Inhaled particles smaller than 4 μm can cause damage to lung tissue, a disease called silicosis. We present an investigation on the use of a Raman microspectrometer for the identification of inclusions in lung tissue. We measured Raman spectra of such inclusions in lung tissue of a patient whose probable cause of death was silicosis. Most of the inclusions we could identify were calcite particles

Buiteveld, H.

Greve, J.

Mud, J.

Mul, F.F.M. de

de Mul, F.F.M.

Greve, Jan

University of Twente Research Information

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Identification of Inclusions in Lung Tissue with a Raman Microprobe H. B U I T E V E L D ,  F. F. M.  D E  M U L ,  J. M U D ,  and J. G R E V E  Twente University of Technology, Department of Applied Physics, P.O. Box 217, 7500 AE Enschede, The Netherlands Inhaled particles smaller than 4 um can cause damage to lung tissue, a disease called silicosis. We present an investigation on the use of a Raman microspectrometer for the identification of inclusions in lung tissue. We measured Raman spectra of such inclusions in lung tissue of a patient whose probable cause of death was silicosis. Most of the in- clusions we could identify were calcite particles. Index Headings: Raman; Microprobe; Silicosis; Lung tissue; Inclusions; Pathology. INTRODUCTION In recent years several techniques have been devel- oped for the identification of small amounts of material. Some of these techniques make use of microbeams (ion, electron, or proton); others use secondary electrons or x-ray scattering. Usually with these microprobes only the nature of the atoms in the sample is determined. 1 Recently a Raman microprobe was developed 2,3 which has the advantage over other microtechniques that chemical identification is feasible without causing dam- age to the sample or the necessity of staining the sample. Out of an increasing number of applications, we mention Received 3 February 1983; revision received 8 June 1983. the identification of airborne particles by Etz 4 and the analysis of silicone inclusions in lymph tissue by Abra- ham)  The results presented here were obtained from a study of lung tissue using a Raman microprobe. The lung tis- sue belonged to a patient who probably died from sili- cosis or pneumoconiosis. This is caused by small air- borne particles embedded in the lung tissue. Some of these particles are removed by macrophages, but others may stay in the lung and cause damage to the tissue. Materials that may cause the disease are silica, silicates, asbestos, or carbonates and related minerals. Identifi- cation of the inclusions in the lung tissue is of impor- tance because this might provide a possibility of deter- mining the source of the pollution which caused the disease. In this way one might prevent other exposures. EXPERIMENTAL Thin (5-15 ttm) paraffinized unstained sections of lung tissue were deposited on normal microscope slides. Be- fore measurement the sections were deparaffinized in xylol, which evaporated afterwards. The sections were analyzed by using polarized light microscopy. This was .304 Volume 38, Number 3, 1984 ooo8-7o2s/84/38o,-o3o452.oo/o APPLIED SPECTROSCOPY © 1984 Society for Applied Spectroscopy 1000 CPS 0 1000 : 1084 281 155 711 1085 281 711 500 1000 CM -I wavelength shift 1085 FIa. 1. A, Spectrum of an inclusion in lung tissue (3 #m). Laser power at  the sample 4 mW; diameter laser spot 1.6 #m; spectral slit width 3 cm-~; scan rate 50 cm-l/min; It,p (1085 cm -1) = 1500 cps. B, Spectrum of a reference calcite particle (10 #m), same conditions as in A; Itop = 3500 cps. done to locate the inclusions in the sample. Once a par- ticle was found, its Raman spectrum was recorded. The measurements were performed using a Raman microspectrometer similar in principle to the one de- scribed by Delhaye? We used a coherent Ar ÷ laser (CR 3) operating at 514.5 nm as a light source. The laser light passed through the epi-illuminator of a Nikon Op- tiphot microscope. A 100× objective (NA = 0.90) was used to focus the beam on the sample (spot diameter 1.6 #m). The same objective was used to collect the scat- tered light and direct it onto an entrance slit of a Jobin- Yvon monochromator (HG 2S). An EMI 9863QB pho- tomultiplier was used for detection. The apparatus was tested with polystyrene spheres of 1- and 10-#m diam- eter (Dow Chemical). A normal polystyrene spectrum was recorded for both types of particles. With other measurements using polystyrene spheres it was possible to estimate that  the effective depth in the particle from which the light was collected lies between 1 and 2 #m. The recording of the Raman spectrum of each sepa- rate particle in the lung tissue was repeated several times to be sure that no sample deformation took place in the laser spot. No effects of heating or deformation could be detected at the power densities used (_-<0.5 x 109 W. m-2). The tissue itself was more sensitive to heating caused by the laser beam. Intensities exceeding 0.5 × 109 W.m -2 caused visible damage to the tissue. A Raman spectrum of the tissue was not recorded because of the high fluorescent background. 212 330 730 I I 500 1000 CM -1 wavelength shift FiG. 2. Spectrum of an inclusion in lung tissue (4 ~m), probably mag- nesite; laser power at  the sample 5 mW; spectral slit width 5 cm-l; scan rate 20 cm-1/min; I,,,p (1084) = 900 cps. RESULTS AND DISCUSSION Because with stained sections the Raman spectrum has a high background caused by fluorescence of the staining dyes, we used unstained specimens. However, with unstained sections the localization of inclusions by birefringence, using polarized light microscopy, can be very difficult. In Fig. 1A the Raman spectrum of one of the types of inclusions in the lung tissue sample under investigation is shown. Raman lines are present at frequency shifts of 155, 281, 711, and 1085 cm 1. This spectrum is in very good agreement with a reference spectrum of calcite 212 A 329 281 155 CaC03 l l i00 200 300 CM -I wavelength shift FIG. 3. Low-frequency spectra of reference MgCO3 and CaCO3 taken from literatureY '8 Other lines of MgCO3 and CaCO~ are at 739 (w) and 711 (m), 1084 (vs), and 1085 (vs), respectively. 