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(8) Koizumi, H.; Yasuda, K. Spectrochim. Acta, Part 8 1 9 7 6 , 3 1 , 237-55. (9) Stephens, R . Manta 1978, 25, 435-40. (10) de Loos-Vollebregt, M. T. C . ; de Gaian. L. Spectrochim. Acta, Part B 1978, 33, 495-512. (11) Grassam, E.; Dawson, J. B.; Ellis, D. J. Ana/yst(London) 1977, 102, 804-18. (12) Koizumi, H; Katayama, M. Phys. L e t t A 1977, 6 4 , 285-6. (13) Koizumi, H.; Katayama, M. Phys. L e t t A 1977, 63, 233-4.
(14) Rann, C . S.; Hambly, A. N. Anal. Chem. 1965, 37, 879-84. (15) Liddeil, P. R . Anal. Chem. 1976, 48, 1931-33. (16) Bower, N. W.; Ingle, J. D., Jr. Anal. Chem. 1977, 4 9 , 574-9,
RECEIVED for review January 23, 1980. Accepted March 13, 1980.
Laser Atomic Absorption Spectrometry for Histochemistry Kimiaki Sumino,
Ryoji Yamamoto, Fumikazu Hatayama, and Shoji Kitamura
Department of Public Health, Faculty of Medicine, Kobe University, Ikuta-ku, Kobe 650, Japan
Harumasa Itoh Nihon Denshi (JEOL) Co., Nakagami, Akishima, Tokyo 196, Japan
A flameless atomic absorption spectrometer with an optical microscope and laser oscillator for determination of metals in various materials in very small target areas has been developed. A microfield of the sample is irradiated by a narrow laser beam under microscopic survey and the element in this area is quickly atomized. The subject element in the vapor is detected by flameless atomic absorption spectrometry and measured by comparing with known amounts of the standard material. I n one application for histochemistry, localization of cadmium in human kidney cortex was quantitatively examined under optical microscopic observation by this instrument. After the method for making standard preparations was established, relatively higher amounts of cadmium were found in the proximal tubules in the cortex and the lesser amounts in distal tubules and the glomerulus.
A microanalytical method for t h e determination of trace metals in desired minute portions of samples, under microscopic survey, has been developed utilizing a flameless atomic absorption technique with a laser beam as an atomization device. The instrument is a modification of a commercial laser microprobe, which can vaporize a sample by laser energy and discharge of spark electrodes, and determine trace metals using emission spectrometry. X-ray fluorescence, microprobe, or ion beams will undoubtedly be used for histochemical analysis of heavy metals in t h e future, b u t no report has yet been found in this field, except the use of the laser microprobe mass analyzer (LAMMA). Microanalysis by LAMMA has been reported in freeze-dried sections of rat kidney (tubulus cells) a t 1 pm in diameter and 1 to 2 pm in thickness, but only light metals like N a , K, a n d Ca were detected ( I ) . Other reports on microanalysis have been found in the field of histochemistry by using the laser microprobe with emission spectrometer (2-4), but only one set of quantitative data was reported on t h e analysis of a heavy metal, cellular gold ( 4 ) . Emission spectrometry is available for the qualitative analysis of t h e components of samples. but its sensitivity, specificity, and quantitative accuracy are generally thought to be less than those of atomic absorption spectrometry. Furthermore, because atomic absorption spectrometry provides precise measurement, ease of use and simple pretreatment of samples, 0003-2700/80/0352-1064$01 OO/O
it has shown great usefulness in trace metal analysis in many fields, particularly in pollution analysis, especially since development of the flameless method. With atomic absorption spectrometry, the measurement of microsamples is possible by using a sampling cup or carbon tube, but direct search for and observation of desired microfields on a sample through a microscope has been completely impossible. A few attempts a t atomic absorption analysis using lasers have been made (5-7), b u t as reviewed by Hieftje e t al. (8), sensitivity remained mediocre, interferences were frequent, and precision was marginal. No microanalysis of a minute sample under direct survey by microscope has been reported. T h e new laser atomic absorption spectrometer with background correction system, therefore, has been developed t o take advantage of the good points of both the laser microprobe and t h e atomic absorption spectrometer, making new experimental techniques possible. T h e description of this apparatus and some basic data with regard to its use, sensitivity, and precision are described first. Thereafter, in one application, this instrument was used t o determine the cadmium within minute portions of t h e kidney cortex in Japanese autopsy cases.
