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Table of Contents
4. HSE/NPL Mark III Test Slide
5. Phase-Shift Angle 3˚and USA
7. Phase Shift and the HSE/NPL and Pickford Slides
8. Visibility Mechanisms of the Pickford Slide and Objective Visibility
9. Visibility Mechanisms of the HSE/NPL Slide
1. The HSE/NPL Test Slide History
During the late 1970’s and early 1980’s, Jean le Guen in the laboratories of the United Kingdom Health and Safety Executive (UK HSE), was far sighted in seeing the need to develop a test to ensure that PCOM’s around the world should have similar detection limits such that the same sizes of airborne asbestos fibres are able to be observed and counted.
The HSE worked in conjunction with the National Physical Laboratory (NPL), and as a result, the HSE/NPL Phase-Contrast Test Slide was designed and developed (Le Guen, J. M. M., Ogden, T. L., Shenton-Taylor, T., Verrill, J. F., ‘The HSE/NPL Phase-Contrast Test Slide’. Ann. Occup. Hyg. Vol 28, No 2, pp 237-247, 1984).
Three versions of the Test Slides were released.
2. The HSE/NPL Mark I Test Slide
Released in approximately 1977, the Mark I HSE/NPL Test Slide comprised seven Bands (known now as blocks 1 to 7) of ridges mounted on a microscope slide, each block comprising 20 ridges separated by 5 µm. The ridges varied from 1.08 µm wide for block 1, to 0.25 µm wide for block 7, and were created by the National Physical Laboratories (NPL) by ruling grooves into a thin aluminium film using a diamond tool.
These grooves were used to create ridges in a replicating resin of refractive index (RI) 1.58 which were mounted onto a glass microscope slide. The height of each ridge was around one tenth of the width, and was designed to simulate the diameters of fine asbestos fibres (Le Guen et al, 1984).
When introduced, the Mark I HSE/NPL Phase-Contrast Test Slide was well received by the scientific community but suffered from a problem where all of the blocks of ridges were visible. Although the resin ridges were designed to produce phase changes similar to those of asbestos fibres, the large refractive index difference between the resin and the surrounding air appears to have caused the Mark I ridges to behave mainly as amplitude objects, thus not being fully able to test microscopes that failed to meet the standard optical requirements specified by the Membrane Filter method (MFM).
3. The HSE/NPL Mark II Test Slide
Soon after the release of the Mark I Slide, it was modified by adding a synthetic resin, Euparal with an RI of 1.485 between the coverslip and the ridges (Ogden et al. 1986). This modification resulted in block 5 being visible and in block 7 being invisible to achieve satisfactory microscope performance.
Geoff Pickford was present at the HSE laboratories soon after this occasion and was then able to test several Mark II Slides in a number of laboratories around the world, assisting in the verification that block 5 was a satisfactory indicator of satisfactory microscope/observer performance (Le Guen et al. 1984, 240). The Mark II Test Slide set a standard throughout the world so that all laboratories testing for airborne asbestos fibres were ‘seeing’ much the same range of asbestos fibre sizes.
A unique and very strong feature of the Mark II HSE/NPL Test Slide is that every Slide is tested by the UK HSE Science and Research Centre, and if satisfactory issued with a Certificate. Slides are now manufactured and supplied only by ULO Optics, and continue to be certified by the HSE. UK HSE’s revision of HSG248 in 2021, has now enabled certification outside of the UK provided that any new Test Slide is proven to be equivalent to blocks 5, 6 and 7 of the HSE/NPL Mark II Test Slide.
4. The HSE/NPL Mark III Test Slide
For an unknown period of time, a Mark III HSE/NPL test slide has been available which is in at least two sub-versions specified as follows, and related to the colour of the Certificate and slide (Crane et al, 2014) and confirmed by verbal communication between the principal author and the UK HSE Science and Research Centre:
- Green Certified Slide (known as the HSE/NPL Mark III (Band 5) Slide) – block 4 is visible, block 5 is visible, block 6 is partially visible, block 7 is invisible
- Red Certified Slide – (known as the HSE/NPL Mark III (Band 4) Slide) block 4 is visible, block 5 is partially visible and block 6 and 7 are invisible This version is not recognised as suitable in the UK and Australia, but is in USA.