7,8 APPLIED SPECTROSCOPY 305 (CaCO3), which was measured with a 10-#m particle of pure calcite (Fig. 1B), and with spectra of calcite re- ported in the literature, s-s The lines at 155, 281,711, and 1085 cm -1 could be assigned to e~, (ex), eg (ex), e~ (int) and alg (int), respectively. 7 A comparison of the intensity ratios of the lines in the inclusion spectrum with those in the reference spectrum indicates that the crystal structure of the small crystallites present in the tissue is the same as in bulk calcite. Most particles that could be located in the tissue were identified as CaCO~. They varied in size between 2 and 4 gm. Another type of inclusion showed Raman lines at 212, 330, 730, and 1084 cm-' (Fig. 2). This spectrum could be identified as being caused by MgCO3 (magnesite), on the basis of the position (see also Fig. 3) and the relative intensities of the lines, as compared with those of other carbonates. ~-s The Raman lines observed in MgCQ at 212, 330, 730, and 1084 cm -~ can be assigned analogously to those of calcite. In both spectra (Figs. 1 and 2) a line centered around 840 cm -1 is present. This line does not belong to the spectra of calcite or magnesite, but  is due to scattering in the microscope objective. According to medical diagnosis, the cause of death could be silicosis due to inhaled quartz particles. We were able to identify calcite, magnesite, and other ma- terial in the present sections; we did not, however, find any quartz particles in these specimens. Later on this was confirmed by x-ray measurement of the tissue. 9 However, in sections of lymph-node tissue from another patient with the same diagnosis, SiO2 inclusions were found. Obviously this tissue contained assembled in- haled particles removed from the lung by macrophages. This will be reported in detail in a following paper. 1° CONCLUSIONS It was shown that by using Raman microspectrometry the character of inhaled particles (diameter 1-4 am) embedded in lung tissue can be revealed. Some prob- lems arise if the sections are stained, because of the flu- orescent background. In unstained sections the localiza- tion of inclusions may give rise to difficulties. Further investigations are in progress. ACKNOWLEDGMENTS This study was supported in part by the S.T.W. (Stichting voor Technische Wetenschappen) and the Pieter Langenhuizen Lamber- tuszoonfonds. The sections were kindly supplied by Dr. J. van der Stadt. Thanks are due to Dr. J. Lankester for helpful discussions. 1. K. F. J. Heinrich, in Proc. Scanning Electron Microscopy Symp., O. Johari, Ed. (Chicago, Illinois, 1977), p. 605. 2. M. Delhaye and P. Dhamelincourt, J. Raman Spect. 3, 33 (1975). 3. G. J. Rosasco and E. S. Etz, Res./Dev. 28, 20 (1977). 4. E. S. Etz, G. J. Rosasco, and W. C. Cunningham, in Environmental Analysis, G. W. Ewing, Ed. (Academic Press, New York, 1977), p. 295. 5. J. L. Abraham and E. S. Etz, Science 206, 716 (1979). 6. W. P. Griffith, in Infrared and Raman Spectroscopy o/Lunar and Terrestral Minerals, C. Karr, Jr., Ed. (Academic Press, New York, 1975), Chap. 12, p. 299. 7. H. N. Rutt and J. H. Nicola, J. Phys. C 7, 4522 (1974). 8. D. Krishnamurti, Proc. Indian Acad. Sci. Sect. A 53, 210 (1956). 9. J. Lankester, personal communication. 10. F. F. M. de Mul, H. Buiteveld, J. Lankester, J. Mud, and J. Greve, "Application of Raman Microscopy in Human Pathology," sub- mitted to Journal of Human Pathology. Raman Spectroscopic Determination of the Integrity of Urea Inclusion Compounds* HECTOR L. CASAL Division of Chemistry, National Research Council of Canada, Ottawa, KIA OR6 Canada Raman spectroscopy is shown to provide a rapid and definitive analysis of the nature of urea inclusion compounds. It is demonstrated that the photodecomposition of included alkanones does not destroy the urea lattice, but the inclusion compounds of n-decane are destroyed by ultra- sonic agitation. Index Headings: Raman spectra; Urea inclusion compounds. Urea forms inclusion compounds with paraffin-like molecules 1 (n-alkanes, fatty acids, etc.). This property has been used successfully to separate linear and branched compounds. 1,2 While pure urea forms tetrag- onal crystals, in the inclusion compounds it crystallizes in an hexagonal lattice forming channels of ~0.5 nm Received 27 June 1983. * Issued as NRCC Publication No. 23084. diameter2 These channels accommodate linear chain molecules permitting free chain rotation to occur. Recently, urea inclusion compounds have been shown to provide a unique environment for photochemical re- actions. The photodecomposition of included n-alka- nones was found to occur with greatly differing conse- quences than is the case for n-alkanones in solution. 4 During the course of these photochemical studies and other investigations on the motions of included mole- cules) it became necessary to determine unequivocally if inclusion of the molecules of interest had indeed oc- curred and if the crystalline lattice had been destroyed during the photochemical reaction. The crystal structures of free urea (tetragonal) and of the inclusion compounds (hexagonal) present hydrogen bonds of different lengths2 As a result, the vibrations (external and internal) of the urea molecules are differ- 308 Volume 38, Number 3, 1984 0003-7028/84/3803-030652.00/9 APPLIED SPECTROSCOPY © 1984 Society for Applied Spectroscopy 

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Identification of inclusions in lung tissue with a Raman microprobe

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Identification of inclusions in lung tissue with a Raman microprobe

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