CYTOCHEMICAL ANALYSIS BY LASER MICROPROBE- EMISSION SPECTROSCOPY *

David Glick Division of Hisrochemistry, Department of Pathology,

Stanford University Medical School Palo Alto, Calif.

At the outset I should like to make i t clear that the work I shall review is the result of the labors of a number of individuals in my laboratory. Credit to the persons responsible for particular aspects of the program will be indicated. The earlier phases of our work on elemental analysis by emission spectroscopy using the laser microprobe as a sampling device, and applications to histochemistry, were covered in earlier reviews (Glick & Rosan, 1966; Glick, 1966). Briefly stated, the principle of the technique has been vaporization of the sample with a laser beam directed through a microscope, discharge of an electric spark set off by the vapor shooting up between charged electrodes positioned above the sample, passage of the light from the spark through a spectrograph, photography of the spectral lines of the elements in the vapor, and densitometric scanning and recording of the line intensities for quantitative evaluation.

With this technique, sampling of selected areas in frozen-dried tissue sections was carried out at the level of SO-2OOp across and 16-2Op deep to provide suf- ficient material for analysis. In this way a number of applications, mentioned in the reviews cited, have been made, e.g., measurements of calcium and magnesium of the order of 10-lo and lop9 moles, respectively, in samples of human stomach taken from serial sections cut parallel to the mucosal surface to obtain quantita- tive histological distributions (Glick, 1966). These earlier studies were carried out with Dr. R. C. Rosan and the current work with Drs. E. S. Beatrice and I. Harding-Barlow.

The use of the cross-excitation from the electric spark was required in the earlier work to intensify the spectral emission. With the relatively large samples needed to give an adequate signal for measurement, the electrodes could be set far enough above the sample to minimize burning of the material surrounding that to be analyzed. Such burning would contribute vapor to that of the sample and interfere with reliable analysis. However, for analysis of samples small enough to be meaningful for most histo- and cytochemical work, i.e., 10 diameter or less, the electrodes had to be brought so close to the sampe (2-3 mm), since so much less vapor was evolved, that burning the surrounding material became a problem. If spark cross-excitation were eliminated, the smaller source of light from only the incandescent vapor generated by the laser strike (FIGIJJ~E 1 ) would necessitate greater efficiency in light collection and spectro- graphic analysis, as well as greater sensitivity in measurement of spectral line intensity. These requirements have now been met and the cross-excitation has been eliminated. Dr. I. Harding-Barlow rearranged the optical system in our Jarrell-Ash spectrograph (Czerny-Turner, 0.75 m. f / 6 . 3 ) with replacement of the original grating by one of higher quantum efficiency and the mirrors by those of higher quality. The external optical system which directs the light into the spectrograph was also changed by removal of the cylindrical lens to rely only

No. XCVI, Studies in Histochemistry. Supported by research grants GM09227, HE06716, SK6AM18, 513 and 5TlGM1413 from the National Institutes of Health, USPHS.

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266 Annals New York Academy of Sciences

L

B s .t: c

Glick: Cytochemical Analysis RD C U Ll

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B A E M R\F Q P

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FIGURE 2. Diagram of laser beam unit. Roof prism (P), Q-switch sealed cuvette containing vanadyl phthalocyanine in benzene (concentration empirically adjusted to give peak power of 10 megawatts in single laser pulses) (Q) , ruby rod and helical flashlamp (R/F), aperture (M) , front mirror, 60% reflecting (E), sealed attenuator cuvette containing copper sulfate solution (0.16-0.34M) ( A ) , beam splitter (B), reflecting prism (RP), microscope objective, 95 X for viewing with 20x ocular, 2 0 ~ for lasing (cement-free, Vickers, or Cooke, Troughton & Simms, (0). sample (S) , laser radiation monitoring apparatus (RD), cooling air, filtered and dried, jet (C) .