EXPERIMENTAL Apparatus. The outline of the apparatus, manufactured by JEOL Co., in accordance with our instructions, is shown in block diagram in Figure 1. The laser is a neodymium glass laser of 6.6 mm rod diameter X 100 mm rod length, rearranged from JEOL laser microprobe JLM-200. The oscillation wavelength is 1.06 pm and the maximum power of the pulse is 2 J, with a water cooling system. The output of the laser can be selected at 0.5, 1.0, 1.5, and 2.0 J, and the diameter of the laser beam can also be controlled, at 0.5 to 7 mm by use of an iris diaphragm. The laser beam can be focused onto any selected areas in a sample by observation with a light microscope. A hollow cathode lamp for atomic absorption and D, lamp for the background correction are provided as light sources. Background Correctivn S y s t e m . The optical pathway constructed specially by JEOL for this system is shown in Figure 2. In the double-beam pathway adopted in this instrument, a dc lighting method was used rather than a pulse lighting method or chopper mirror, because it was considered that the simultaneous background correction was highly significant, in order to catch extremely high-speed phenomena caused by laser vaporization. Both beams become integrated into one by the partially coated mirror which is 5 cm x 7 cm with alternate slits of 0.5 cm, and pass through the atom cloud which is created by the laser shot. C 1980 American Chemical Society
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Figure 1. Block diagram of laser atomic absorption spectrometer. HCL: hollow cathode lamp, D,: deuterium lamp, D: diaphragm iris, ST: stage for sample or metal heater, HM: half mirror, M.H.: metal heater, P.S.: power supply, G.T.: glass tunnel
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Figure 2. Background correction system using D, lamp. (A) Laser oscillator, (B) microscope, (H) hollow cathode lamp, (L) condenser lens, (M) mirror, (Hm)half mirror, (Tm) triangular mirror, (Pm) photomultiplier tube, (G)grating, (S,) entrance slit, (S,) exit slit
At that time, both beams have a portion of their original energy absorbed by the atom vapor and smoke, and they are then projected through the entrance slit into a usual spectrophotometer of the Czerny-Turner type. The beams dispersed by the grating (50 X 50 mm with 1200 lines/mm) are spread further by two total mirrors, separated by a triangular mirror, projected into two photomultipliers (side-on type, R106 manufactured by Hamamatsu T V Co.) through the exit slit of 0.08- or 0.14-mm width (corresponding to 0.7- and 1.3-nm wavelength width, respectively) for the hollow cathode beam, and 1.0-mm width (corresponding to 10 nm) for the D2 beam, and finally become electric signals. Each final signal is received by a photomultiplier, amplified, and recorded by a three-pen high-speed recorder (0.16 s/250 mm), which can follow three signals from the hollow cathode beam, D, beam, and the difference between them, i.e., net absorption signal, directly or after logarithmic transfer. If the recorder cannot keep up with the signals, they are automatically stored in a data memory attachment and later recorded in a n output showing net absorbance as net peak profile, peak height, or peak integrated value. Auxiliary Attachments. This system has two auxiliary attachments, besides the laser oscillator and microscope. The first is a metal heater of tungsten or tantalum, which is modified from the usual metal flameless atomizer, AA-HMA01 by JEOL, and placed as a substitute for the stage of the microscope. I t can be used for preheating a hard sample which is placed on the metal heater, and the laser beam can be emitted a t any time in the drying, charring, or atomizing stage of the flameless method. This device was not finally used in these experiments. The second is a pair of spark electrodes, modified from those of the laser microprobe, JLM-PO0 by JEOL. The diameter of the electrodes was
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reduced to 0.2 mm, and they were arranged above the stage of the microscope. These are for additional heating of a sample, and a spark discharge is initiated between electrodes through the atomic atmosphere when the sample is atomized by the laser beam. The effect of using the spark electrodes was tested. For example, over 5 times higher signals were obtained by using them than by not using them, for iron and chromium analysis in a razor blade, but in the analysis of cadmium in soft tissues in this study, no additional quantitative effect was gained, because the laser beam was enough to completely vaporize cadmium. Samples. Test Samples. To test the precision, reproducibility, and sensitivity of this device, an NBS sample of Cartridge Brass B type (C-1101) for zinc analysis and standard materials made by a tablet compressor for infrared spectrometry, which consisted