Phase Contrast Test Slide

Description and Manufacture

The Phase Contrast Test Slide was developed by the Health and Safety Laboratory (HSL), on behalf of the Health and Safety Executive (HSE), in collaboration with ULO Optics (formerly PTR Optics) and the National Physical Laboratory (NPL). The slide is known as HSE/NPL Test Slide. Its purpose is to provide a reproducible phase object to check microscope system performance prior to counting asbestos fibre samples.

The master engraving was produced by the NPL using a ruling engine. It consists of seven bands of lines, with 20 lines in each band, progressively reducing in width and depth, from 1.1μm to 0.25μm wide. The lines were produced using a V shaped inscriber with a depth to width ratio of approximately 1:10. The bands are separated by 20μm gaps. A test zone is delineated by a rectangle bounded by deep groves, which can be viewed using a microscope with x100 magnification in either dark field or phase contrast mode. Figure 1 illustrates the bands of lines on a test slide and Table 1 gives the widths of the lines within each band and the associated phase contrast induced.

Figure 1: An illustration of the bands of lines that form the basis of the HSE/NPL Mark III Test Slide

Table 1: The width of the lines within each band and the associated phase contrast introduced when green light (Lambda = 530nm) passes through a line.

Each of the HSE standard test slides is an epoxy replica of the NPL Master. An impression of the lines is produced in a resin of refractive index 1.58 and is mounted on a standard glass microscope slide (76 x 25 x 1.2mm). This impression is sealed in another resin of refractive index 1.485 and covered with a standard (0.17mm thick) glass coverslip. To maintain a steady level of quality between slides HSL assesses each one. Only the test zone bound by the four sets of intense lines (Figure 1) is examined assess the contrast of the band 4, 5, 6 & 7 and inspected for dust deposits that may cause light or dark spots. The whole slide is inspected for cracks, splits and other defaults in manufacture. Although these defects cannot be eliminated, all slides are considered to be satisfactory.

HSL certifies the test slides as satisfactory for use to set up phase contrast microscope and categorises them into three sets:

1. those that have band 4 fully visible and band 5 partially visible (red documents)
2. those that have band 5 fully visible and band 6 partially visible (green documents)
3. those that have band 6 fully visible and band 7 partially visible (yellow documents)

Each slide is allocated a unique number and this is shown on the accompanying certificate. Records and computer images of each slide inspected are kept by HSL. Anyone experiencing difficulty in using the test slide may contact the Minerals and Fibres Section of the HSL.