nance with the coil. For the 40.68 MHz unit, the initial rf power was set near 1 kW and the vacuum-capacitor tuning knob set close to the resonance point. Plasma ignition was then attempted as the air-capacitor value was slowly varied. When resonance was close, as evi- denced by the presence of a filamentary discharge in the ICP torch, the vacuum-capacitor value was changed slightly to improve the coupling. After the first ignition has been achieved, the plasma can be re-ignited in a few seconds. A similar procedure was used with the 27.12 MHz unit.

5. A.L. Gray, R. S. Houk, and J. G. Williams, J. Anal. Atomic Spectrom. 2, 13 (1987).

6. J. M. Mermet, 1988 Pit tsburgh Conference, New Orleans (1988), Paper 004.

7. D. J. Douglas, personal communication (1988).

A U T O T U N I N G IN PHASE M A T C H I N G

Usually, the modifications described above will cause a phase-matching problem in the autotuning network. This problem arises because the original feedback net- work receives a signal of the improper phase or polarity from the modified unit. As a result, the autotuning sys- tem fails to function normally. Physically, the phase dif- ference could be as much as 180 ° with the modified cir- cuit.

A straightforward way to correct the signal phase is implemented by adding a delay cable. This delay cable (labeled D in Figs. 1 and 2) can be added between the phase-monitor unit (labeled P) and the tuning capaci- tors. The length of the cable should be between a quarter and a half wavelength of the rf source, corresponding to 90-180 ° of the phase delay.

Correcting for this autotuning phase error was possible in the 27.12 MHz unit also by switching the connections to the load coil. The upper end of the load coil was connected to the capacitor C2 in the original design but was switched to the capacitor C1 in the modified unit. With this small change, the autotuning mechanism for capacitor C1 was found to function properly, and the plasma was ignited with this capacitor autotuned. This improvement made the ignition procedure as simple as that experienced with the original system.

Unfortunately, this method could not be applied to the 40.68 MHz unit. For that system, a change in the polarity of the feedback signal was found to be necessary. The schematic diagram for a circuit to accomplish this task is illustrated in Fig. 3. By passing the dc phase signal (V) through a voltage inverter (T, laboratory construct- ed), we inverted its polarity, to produce a feedback signal that was virtually identical to that obtained from delay- ing the phase. It was concluded that both methods pro- vide good control over the phase-matching network. However, reversing the phase-signal polarity is recom- mended because of its simplicity.

ACKNOWLEDGMENTS

The authors wish to thank D. Douglas for his valuable comments during the course of this work. This research was supported in part by the National Science Foundation through Grant CHE 87-22639, by American Cyanamid, and by Leco Corp.

Infrared Microspectroscopy of Human Tissue*

T I M O T H Y J . O ' L E A R Y , t W A L T E R F. E N G L E R , and K A T H L E E N M. V E N T R E Department of Cellular Pathology, Armed Forces In- stitute of Pathology, Washington, D.C. 20306-6000

Index Headings: Infrared; Analytical methods; Reflectance spectros- copy; Spectroscopic techniques.

INTRODUCTION

Infrared spectroscopy is a convenient tool for deter- mining the molecular conformations of proteins, lipids, and nucleic acids, 1 as well as for identifying unknown chemical compounds. 2 Previously, we have shown that infrared spectroscopy may be used to characterize the conformation of an abnormal protein found in pathologic specimens of medullary carcinoma of the thyroid2 The technique described in that paper is of limited general utility, however, because it requires that the abnormal protein deposit constitute the vast majority of the tissue specimen examined by infrared spectroscopy. In addi- tion, it requires that the specimen be mounted on a rel- atively expensive CaF2 crystal. Since it is customary in diagnostic pathology to retain such mounted specimens in perpetuity, a new crystal would be required for each specimen, resulting in relatively high sample preparation costs. The recent availability of high-optical-quality in- frared microscopes eliminates the requirement that the abnormal material of interest constitute most of the spec- imen to be examined. 4 In this note we describe a method for preparing samples which drastically reduces the cost of preparing a permanent specimen while, at the same time, making it somewhat easier to acquire optimal spec- tra.