According to Crane et al (2014) a Yellow Certificated Slide was also available but because all blocks were visible, was not acceptable in USA. This Slide should not be acceptable for any airborne asbestos fibre analysis work, because it allows the acceptance of inadequately performing microscopes or observers.
5. Phase-Shift Angle 3˚ & USA
USA’s Code of Federal Regulations (CFRs) on Asbestos (1910.1001) prescribes actions to be taken when dealing with asbestos.
Appendix A of this CFR, describes mandatory procedures for analysing air samples for asbestos, and refer to the ‘most current version of OSHA method ID-160, or the most current version of NIOSH Method 7400’.
- Section 10 of Appendix A of CFR 1910-1001, refers to ‘The phase-shift detection limit of the microscope shall be about 3 degrees measured using the HSE phase shift test slide as outlined below’, and then requires microscopes to be able to achieve stated criteria of subjective visibility.
- OSHA ID 160 refers to a ‘Phase shift test slide, mark II’ and makes no reference to phase shift angle.
- Section 10 b, of Issue 2 (15 Aug 1994) NIOSH Method 7400 states ‘Check the phase-shift detection limit of the microscope periodically for each analyst/microscope combination’, and then refers to the use of the HSE/NPL Mark II phase-contrast test slide, without any reference to ‘3 degrees’, but does refer to a paper by Rooker et al (1982).
- However, Issue 3 (14 June 2019) only refers to a ‘phase-shift test slide’, further specified by Reference 19, OSHA Fact Sheet, by Daniel Crane and Martin Harper, ‘The Phase Shift Test Slide Interpretation’, 2011
Appendix B of CFR 1910.1001, describes non-mandatory requirements for asbestos sampling and analysis, which includes the use of the HSE/NPL Mark II Test Slide, but no mention of magnitude of phase shift.
Based on knowledge then available, excellent collaboration between OSHA and NIOSH produced a Fact Sheet, Crane and Harper (2011) which states ‘Should a future technology or product become available for the purpose of assessing phase-shift, it should be acceptable to the extent that it could bracket or measure the phase shift of a microscope to assure that it is operating at about 3 degrees of phase shift.
The CFR, NIOSH and OSHA membrane filter methods have different visibility requirements as follows:
- Phase shift detection limit shall be about 3 degrees
- The slide consists of seven sets of grooved lines (ca. 20 grooves to each block) in descending order of visibility from sets 1 to 7, seven being the least visible. The requirements for asbestos counting are that the microscope optics must resolve the grooved lines in set 3 completely, although they may appear somewhat faint, and that the grooved lines in sets 6 and 7 must be invisible. Sets 4 and 5 must be at least partially visible but may vary slightly in visibility between microscopes. A microscope that fails to meet these requirements has either too low or too high a resolution to be used for asbestos counting.
6. Explanation of Phase shift
To put phase shift into context in terms of airborne asbestos analysis, it is best to first point out that some airborne asbestos fibres (particularly crocidolite and to a lesser extent, amosite asbestos) captured on a membrane filter (MF) can be observed under bright field light microscope conditions. In microscopy, these fibres are known as ‘amplitude objects’, which generally apply to coloured, translucent or opaque objects.
However, many fine asbestos fibres (especially chrysotile) have a refractive index close to that of the membrane filter; are essentially transparent; and cannot be observed using bright field microscopy. They are known as ‘phase objects’.
The Phase Contrast Optical Microscope (PCOM) has been adopted from the biological field because of its ability to convert phase objects into amplitude objects by means of the partial destructive interference of light which renders transparent objects visible. In practical terms, phase contrast uses the difference in refractive indices between the fibres and the surrounding membrane filter, coupled with the thickness of the fibre in order to produce an amplitude object that is visible.