P M T o s c

S L P

ANALYTICAL SYSTEM FIGURE 3. Diagram of spectrographic and recording system. Incandescent vapor, "plasma,"

light source (P), quartz collecting lens (L), spectrograph entrance slit ( S ) mirrors (M,M,) and grating (G). photomultiplier tubes mounted on spectrograph at position of a spectral line and background respectively (PMT) , and oscilloscope or other recorder (OSC).

268 Annals New York Academy of Sciences

on the biconvex spherical quartz lens to direct the light into the spectrograph. These optical changes provided a gain in sensitivity of about 10 times.

Greater additional gain was afforded by replacement of photographic record- ing and densitometry of the spectral lines by direct photomultiplier read-out of line intensity, as arranged by Dr. E. S. Beatrice. The 100-time gain obtained in this way was increased 100 to 1,000 times more by use of a preamplifier in the circuit.

Another instrumental modification was the redesign of the laser source and the addition of a monitoring system to control sampling and to make successive laser shots reproducible ( * 5 % ) . This was carried out by Dr. N. A. Peppers and coworkers through an arrangement with the Stanford Research Institute, Menlo Park, California. The new instrumental set-up is illustrated by the diagrams (FIGURES 2, 3), and the linear relationship between the controlled energy output of the laser and the diameter of the area sampled is shown in (FIGURE 4).

As shown by oscillograph recording, the single-pulsed laser flash occurs in about 10 nsec and the incandescence of the vapor lasts about 4 psec. In contrast, the flash, usually consisting of 4 pulses, occurred about 1 psec when cross- excitation was used and the light lasted about over 300 psec (FIGURE 5 ) .

Using the newly designed and improved system, tests of the detection capabil- ity for several elements were made on single cells (TABLE 1) and, for a greater number of elements, on individual human kidney glomeruli (TABLE 2). Data in the literature from macro analyses were calculated to concentrations in g/p3 for comparison with the laser-emission data calculated similarly from measurements on approximately 150 p3 samples of single cells or on 100 p3 of structures in tissue sections. The actual quantities of the metals detected in individual laser

UJ 35 t L A S E R O U T P U T V S S P O T

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m i c r o n s FIGURE 4. Relationship between diameter of area sampled and beam energy from laser

source.

Glick: Cytochemical Analysis 269

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FIGURE 5. Diagram of oscillograph recording of laser strike with (top) and without (bottom) spark cross-excitation.

samplings ( 5 - 1 0 ~ diameter, I - 3 ~ depth - shape of the volume sampled approxi- mately a section of a hemisphere) ranged from grams. The a p pearance of cells, before and after the laser sampling, is illustrated in FIGURES 6-9. A reliable and convenient method of standardization for the elements of interest in cytological samples is still a major problem remaining to be solved.

In the earlier work cited by Glick, 1966, the considerable variation in energy delivered in separate laser shots, and the lack of monitoring, made sampling in- determinate. An attempt was made to provide a silver standard for comparison of spectra from separate analyses by placing the sample on the emulsion of photo- graphic film (Kodak 649-0), vaporizing underlying emulsion along with the

to

270 Annals New York Academy of Sciences TABLE 1

ELEMENT DETECTION CAPABILITY TESTS ON SINGLE CELLS

Cell Estimated

(Calculated as g / p 3 ) Laser Detection Limit (g)* Published Values Species Element

Whole erythrocyte Human Fe 0.7 x IO-l7 (Bowen) < 10-18 Whole leukocyte Human Zn 0.33 x 10-le (Frederick eta / . ) Spermatazoon head Rat Zn 0.64 x (Hall ef a!.) 10-15 Parenchymal cell Cytoplasm (liver) Human C a 0.71 X 10-l8 (Tipton & Cook) 10-17 Parenchymal cell Cytoplasm (liver) Human Fe 1.40 X 10-19 (Tipton & Cook) 1 0 4 7

Parenchymal cell Cytoplasm (liver) Human Mg 0.81 X lO-l9 (Tipton & Cook) 10-18

Estimated, using approximately a 150 CL” sample, by Dr. 1. Harding-Barlow.