1. D. J. Douglas and J. B. French, Spectrochim. Acta 41B, 197 (1986). 2. D. J. Douglas, Can. J. Spectrosc., "Some Current Perspectives on

ICP-MS," in press, 1988. 3. R. S. Houk, V. A. Fassel, G. D. Flesch, H. J. Svec, A. L. Gray, and

C. E. Taylor, Anal. Chem. 52, 2283 (1980). 4. A. L. Gray, J. Anal. Atomic Spectrom. 1, 247 (1986).

Received 28 December 1988. * This work was supported in part by a grant from the American Reg-

istry of Pathology. The opinions expressed herein are the private views of the authors and are not to be construed as official or as representing the views of the Department of the Army or the De- par tment of Defense.

t Author to whom correspond should be sent.

Volume 43, Number 6, 1989 0003-7028/89/4306-109552.00/0 APPLIED SPECTROSCOPY 1095 © 1989 Society for Applied Spectroscopy

0.800-

(9 0.60G z

<

0.4011 L 1500 16~50 1800

FREQUENCY(cm" 1 ) FIG. 2. Infrared spectrum of a 67-#m-diameter region of the specimen shown in Fig. 1A, showing the amide I and amide II spectral regions. Spectrum was obtained at 4 cm -1 resolution, with 1600 coadditions.

D I S C U S S I O N

Tissue is fixed in 10% pH 7.0 phosphate buffered for- malin, then dehydra ted by being passed through a series of graded alcohol/water mixtures (80, 95, and 100 % eth- anol, by volume). The tissue is then placed in xylene and infi l t rated with a low-melting paraffin (Paraplast) . This is a s tandard histologic procedure which may be carried out by most histology laboratories. The paraff in-embed- ded tissue is cut with a ro tary microtome in 3-5-gin- thick sections, and floated from a 40°C water ba th onto the specially prepared glass microscope slides which are described below. The sections are allowed to dry over- night, then are deparaffinized in xylene, r ehydra ted by being passed through graded alcohols (100, 95, 80, and 50 % ) and distilled water, and then stained, typical ly with the use of hematoxyl in and eosin. Finally, they are once again dehydra ted by being passed through the graded alcohols and xylene. These procedures are described in many histology manuals. ~,6

One prepares reflective microscope slides by first acid cleaning the 1 × 3 in. glass slides. The slides are then baked at 50°C for one hour. Following this, the slides are covered with a reflective coating of gold or a 60/40 (mole/ mole) gold/pal ladium alloy. The metal is pu t on the slide by sput ter ing a gold or gold/pal ladium anode in a flushed argon a tmosphere (0.1 Torr) in an E M S C O P E SC500 coater (Emscope Laboratories , Ltd. , Kent , England) run- ning with a 10-mA current . Metal is sput te red onto the slide unti l i t is nearly total ly reflective, with a film thick-

FIG. 1A. Optical appearance of a medullary carcinoma of the thyroid, prepared and mounted on a gold plated slide as described in text and viewed through 10x refracting lens (original magnification 200x). Preparation method results in images that are somewhat "fuzzier" than those usually observed with transmission light microscopy. FIG. lB. Specimen from Fig. 1A viewed through 15 x Cassegrain ob- jective (original magnification 300 × ). Images with Cassegrain lenses are even "fuzzier" than those with the refracting lens. FIG. 1C. Specimen from Fig. 1A viewed through diaphragm and 15 x Cassegrain objective allowing 67-#m region for viewing and spectrum acquisition (original magnification 300 x ).

1096 Volume 43, Number 6, 1989

ness of approximately 400 nm. This sputtering procedure is very similar to that used to coat tissue for scanning electron microscopy, and it uses the same equipment. The gold or gold/palladium-coated slides are again placed in a 50 mg/mL solution of high-molecular-weight poly- L-lysine for 30 min to one hour, then baked at 50°C for one hour. The poly-L-lysine coating helps to ensure that the relatively fragile gold or alloy layer remains on the microscope slide and provides a layer to which tissue is strongly adherent. The layer of poly-L-lysine deposited on the metallic surface of the coated microscope slide is essentially monomolecular, and does not contribute an observable spectrum under the conditions described here. The total cost for preparing a microscope slide in this way is trivial (much less than one dollar per slide) if a sputter coater is available.

