ASBESTOS by TEM: METHOD 7402, Issue 2, dated 15 August 1994 - Page 4 of 7
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Cut a V-notch on the edge of the foam and filter paper. Use the V-notch as a reservoir for adding solvent. NOTE: The wicking substrate should be thin enough to fit into the petri dish without touching the lid. Place the TEM grid on the filter or lens paper. Label the grids by marking with a pencil on the filter paper or by putting registration marks on the petri dish halves and marking with a waterproof marker on the dish lid. In a fume hood, fill the dish with acetone until the wicking substrate is saturated. NOTE: The level of acetone should be just high enough to saturate the filter paper without creating puddles. Remove about a quarter section of the carbon-coated filter from the glass slide using a surgical knife and tweezers. Carefully place the excised filter, carbon side down, on the appropriately-labeled grid in the acetone-saturated petri dish. When all filter sections have been transferred, slowly add more solvent to the wedge-shaped trough to raise the acetone level as high as possible without disturbing the sample preparations. Cover the petri dish. Elevate one side of the petri dish by placing a slide under it (allowing drops of condensed acetone to form near the edge rather than in the center where they would drip onto the grid preparation).
CALIBRATION AND QUALITY CONTROL: 14.
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Determine the TEM magnification on the fluorescent screen: a. Define a field of view on the fluorescent screen either by markings or physical boundaries. NOTE: The field of view must be measurable or previously inscribed with a scale or concentric circles (all scales should be metric) [7]. b. Insert a diffraction grating replica into the specimen holder and place into the microscope. Orient the replica so that the grating lines fall perpendicular to the scale on the TEM fluorescent screen. Ensure that goniometer stage tilt is zero. c. Adjust microscope magnification to 10,000X. Measure the distance (mm) between the same relative positions (e.g., between left edges) of two widely-separated lines on the grating replica. Count the number of spaces between the lines. NOTE: On most microscopes the magnification is substantially constant only within the central 8- to 10-cm diameter region of the fluorescent screen. d. Calculate the true magnification (M) on the fluorescent screen:
where: X = total distance (mm) between the two grating lines; G = calibration constant of the grating replica (lines/mm); Y = number of grating replica spaces counted e. After calibration, note the apparent sizes of 0.25 and 5.0 µm on the fluorescent screen. (These dimensions are the boundary limits for counting asbestos fibers by phase contrast microscopy.) Measure 20 grid openings at random on a 200-mesh copper grid by placing a grid on a glass slide and examining it under the PCM. Use the Walton-Beckett graticule to measure the grid opening dimensions. Calculate an average graticule field dimension from the data and use this number to calculate the graticule field area for an average grid opening. NOTE: A grid opening is considered as one graticule field. Obtain reference selected area electron diffraction (SAED) or microdiffraction patterns from standard asbestos materials prepared for TEM analysis. NOTE: This is a visual reference technique. No quantitative SAED analysis is required [7]. Microdiffraction may produce clearer patterns on very small fibers or fibers partially obscured by other material. a. Set the specimen holder at zero tilt.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94