of 5% carbon, 95% flour, and known trace amounts of cadmium, were used. In these tests, it is absolutely necessary that the surface of the materials is flat and the matrices are uniform a t microscopic level, since if samples are rough, the effects of successive laser shots and the sample volumes would all be different and impossible to measure precisely. Standard Preparations for Histochemistr2. In any microanalytical study, the method for making standard preparations must first be established for the precise evaluation of cadmium amounts in sample preparations. An attempt a t making standards for Fe, Co, Ni, Cu, and Zn in laser microprobe emission spectrometry of biological materials has been reported (9). However, since the standards were synthesized from a homogenate of rabbit muscle tissue by adding standard solutions, then ashing and compressing it, difficult problems remain in comparing ashed materials for measuring target elements in histological preparations. Making standard preparations is an extremely difficult problem because the standard material needs physical properties and matrices similar to the sample to be analyzed, for example, the ability to absorb laser energy, and even the ability to receive staining. Moreover, the material cannot be used as a standard preparation unless microscopically smooth and uniform. In this study, standard preparations were made of supernatant fluid from human kidney cortex similar to the sample materials. Previous research has shown similar cases included lC-94 pg/g of cadmium in whole kidney of Japanese (10). (1)The parts of adult renal cortex (male) were sampled within 5 h after death by accident, and homogenated ultrasonically. (2) After centrifugation at lo5 G, supernatant sap fluid was obtained and the cadmium content of this solution was measured by the usual flameless method. (3) A rectangular piece of vinyl tape (0.2 mm thick) had a 2 X 5 cm section cut out, and it was fixed on the object glass to contain the liquid sample and give it a uniform thickness. Fifty pL of the supernatant solution were poured carefully and evenly into this container, to obtain a sample solution calculated to be 0.05 mm in depth. This sample was dried at room temperature. (4) The glass was weighed before and after this operation, and the thickness of the standard preparation was calculated and measured. (The glass weight before the solution was added was compared to the weight after drying of the sample. The latter weight averaged 3-4 mg heavier, from which it was possible to calculate the thickness as 3-4 pm in each of 10 cases. The net thickness of 3-4 pm was accurately measured by using the micrometer in the microscope.) (5) Methylene blue (0.2%)-gelatin (0.5% ) water solution was dropped onto the standard film to stain. Thus, thin and flat films with different concentrations from different cortex fluids could be obtained. S a m p l e Preparations f o r Histochemistry. Another problem is in the method of sample preparation, because in the usual method (formalin fixation, dehydration, clearing by alcohol, paraffin embedding, staining, washing, dehydration, and mounting etc.), there is a loss of water-soluble cadmium which cannot be disregarded. The loss was found directly in solutions dropped on the sliced material on the object glass, by using a Zeeman flameless absorption spectrometer (170-70 by Hitachi Co.), at each stage when water was used in the preparation procedure. Even if a cryostat involving a microtome was used to secure thin frozen sections, cadmium effluence was also found in the stage of washing slides with water. Murakami et al. have reported a better method for use with light and the electron microscope, adding tannic acid as a fixative containing paraformaldehyde and glutaraldehyde buffered with phosphate, which reduced the loss of cadmium to
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Figure 3. (A) Reproducibility in Zn microanalysis of NBS test material including 30.2 YO Zn according to Provisional Certificate of Analysis. Conditions: Laser output, 0.6 J; objective lens, X20; crater size, 10 p m in diameter; sensitivity of data memory was set very low. (8)Calibration peaks of Cd in standard materials made of carbon powder and flour. Conditions: Laser output, 2.0 J; objective lens, X20; crater size, 160 pm in diameter; peak profiles were traced directly by a high-speed recorder at 0.5 Abs./full scale
