 100is more realistic, and M ø 1000 would be needed in orderto be fully equivalent to the FIC instrument in which acomplete Raman spectrum (with 3-cm2 1 resolution) iscollected from each x-y image resolution element.

The limiting collection speed of an FIC instrumentmay be estimated by assuming an optical damage thresh-old for a typical sample of 0.5± 5 mW/m m2 (althoughsome materials may have signi® cantly higher damagethresholds in the near-IR). Thus an excitation laser inten-sity of 0.1± 1 mW/m m2 at the sample is assumed. If inaddition an imaging resolution element of 1 m m2 is de-sired and a collection time of 10 s/mW is required inorder to collect a Raman spectrum of 10:1 signal-to-noiseratio (which is reasonable for a 10-m m-thick non¯ uores-cent Raman scattering sample), then it should take be-tween 10 and 100 s of the integration time to collect acomplete 3D data cube. Note that speeds of 1 s, or evenfaster, may be obtainable if only 10-m m spatial resolutionis required or if one uses materials with higher damagethresholds (and higher excitation laser power is avail-able). Furthermore, imaging applications with higher in-trinsic signal intensity, such as ¯ uorescence, color iden-ti® cation, or UV/vis/NIR absorption, could be performedon subsecond or even millisecond time scales.

1848 Volume 51, Number 12, 1997

Our preliminary Raman imaging measurements of amixed-salt sample demonstrate the feasibility of the FICtechnique but are far from representing the best possibleperformance obtainable with an FIC instrument. Thislimiting performance is determined by the optical param-eters and dimensions of the microscope, ® ber-bundle,spectrograph, and CCD detector.

The ® ber bundle and spectrograph used in this prelim-inary study have optical transmissions of about 40% and50%, for a total optical throughput of about 20% (whichis comparable with the 17% transmission of multi-stageliquid crystal tunable ® lter used in recent TFI Ramanstudies).10 The ® ber-bundle and spectrograph transmis-sion could theoretically exceed 70% each, for a totalthroughput of nearly 50%. It is furthermore not incon-ceivable that even higher throughput could be obtainedby rastering the excitation laser through discreet pointsin the sample, which are optimally coupled to individualcollection ® bers.

The spatial resolution of the FIC method is determinedby the size of each optical ® ber and the image magni® -cation. It is thus quite feasible to obtain diffraction-lim-ited resolution ( , 1 m m) by using ® bers of 50-m m to 200-m m core diameter and image magni® cation of 1003 to4003 . Of course, lower resolution images may also bereadily obtained (as demonstrated above) with a corre-spondingly larger total image area.

The maximum number of spatial resolution elementsthat may be simultaneously resolved is limited by thediameter of each ® ber and the size (height) of the CCDdetector. Assuming a spectrograph magni® cation of m, aCCD height of n pixels, each of height a, and ® bers ofdiameter d, the total number of resolution elements is(na)/(md). Thus 500 image resolution elements may beobtained by using a ® ber bundle with 50-m m-diameter® bers, a spectrograph of unit magni® cation, and a CCDof 25-mm (1-in.) height and 25-m m pixel size. The the-oretical maximum number of image resolution elementsmay be as high as 2000 when a CCD of 50-mm heightand ® bers of 25-m m diameter (core plus cladding) areused, although constructing such a system would requireexceptionally precise imaging and ® ber-bundle speci® -cations.

CONCLUSION

In summary, the feasibility of performing simultaneousspectroscopic imaging by using ® ber-bundle image com-pression has been demonstrated. Theoretical 3D datacube collection speeds obtainable with this technique canbe as fast as 1 s for Raman imaging or as fast as 1 msfor ¯ uorescence and UV-vis color or absorption appli-cations. The imaging resolution may be diffraction-lim-ited with hundreds to thousands of simultaneously im-aged resolution elements. Thus the FIC technique repre-sents a signi® cant advance in spectroscopic imaging ef-® ciency and performance, opening up new horizons inreal-time microchemical image analysis.

ACKNOWLEDGMENTS

Support for this work from the Of® ce of Naval Research and Hewlett-Packard is gratefully acknowledged.

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