Phase Shift Angle (ϕ ˚) is defined as OPD x 360 ÷ λ
Where:
- OPD = Optical Path Difference = (n2 – n1) x t
- n2 = refractive index of the fibre
- n1 = refractive index of the material surrounding the fibre
- t = diameter of fibre (nm)
- λ = light wavelength (nm), usually 500 or 550 nm
7. Phase Shift and the HSE/NPL Slide
The design of the HSE/NPL test slide attempted to simulate fine asbestos fibres that were either just visible, or clearly not visible when using a ‘standard’ phase contrast light microscope used for airborne asbestos fibre analysis. This approach was later supported by work conducted by Rooker et al. (1982), who measured the phase shifts exhibited by real fibres with varying fibre diameters, and varying refractive index differences between the fibres and the mounting medium. He established that ‘fibres down to about 0.15 µm diameter are visible with good optics provided the phase shift exceeds about 4˚’. It was further explained that ‘…the detection limit with good optics is about 3˚ to 4˚’. When calculating the phase shifts of Mark II and Mark III (green) slides based on the maximum ridge height and the refractive index differences between the ridge material and the mounting medium shows that it is unlikely that block 5 would be visible. When re-calculating the phase shifts based on realistic detection limits using Rooker’s 0.15 µm, it is unlikely that blocks 4 and 5 would be visible, when in fact they are visible. Table S1 shows phase shift angles calculated for the HSE/NPL Mark II and Mark III (Band 5) test slides based on 530nm light wavelength, and on the ridge height of the phase objects as follows:- Maximum ridge height
- Taking into account the detection limit (DL) of the microscope, namely that width of a fibre below which the PCOM cannot detect. As worst case, this should be applied to amplitude objects which are more visible than phase objects. If the DL is based on Rooker’s estimate of 0.15 µm, the Phase Shift angles are significantly reduced as shown.
Table S1 – Phase Shift Angles for HSE/NPL Mark II Slides
| Block No. | Phase Shift Angle (˚) | |
|---|---|---|
| Using Maximum Ridge Height | Ridge Height Using DL = 150 nm | |
| 1 | 7.0 | 6.0 |
| 2 | 5.0 | 4.0 |
| 3 | 4.1 | 3.2 |
| 4 | 3.4 | 2.5 |
| 5 | 2.8 | 1.9 |
| 6 | 2.3 | 1.4 |
| 7 | 1.6 | 0.6 |
- For (a), ‘maximum ridge height’ – and based on a minimum phase shift of 3˚, Rooker’s (1982) findings show that block 4 could be visible, and blocks 5 to 7 invisible.
- For (b), ‘ridge height using a DL of 150 nm’ – and based on a minimum phase shift of 3˚, it appears that block 3 could be visible, and blocks 4 to 7 invisible.
Table S2 – Phase Shift Angles for Pickford Slide
| Set No. | Phase Shift Angle (˚) |
|---|---|
| A | 1.40 |
| B | 0.49 |
| C | 0.24 |
| D | 0.19 |
8. Visibility Mechanisms of the Pickford Slide and Objective Visibility
The Pickford Test Slide, which has been designed to be an alternative to the HSE/NPL Mark II Test Slide, is of rugged stainless-steel construction dimensionally similar to a standard microscope slide, and houses four ‘chips’ side by side, labelled Sets A, B, C, and D. Each chip is 0.5 mm thick pure quartz, with a set of 20 etched grooves 1 µm wide and 100 µm long. The grooves range from 90 nm deep for Set A, down to 5 nm deep for Set D.
On each chip, the set of grooves is surrounded by metal deposits forming a series of circular and radial guidelines to assist observers in locating the grooves. The most visible set of phase objects (Set A) is designed to reveal the nature of the grooves to an observer and has the same function as blocks 1 to 4 of the HSE/NPL Mark II Slide. Sets B, C, and D are equivalent to blocks 5, 6, and 7 of the HSE/NPL Mark II Slide.
An objective visibility measurement was developed using ‘grey levels’ (GLs), based on an 8-bit digital system (i.e., 256 individual levels), using a PCOM, a stable high-quality microscope camera, and image processing software (ImageJ).
The following diagrams show plots of GL (Y axis) and Pixels (X axis) of each of the four sets of grooves (Sets A, B, C, and D) in the Pickford Test Slide. The red circles show the background (lighter) GLs between the grooves, and the blue circles show the darker GLs of the grooves themselves.
I have defined percent Grey Level Differences (%GLD) = 100 x (B – P) ÷ B,
where B = the GL of the background of the image, i.e. in-between the ridges or grooves P = the GL of the ridge or groove
The %GLD’s of the Pickford Test Slide were found to be linear with respect the etched groove depth, as shown in the following graph:
For phase contrast microscopy applying to phase objects near the limit of visibility, the relationship between visibility and %GLD is linear.