as low as 6.570 (11). But in our research, when this fixative solution was dropped onto a renal cortex section on the slide glass. sectioned in a cryostat, more than 50% leakage of the cadmium into the solution occurred. Therefore, a method for making sample preparations without cadmium loss was devised. (1)After taking out the kidney cortex within 5 h after death, a piece of tissue was frozen in dry ice-n-pentane solution and a slice, 6-pm thick, was sectioned in a cryostat (Kryotom WK 1150 by R. Jung Co., West Germany). ( 2 ) The piece of tissue was fixed on a slide glass by dropping on it 0.1% phenol alcohol solution and drying at room temperature. (Little cadmium was eluted into the phenol alcohol solution; therefore it can be assumed that the distribution of the cadmium was almost unchanged.) (3) The sample preparation was stained with the methylene blue-gelatin solution mentioned above and dried. After drying, the thickness of the sections was approximately 4 pm with the micrometer of the microscope. Procedure. It is necessary first to determine the desired crater size for the particular sample material and target element. The size of the crater (several pm to 1 mm) depends upon sample thickness, quality of the material, such as density, color, and heat conductivity, the laser power applied to the sample, and the laser diameter, controlled by the iris diaphragm and the magnification of the objective lens of the microscope. If a sample is thin enough to vaporize completely and of uniform thickness, differences in sample volume depend only on the crater diameters. Also, if no peak is found by a second laser shot t o the same crater area produced by the first shot, this shows that the element in the crater is thoroughly atomized. In the case of cadmium analysis in sample sections in this study, no peaks were found by the second laser shot. Therefore, in the calculation of the amounts of cadmium in the sample craters in this type case, the amounts of cadmium are proportional to the square of the radius (Le., crater area). So the final concentration of the target element in a crater can be calculated by comparing both the area and the absorbance signal from the sample crater with those of a standard crater with the same thickness as the sample. To get good precision and reproducibility, or more sensitivity, the low-concentration samples in this experiment were covered by a small glass tunnel (1 X 2 cm and 5 mm in height as shown in Figure 1,designed for this purpose and based on the graphite tube in the flameless method), to maintain the atomic atmosphere as long as possible.
RESULTS AND DISCUSSION
As t o the sensitivity of this device to various elements, the elements which are known to be detected with high sensitivity
in the usual atomic absorption method, such as cadmium, mercury, or zinc, were also measured with high sensitivity by this instrument as described below. In the case of elements which require high temperature for atomization if in low concentration, such as copper, iron, or chromium, it is necessary to use a spark discharge to increase t h e temperature as mentioned before. In materials which had a transparent body or include a high percentage of water, their sensitivity became extremely low because of weak absorption of the laser beam. T h e background correction system adopted in this instrument was effective enough to overcome the absorbance of heavy concentrations of smoke from outside, corresponding t o 1.0 absorbance, much greater t h a n t h a t produced by t h e laser beam, in practice. Figure 3A shows the analytical results for zinc in a n NBS test sample including 30.2% of zinc. T h e relative standard deviation (rsd) was 3.070, so the precision was enough to use this instrument practically. T h e result showed t h a t net absorbance could be recorded quantitatively, if a standard material with a homogeneous distribution of the element was obtained. Figure 3B shows the microanalysis of cadmium in standard test materials made by a tablet compressor, which consisted of 5% carbon, 95% flour, and known trace amounts of cadmium. T h e net absorbance (rsd) figures were 0.51 absorbance (11%)in 5 ppm, 0.101 (5.7%) in 10 ppm, and 0.227 (1170)in 20 ppm. This showed t h a t each absorbance peak corresponded to t h e known concentration of cadmium, a n d peak heights showed good linearity. Using this method of making a standard material, it was possible to determine less than 10 ppm in materials when craters were 40 pm in diameter, in the cases of Cd, Hg, and Zn. For these elements, the detection sensitivity was sufficient to measure less than 0.1 pg, determined by calculating the crater volume vaporized by t h e laser shot. With regard to the characteristic localization of cadmium in kidney, autoradiographical studies have been examined and found to show a higher accumulation in the cortex than in the medulla of mice administered radiocadmium (12, 13),b u t no quantitative determination has been made in local cortex areas. Figure 4A shows the standard calibration peaks of cadmium in standard preparations which were made from supernatant
