PART IV. MICROBEAM TECHNIQUES

LASER MICROPROBE-EMISSION SPECTROSCOPY *

David Glick, Ph.D., Kenneth W. Marich, B.S., Peter W. Carr, Ph.D. and Edwin S . Beatrice, M.D.

Division of Histochemistry, Department o f Pathology Stanford University Medical School

Stanford, Calif.

At a conference held in 1967, the state of development of our laser micro- probe system for elemental analysis of microscopic biological samples was reviewed.' This paper is concerned with a summary of the more recent advances in our on-going program of technological development of the instrumentation and the study of factors that influence the spectral emission of elements in a biological matrix. Application to biological and medical problems has been set aside for the time being until the basis for sound analytical work has been established.

The technique followed consists of selecting of the sample to be analyzed by viewing through a microscope, positioning the sample in the optical axis on the microscope stage, flashing a Q-switched ruby laser beam through the microscope to vaporize the sample, collecting and passing light from the incandescent vapor into a spectrograph, and recording intensities of particular spectral lines by photography, or, with much greater sensitivity, by photomultiplier readout.

Through a collaborative effort with the Stanford Research Institute, a major advance was made in the design of a laser unit which (1 ) provided a highly stable output that could be monitored to record the actual energy delivered in each laser shot, (2) utilized a sensitive photomultiplier system for measurement of the spectral line intensities obtained with the f/6.3 spectrograph, and (3) incorporated high quality optical components in both the light collection system and the spectrograph.2 These improvements extended the resolution of sampling below the limit of the light microscope, although the detection and measurement capability still do not match that of the sampling. As a test of the precision, replicate measurements of magnesium in air-dried 0.1 Pliter samples of 17 different human blood sera (each sample totally vaporized) gave coefficients of variation of 4.3% for laser energy delivered, 3.9% for spectral signal and 3.0% for the ratio of the latter to the former.

One of the bothersome features of earlier equipment was the need to increase spectral intensity by cross-excitation when the detection system was not suffi- ciently sensitive, or when the intensity was too low due to the presence of too little of the element to be analyzed in the sample. Intensification was achieved by positioning electrodes above the sample so that the vapor formed from the latter shot up between the charged electrodes and set off a spark which further excited the atoms to greater spectral emission. A critical difficulty with the original tech- nique was that the spark burned an area surrounding the sample, and thus added extraneous material. An electrode system was developed in our laboratory that enabled discrete sampling in the range of 10-25~ crater diameters by modifica- tion of the shape of the carbon electrode tips and reduction of the electrode

No. CVI, Studies in Histochemistry. Supported by research grants nos. GM 16181, GM 09227, HE 06716, and 5 K6AM18,513 from the National Institutes of Health, United States Public Health Service.

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Bovine albumin Sucrose

Annals New York Academy of Sciences

88 80 33 81 58 34

gap and spark energy.3 Although this did much to lessen the undesirability of cross-excitation in analysis of a microscopic sample surrounded by other mate- rial, it was clear that if the cross-excitation could be eliminated entirely, con- siderable advantage would be gained. To do this, however, requires more sensitive detection capability under conditions in which a favorable ratio of signal to noise is maintained. Our efforts in this regard have progressed.

Another technical problem that developed concerned simultaneous measure- ment of line intensity and the immediately adjacent background. A polychroma- tor device was built to separate the light from a line from the area adjacent to it far enough to accommodate two photomultiplier tubes for measurement of the separate signals. The construction and use of this device was described by Beatrice and G l i ~ k . ~ Essentially it consists of a nickel-stainless-steel 60" prism, aluminized for high reflectance in the ultraviolet region with a 3 0 ~ edge to separate the two light beams. Pairs of slits, photoetched in a beryllium-steel plate 2 5 ~ thick, are used to isolate individual spectral lines and equal adjacent areas. Any given pair of slits is centered in front of the prism edge. Each of the two beams reflected from its respective side of the prism is again reflected by an aluminized stainless-steel mirror to its photomultiplier tube (Amperex, XP 1118). Micrometer adjustments of all essential parts are provided to allow pre- cise optical alignment of the components.

Although this polychromator was designed for a single spectral line and its background, it would be highly desirable to have simultaneous measurements of a number of lines and backgrounds for determinations of a group of elements in the same sample. Work is proceeding on such a device.

Any quantitative application of the laser microprobe requires correction for the so-called matrix effect. This is the influence on the spectral emission of the element to be analyzed caused by the other components of the sample. An ex- ample of the matrix effect is shown in TABLE 1. The magnitude of the suppression of the emission of calcium and magnesium by organic matter (albumin) is strik-

TABLE 1 MATRIX EFFECT OF 7% BOVINE ALBUMIN ON SPECTRAL EMISSION OF CALCIUM AND MAGNESIUM.

Elementt Concentration (mg/100 mg)

1 2 10

Ca 75 92 91 Mg I1 I1 91

Values expressed as % suppression in emission. t Dried 40-111 samples of Specpure@ CaCO, and MgO (Johnson, Matthey & Co. Ltd., Lon-

don) were used.

TABLE 2 MATRIX EFFECT ON SPECTRAL EMISSION OF SILVER*

Concentration (%)

7 1.4 0.3 Matrix Substance

Glick et al.: Laser Spectroscopy 509

ing. The effect is also apparent in TABLE 2. Suitable standardization will involve utilization of known concentrations of the elements in a mixture like that of the unknown, or it may prove more feasible in some instances to employ ratios of elements in the unknown when one of the elements can be used as a reference quantity. Work is being continued on this problem as well as on the others discussed.

References

1 . GLICK, D. Cytochemical analysis by laser microprobe-emission spectroscopy. Ann. N. Y. Acad. Sci. 157( 1 ) : 265-274.

2. PEPPERS, N. A., E. J. SCRIHNER, L. E. ALTERTON, R. C. HONEY, E. S. BEATRICE, 1. HARDING- BARLOW, R. C. ROSAN & D. CLICK. 1968. Q-Switched ruby laser for emission micro- spectroscopic elemental analysis. Anal. Chem. 40: 1178-1182.

1969. Electric spark cross-excitation in laser niicroprobe-emission spectroscopy for samples of 10-25 micron diametei. Appl. Spect. 23: 257-259.

4. BEATRICE, E. S. & D. GLICK. 1969. A direct reading polychromator for emission spectroscopy. Appl. Spect. 23: 260-263

1969.

3. BEATRICE, E. S., 1. HARDING-BARLOW & D. GLICK.