TABLE 2 ELEMENT DETECTION CAPABILITY TESTS ON HUMAN KIDNEY GLOMERULI

Element Line Concentration Estimated

in Kidney Detection Limitf B/L3*

Mg 2803 1.2 x 10-’8** < 10-14

Ca 3934 F e 3020 Zn 2139

8.6 x lo-”** < 10-15 8.6 x 10-17 10-15 4.7 x 10-17 10-15

Cd c u Mn

2288 3247 2576

9.1 x 10-18 2.0 x 10-18 < lo-” 1.2 x 10-18 < 10-18

Al Pb Mo

3962 7.5 x 10-19

3798 4.4 x 10-19 2170 7.0 x 1O-l0

10-17 10-17 1 0 4 7

Sn Sr Cr

3175 4078 4254

9.0 x lO-*O 2.9 x 1.8 x

10-18 10-1* 10-18

Ag 3281 2.0 x lo-” 10-19

Harding-Barlow (1961). ** Tipton and Cook (1963). i Estimated, using a loop3 sample from a kidney glomerulus, by Dr. 1. Harding-Barlow.

sample, and using the spectral lines of the silver whose concentration in the emulsion was previously determined.

With the controlled and reproducible sampling now attained with our improved instrumentation, the known laser energy employed in a given sampling should serve to define the amount of the material sampled. From this amount and the known concentration in the sample of a suitable element that can act as a reference, it should be possible to standardize internally.

If an external standard is used, it must provide compensation for the “matrix” effect, i.e., the influence on spectral emission of the “background” material in the sample. It is the “matrix” effect that precludes use of pure metal standards.

Glick: Cytochemical Analysis 27 1

FIGURE 6. One human erythrocyte lasered out of a group of three in an air-dried blood smear.

FIGURE 7. cell in center.

Nuclei laser-sampled in three air-dried human liver cells in an imprint, unsampled

272 Annals New York Academy of Sciences

FIGURE 8. Appearance before (top) and after (bottom) laser sampling of two leucocytes (top and bottom) and a group of three erythrocytes (upper right) in an air-dried human blood smear.

Glick: Cytochemical Analysis 273

274 Annals New York Academy of Sciences

Therefore, a suitable external standard must have a general composition similar to that of the unknown sample, and it must contain known concentrations of the elements to be analyzed, e.g. by incorporation of known concentrations of ele- ments in a suitable matrix material such as blood plasma.

References BOWEN, M. J. 1963. Trace elements in mammalian blood. U. K. Atomic Energy Corn.

Report, AERE-R 4196, H. M. Stationery Office, London. FREDERICKS, R. E., K. R. TANAKA & W. N. VALENTINE. 1964. Variations of human blood

cell zinc in disease. J. Clin. Invest. 43: 304-31s. GLICK, D. The laser microprobe. Its use for elemental analysis in histochemistry. J.

Histochem. Cytochem. 14: 862-868. GLICK, D. & R. C. ROSAN. Laser microprobe for elemental microanalysis, application

in histochemistry. Microchem. J. 10: 393-401. HALL, T. A., A. J. HALE & V. R. SWITSUR. 1966. Some applications of microprobe analysis

in biology and medicine. In The Electron Microprobe. T. D. McKinley, K. I-'. J. Heinrizh & D. B. Wittry, Eds. : 805-833. Jbhn Wiley & Sons. N. Y.

Studies on the trace element content of human tissues. Ph.D. Thesis, Capetown University Press. Capetown, South Africa.

1963. Trace elements in human tissue 11. Health Physics 9:

1966.

1966.

HARDING-BARLOW, I.

TIPTON, I. H. & M. J . COOK.

1961.

103-14s.