Anal. Chsm. 1985. 57,440-443
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Comparison of Laser and Ion Microprobe Detection Sensitivity for Lead in Biological Microanalysis R i c h a r d W. Linton* and Scott R. B r y a n Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27514
Peter F. Schmidt Institute for Medical Physics, Westfalische Wilhelms University, Hufferstrasse 68, 0-4400 Munster, West Germany
Dieter P. Griffis Engineering Research Services Division, North Carolina State University, Raleigh, North Carolina 27709
Lead detection sensitivity in doped epoxy standards was compared for the Leybold Heraeus LAMMA 500 laser microprobe V.I the Cameca IMS-31 ion microscope (modified for high sensitivity digital imaging). For comparable lateral spatial resolutions of about 1 pm, the LAMMA relative detection limit of 5 pg/g is superior to that of the IMS-3f by more than 2 orders of magnttude. However, the analytical volume of the latter (0.02 pm3) was only about 0.05 that of LAMMA measurement as the result of differences in analysis depth (300 A vs. 5000 A). Both higher ion sputtering rates and greater analysis depths may be used to Improve the relative Pb detection limit for the IMS-3f. The estimated useful ion yield, however, remains about 2 orders of magnitude better in vs. 5 X This is consistent with LAMMA ( 3 X experimental LAMMA studies showing enhanced Pb sensttivtty compared to LTE calculations. Dtfferences in sensitivity also are illustrated In a comparative study of Pb distributions in atherosclerotic human aorta.
The recent environmental mobilization of P b has resulted in chronic exposures and progressive accumulation at trace levels in human tissue. Consequent toxic effects in part are related to the inhibition of specific enzyme activities ( I ) . Since the bulk P b levels in various human soft tissues are typically in the range of 0.1-10 ppm (2),analytical techniques with trace level detection are essential. Further, to examine the specific interactions of P b with tissue compartments, microscopic techniques are desired t o localize P b distributions a t the cellular and subcellular level. For example, one such study under investigation involves the concentration distribution mapping of P b in human aortas and its relationship t o the degree of atherosclerosis ( 3 ) . To meet the analytical criteria above, microprobe mass spectrometric techniques are possible techniques of choice. Focused laser (4) or ion beam ( 5 ) irradiation of biological tissue sections enables spatial resolutions on the order of 1 pm in the plane of the section t o be obtained. This study compares two modern commercial mass spectrometers for microscopic P b detection in biological standards. T o our knowledge it presents the most detailed comparison of sensitivities involving measurements of identical standards applicable to biological microanalysis. The laser microprobe (6) involves high power density, pulsed laser beam irradiation with the subsequent detection of ions from the small vaporized volume using a time of flight mass spectrometer (Leybold Heraeus LAMMA 500). The ion microscope ( 7 ) provides stigmatic imaging of the specimen enabling secondary ion images to be transferred via a double focusing mass spec-
trometer to an imaging microchannel plate detector (Cameca IMS-30. Details of the instrumentation employed can be found elsewhere (6-8). EXPERIMENTAL S E C T I O N Biological Specimen Preparation. Conventional epoxy (Epon) thin sections (0.5 pm) were prepared after doping the epoxy with known concentrations of lead acetate ( 3 ) . Prior laser microprobe experiments have demonstrated that a high degree of Pb homogeneity is achieved in the standards (3, 9). A blank specimen and a graded series of standards ranging from about 5 to 75 pg/g Pb were characterized. The thin sections were mounted on high-purity Cu TEM grids for both laser and ion microprobe examination. Human aortal tissue was dehydrated in 80% and 96% ethanol solutions prior to embedding and sectioning ( 3 ) . Finders grids and light microscopy were used to identify regions of interest for analytical investigations related to studies of atherosclerosis. Laser Microprobe Mass Spectrometry (LAMMA). A Leybold Heraeus LAMMA-500 was employed (6). A 10-ns pulsed Nd-YAG laser, frequency quadrupled to a wavelength of 265 nm, was used at a power density of about 1O'O W/cm2. The laser was focused on the specimen through an optical microscope having UV-transparent lenses with the aid of a low power He-Ne pilot laser. Laser perforations on the order of 1 to 1.5 pm diameter (verified by SEM measurement) were observed for Epon sections of 0.5 pm thickness. Ions were extracted into the time-of-flight mass spectrometer drift tube using an ion optical einzel lens. The detection system was the standard Cu-Be secondary electron multiplier/preamplifier coupled to a nominal 8-bit transient wave form digitizer (Biomation 8100) with 2048 memory channels. Ion Microanalysis (IM). A Cameca IMS-3f ion microscope was employed using primary and secondary ion optical parameters typical of those used t o obtain about 1 pm lateral resolution in secondary ion imaging studies of thin biological tissue sections ( 5 ) . A mass filtered, 10.5-keV primary beam of Oz+ was used. Primary beam current was estimated to be 400 nA with a Faraday cup detector. A 50 pm diameter primary beam was rastered over an area of 250 pm X 250 pm. The secondary ion extraction was confined to a circular area of approximately 150pm in the center of the sputtered area. Sputter rates of 10 A/s were estimated by measurement of the elapsed times to sputter through Epon films of known thickness on TEM grids as viewed through a reflected light microscope. Detection of secondary ions over the entire 150 pm image field was accomplished with a conventional electron multiplier tube (EMT). High sensitivity secondary ion images were obtained by using a dual microchannel plate (DMCP) (Charles Evans and Associates, San Mateo, CA) coupled to a custom digital imaging system with a charge injection devide (CID) camera to encode information from the phosphor screen (10). Digital images of the 150 pm image field contained 25 400 pixels or about 0.7 pm2/pixel. R E S U L T S A N D DISCUSSION Lead Detection Sensitivity Comparisons. The relative figures of merit for the laser microprobe (LAMMA) and ion
0003-2700/85/0357-0440$01.50/0Q 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 2, FEBRUARY 1985
Table I. Lead (zOsPb+)Detection Comparisons, Laser Microprobe (LAMMA) and Ion Microanalysis (IM)" feature approx lateral resolution, wm analysis time, s area vaporized, Fm2 analyzed area, pm2 depth vaporized, nm analyzed volume, 1m3 relative Pb detection limit, Fg/g absolute Pb detection limit, g 208Pbatoms in the analyzed volume at the detection limit useful yield of Pb+: 7"
LAMMA 1