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Figure 6. Peak detection of cadmium in microfields (50 p m in diameter) of human kidney cortex from t h e same autopsy as in Figure 5. I: glomerular area, 11: proximal tubulus, 111: distal tubular area. (A), (E), and (C) correspond to Figure 5. Time scale: 10 s/div. Net p e a k profiles were output from the data memory with high sensitivity
Flgure 4. (A) Standard calibration peaks of cadmium by laser atomic absorption spectrometer. These preparations from human cortex solution had 33 ppm (A), 7 7 ppm (B) and 145 ppm (C) of cadmium at 4 p m thick. Crater diameter: ( A ) is 160 p m , (E) 160 prn and (C) 120 Fm. Time scale: 10 s/div. Net peak profiles were output from a data memory attachment after subtracting each background. (E) Standard working curve of cadmium concentration. This is obtained from the peak heights (absorbances)in Figure 4A by calculations coinciding with the conditions in Figure 6: crater diameter, 50 pm. SE = standard error
detected in various areas of kidney cortex are shown in Figure 6. The amounts of cadmium in Portion A in Figure 5 (glomerulus part) was measured a t 55 ppm, in B (proximal tubulous portion) 90 ppm, and in C (peripheral tubulous part) 55 ppm, respectively, by comparing peak heights (i.e., absorbance) with the standard working curve in Figure 4B. T h e three peaks in each different part were varied because the numbers of cells varied in the different craters even though the functional areas were the same. T h e average and standard deviation of cadmium amounts in three functionally different areas were 45 f 8 p p m in glomerulus (I in Figure 6), 80 f 30 p p m in proximal tubulus (11) a n d 60 f 6 p p m in peripheral tubulus (III), respectively. Analysis after acid digestion showed t h a t this kidney had 43 ppm of cadmium in the total cortex in wet base. Another case showed about 70, 140, and 90 ppm in the correspondent parts, and 90 p p m in the total cortex. These results and Figure 6 show t h a t the cadmium in the cortex is highest in the proximal tubulous portion, roughly about half as much in t h e glomerulus part, and less than two thirds as much as in the proximal portion, in the distal tubulous portion. However, it is not yet certain that cadmium in these portions has any significant meaning regarding kidney impairment by cadmium.
ACKNOWLEDGMENT We thank E. C. Woods for helpful suggestions. Flgure 5. Craters in human kidney cortex (containing 43 p g / g of cadmium in total cortex in wet base) which were created by laser shots. (A) Glomerulus area, (E) proximal tubulus, (C) distal tubulus. This sample was made by sectioning by cryotome and direct staining with gelatin-
methylene blue solution without any washing, to prevent cadmium loss solutions from three different autopsy cases, in which cadmium concentrations had been measured previously. Figure 4B shows t h e standard working curve made from Figure 4A by taking into due consideration the crater diameter and absorbance peak height, as the concentration of cadmium was easily calculated under t h e conditions in Figures 5 and 6. Reproducibility and linearity of calibration were shown for t h e quantitative determinations of cadmium within minute portions. Peak heights from known amounts of cadmium can be used as standards by comparing and calculating from the diameters of t h e craters when they differ. T h e photograph in Figure 5 shows a sample of human kidney cortex which was radiated by a laser. Only three craters are shown, of 50-pm diameter, with 120 times magnification. T h e three craters are in different parts of the kidney cortex. Tissue has been vaporized almost completely by the laser shot and the amounts of cadmium in those craters are shown as peak heights in Figure 6. The peaks of cadmium
LITERATURE CITED R . Kaufman, F. Hillenkamp, R . Nitsche, M. Schurrnann, and E. Unsold, J . Microscopie Biol. Cell., 22, 389 (1975). W. J. Treytl, J. B. Orenberg, K. W. Marich, A. J. Saffir, and D. Glick, Anal. Chem., 44, 1903 (1972). K. Mietkiewski, J. B. Warchol. and 8 . Arcirnowicz, Acta Histochem., 42, 95 (1972). D. Glick and K. W. Marich, Clin. Chem., 21, 1238 (1975). J. P. Matousek and B. J. Orr, Spectrochim. Acta, Part B , 31, 475 (1976). T. Kantor, L. Polos, P. Fodor, and E. Pungor, Talanta, 23, 585 (1976). T. Ishizuka, Y . Uwamino. and H. Sunahara, Anal. Chem., 49, 1939 ( 1977). G. M. Hieftje and T. R. Copeiand, Anal. Chem., 5 0 , :300R (1978). E. Giovannini. G. B. Principato, and F. Rondelli. Anal. Chem., 48, 1517 (1976). K . Sumino, K. Hayakawa, Y . Shibata, and S. Kitarnura, Arch. Envlron. Health, 30, 487 (1975). M. Murakami, K. Hirosawa, S. Suzuki, and H. Katsunurna, Ind. Health, 13, 123 (1975). M. Berlin, L. Hamrnarstrom, and A. 8 . Maunsbach, Acta Radio/., 2 , 72 (1963). M. Murakami, S. Suzuki, K . Hirosawa, and H. Katsunuma, Ind. Health, 15, 149 (1977).
RECEIVED for review June 19,1979. Accepted March 10, 1980. This study was supported by a grant in aid for scientific research from the Ministry of Education of Japan (No. 144043).