Both optical examination and infrared microspectros- copy have been carried out on various tissues with an IR-Plan microscope (Spectra Tech) interfaced to a Bo- mem DA3.2 spectrometer (Bomem Inc., Quebec, Cana- da) equipped with a 0.25-mm narrow-band MCT detec- tor. Prior to optical examination or spectroscopy, the tissue should be covered with an index matching liquid, such as Fluorolube or Nujol (both available from Aldrich Chemical Company), which is infrared-transparent in the region of interest. The histologic appearance of the tissue viewed in reflectance mode (Fig. 1) is similar to its appearance when mounted on an ordinary glass mi- croscope slide, covered with a glass coverslip and viewed in transmission mode, except that the image is "fuzzier," especially when one is using the Cassegrain lenses (Fig. 1B and 1C). Reference spectra can be obtained with the use of a portion of the microscope slide which is not covered by tissue. Very high-quality infrared spectra of small tissue regions are easily obtained, as illustrated by Fig. 2, which shows the amide I and amide II features of a highly cellular region--a 67-#m-diameter region of a medullary carcinoma of the thyroid in which little or no abnormal protein deposition is apparent by visual ex- amination. Because spectra are obtained on very thin sections in the reflectance mode, optimal focus condi- tions for the infrared and optical regions are identical, and sample thickness compensation has not been re- quired. This contrasts strongly with the situation that pertains in the transmission mode, with the use of CaF2 or BaF2 plates, where great attention to sample thickness compensation is required in order to obtain high-quality spectra.

In summary, a method for coating glass microscope slides, preparing and mounting histologic sections, and obtaining infrared spectra from them is described. The method is simple and inexpensive, and it gives prepa- rations which yield high-quality infrared spectra on mi- croscopic samples.

1. H. Susi, "Infrared Spectra of Biological Macromolecules and Related Systems," in Structure and Stability of Biological Macromolecules, S. M. Timashjef and G. Fasman, Eds. (Marcel Dekker, New York, 1969), Part II, p. 576.

2. D. J. Pasto and C. R. Johnson, Organic Structure Determination (Prentice-Hall, Englewood Cliffs, New Jersey, 1969).

3. T. J. O'Leary and I. W. Levin, Lab. Invest. 53, 240 (1985). 4. H. Ishida, R. Kamoto, S. Uchida, A. Ishitani, Y. Ozaki, K. Irimaya,

E. Tsukie, K. Shibata, F. Isihara, and H. Kameda, Appl. Spectrosc. 41, 407 (1987).

5. M. C. Bowling, Histopathology Laboratory Procedures, Public Health Service Publication 1595 (Government Printing Office, Washington, D.C., 1978).

6. B. J. Coolidge and R. M. Howard, Animal Histology Procedures, NIH Publication 80-275 (Government Printing Office, Washington, D.C., 1979).

Near-Infrared Surface-Enhanced Raman Spectra of 3-Picoline and 3-Chloropyridine on a Copper Electrode

S. M. A N G E L * and D. D. A R C H I B A L D Environmental Sciences Division, Lawrence Liver- more National Laboratory, Livermore, California 94550 (S.M.A.); and Department o[ Chemistry BG- 10, University o[ Washington, Seattle, Washington 98195 (D.D.A.)

Index Headings: SERS; FT-IR; FT-Raman; Raman; NIR Raman; Cop- per electrodes.

INTRODUCTION

SERS is well suited to detecting and fingerprinting environmental contaminants such as polychlorinated bi- phenyls. For this reason, we have been investigating SERS for identifying and quantifying the individual compo- nents of mixtures of these and related compounds. Ini- tially, very simple monosubstituted aromatic compounds are being studied. One of the simplest of these is 3-chloropyridine (CP). This compound has been found to give very intense NIR-SER spectra on copper colloids and on copper electrodes; these results have been pre- viously reported. 1 In this paper we report the NIR-SER spectrum of 3-picoline on a copper electrode as well as the NIR-SER spectra of a mixture of 3-picoline and 3-chloropyridine on a copper electrode at several differ- ent potentials. Electrode potential effects on SER spec- tra have been mentioned in many reports, 2-6 but there have been very few studies that have dealt specifically with these effects, and fewer still that have dealt with the SER spectra of mixtures. 7,s Most of the qualitative features of SERS are explained by two models, an elec- tromagnetic model and a charge-transfer model. 9 Vari-

Received 27 January 1989. * Author to whom correspondence should be sent.

Volume 43, Number 6, 1989 0003-7028/89/4306-109752.00/0 APPLIED SPECTROSCOPY 109"I © 1989 Society for Applied Spectroscopy