There is a linear relationship of OPD (and therefore phase shift angle) with groove depth. In other words, if the OPD or phase shift angle is doubled, the objective visibility also doubles. Within the limits of normal measurement variabilities, this relationship applies to all of the Sets of Pickford Slides, thus showing the etched grooves to be pure phase objects.
9. Visibility Mechanisms of the HSE/NPL Mark II Slide
Plotting the objective visibility (i.e. %GLD) versus the maximum Ridge Height (which is theoretically equivalent to OPD because the RI difference between ridge and mounting material remains constant for all ridges) for the HSE/NPL Slide showed the relationship to be markedly non-linear, as shown in the following graph.
A number of different forms of visibility mechanisms were developed and investigated, but all failed to explain the non-linear behaviour of the ridges of the HSE/NPL Slide either with respect to phase angles or %GLDs. It is possible that the triangular fashion of the ridges, including the ‘pushed up sides’ illustrated by Le Guen (1984, Figure 1 and p. 240), and further explained that ‘the Mark I ridges have turned out to be far from simple phase objects the equivalence was probably never completely true in any case’.
10. Visibility Based on Subjective Perception
It has been shown that the concept of phase shift angle is not relevant to HSE/NPL or Pickford Test Slides.
Extensive experimentation has also shown that even a good objective visibility test (based on grey level measurements of the phase objects) can really only assist during the production stages of a Test Slide.
Subjective visibility is the gold standard to determine whether a Test Slide is acceptable or not, and is used on all assembled Pickford Test Slides to ensure that they are equivalent to the HSE/NPL Mark II Slides, and is also used by the UK HSE for Certification of all HSE/NPL Mark II Slides.
All Pickford Test Slides are therefore certified by the Southern Cross University (Australia) as being equivalent in subjective visual performance to that of the HSE/NPL Mark II Test Slide, which satisfies UK HSE requirements as stated in document HSG248, 2021, Section A1.37.
11. Certification and Metrological Traceability
During 5 of the 7 year development of an alternative Test Slide, a number of them were submitted to the United Kingdom Health and Safety Executive (UK HSE) Science and Research Centre, who then offered to certify them. However, it was decided to try and keep certification within Australia.
It should be noted that from the late 1970’s to date, the UK HSE has found that subjective visibility is not only the gold standard, but is the only standard for certification.
In this field, metrological traceability does not apply. For example, over years, several high level approaches to National Measurement Institute (NMI) were made to certify an alternative Test Slide. NMI found that the area of certification was not able to be handled by them because nothing tangible was actually being measured, such as length, mass, etc that could be related reliably and directly to visibility. NMI obviously can measure and certify groove depth and refractive index, but not the human act of being able to detect them visually. Further, even though the Australian National Fabrication Facility (ANFF) attached to the UNSW provides comprehensive groove depth data of each quartz wafer (which contains 46 Chips) which is useful as a quality control measure, and this is only indicative of subjective visibility.
In the absence of NMI, an Australian university which embodies one of the largest environmental and research laboratories (Southern Cross University Environmental Analysis Laboratory and Analytical Research Laboratory) conducts certification of the alternative Test Slide. The University Laboratory operates under ISO/IEC 17025, and is NATA accredited in 35 different areas, see https://nata.com.au/accredited-organisation/southern-cross-university-14960-15180/?highlight=environmental%20analysis%20laboratory.
Whilst detailed information concerning the protocols, checks and balances remain privy to the University, they embody solid and rigorous steps to ensure that certification standards are able to be maintained over years of time.
Before even being presented for certification, a series of tests are conducted on the nano-fabricated quartz chips and again when the Slides have been assembled. These include subjective visual testing on two phase contrast microscopes (Zeiss Standard 14, and Olympus CX41), and also objective visibility testing using an 8-bit grey level system that has been validated by using hundreds of Pickford Test Slides and 10 or more HSE/NPL Mark II Test Slides.
Only batches of 10 Test Slides are presented for certification at any one time, each batch commences with the microscope being set up for Khoeler Illumination and optimum phase contrast conditions; the microscope and observer are tested with a certified HSE/NPL Mark II Slide before and after the batch; and the microscope and observer are tested with several ‘Master’ Pickford Test Slides before and after each batch.
The Master Pickford Test Slides are a combination of Slides that passed the examination of the head Certifier for the UK HSE as being certifiable.
In addition, as part of the quality control process, the University has access to several Pickford Test Slides that have several different minor defects that have caused them to fail certification.
