Counting the dots:

Giving microscopes a 'workout' using
diatom test slides

by Dave Walker, UK
Underlined words are defined in the glossary



 
 
 

Diatoms are a popular subject for many amateur microscopists. Living specimens are particularly attractive to observe and a number of Micscape articles have described and illustrated them. (See links in Appendix).

The diatoms are a phylum of algae (protists) with silica shells which have well defined markings. The markings aren't always evident in living species but prepared slides of the empty shells are available where this detail can be clearly seen. Depending on the species the fineness of the markings varies and some species have markings close to the resolution limit of the optical microscope. Therefore diatoms are a favourite subject for assessing various aspects of the optical performance of a microscope. These include:

A number of microscope slide suppliers can offer either test sets or individual species (see Appendix).

As an illustration, here's how my two microscopes faired. My main 'scope is a typical budget model - the Russian Biolam 'R' stand (ca. 1973) with achromatic objectives but I also own a Cooke, Troughton and Simms (CTS) M2000 (ca. 1945) with achromatic phase objectives which is probably comparable to a modern budget to mid-priced stand.

The species names are a bit of a mouthful but those below are some of the most popular and will be present in most test sets or available separately.

The images were captured directly from a cheap B&W security video camera and 'Snappy 2.0' capture card so the images won't be as crisp as a 35mm slide or visual view. Note that the green filter (and infra red filter) used gives a rather flat image.

The rough sketches are not to scale and just show some of the main features.
(An excuse for my poor drawing skills!).
 
 

Stauroneis phoenicenteron - a good test for low to mid power objectives
Approx. 150 mm long, lines of dots ca. 0.72 mm apart.
  • a 10x - 20x objective should give a crisp image of the diatom's main features and show the unresolved dots as fine lines
  • a 40x dry objective should resolve the lines into rows of dots
  • if the diatoms are well mounted - good for checking field flatness and focus with a 40x objective
Right: the CTS 20x N.A. 0.45 objective was used right but the Russian 20x N.A. 0.40 gives a comparable view. The camera just captures a hint of the lines from the clearer visual field.
Left: the Russian dry 40x N.A. 0.65 objective with normal brightfield lighting resolves the dots. Although not as crisply as the CTS 45x objective.

Pleurosigma angulatum - a classic test object for mid powers
Aprox. 150 mm long, lines of dots ca. 0.53 mm apart.
  • a good subject to show that contrast as well as resolution is important in microscopy 
  • a 40x dry achromatic objective should resolve the dots but contrast enhancement often needed. Good for assessing lighting techniques (e.g. oblique or darkfield) 
  • with a 60x+ objective the dots should be clearly resolved
  • poor optics or setups may just show fine hatchings of lines

Right: the Russian 40x N.A 0.65 dry objective in brightfield has sufficient resolution but can't reveal the detail as the contrast is poor. The line down the centre (the raphe) is clearly seen.
Left: the same Russian 40x objective with oblique lighting to enhance contrast does reveal the dots (arrowed). The Russian aplanatic oblique condenser was used with 5mm offset but a card circle with notch cut out for the condenser filter tray can achieve a similar effect.
Right: the CTS 45x N.A. 0.95 oil fluorite objective clearly resolves the dots in brightfield with no oblique lighting. Note the contrast reversal effect, which is often seen with diatoms as the plane of focus changes. i.e. the dots are dark with pale background at the edge (and centre line) but the reverse away from the edge.
Left: the same CTS objective with phase contrast gives a clearer image.
 

Surirella gemma - a test for mid to higher powers
Approx. 100 mm long, lines of dots ca. 0.5 mm apart.
  • a 40x objective should give crisp views of the diatom and the heavier white borders
  • the finer lines may also be seen
  • a good 40x objective should resolve the lines to dots, but if not a 60X+ objective should clearly resolve the dots
The Russian dry 40x and the CTS 45x oil objective both show the fine lines and the CTS is also good enough to resolve the lines to dots (shown right, clearer visually than this image capture).
Left: at 60x+ the dots should be well resolved. The CTS 95x objective does this well. Focusing in and out gives a black and white contrast reversal effect.


 

Nitzschia obtusa - one of the easier tests for highest powers
Approx. 200 mm long, lines of dots ca. 0.3 mm apart.
  • a long straight species good for assessing field flatness at higher powers if the specimens are well mounted
  • a 95x - 100x objective should clearly resolve the dots
Right: the dots are clearly resolved with the CTS 95x N.A. 1.3 fluorite. Also shows good field flatness as the depth of field is minute at this magnification.

Frustulia rhomboides var. saxonica -
the 'going gets tough' for higher powers
Approx. 50 mm long, lines of dots ca. 0.3 mm apart.

Quite a handful as a species name and quite a handful as a test subject, as this is at the limit of the Russian and CTS microscopes' capabilities.

Note added 2002: Thank you to Frithjof Sterrenburg who kindly pointed out the change of taxonomy of F. rhomboides. Read Frithjof's fascinating Micscape article 'A second look at some well known test diatoms' where this is discussed.

Right: with the CTS 95x objective and using the better corrected Russian aplanatic N.A. 1.3 condenser (the CTS phase condenser is only N.A 1.0) and oiling the slide to the condenser, the lines (perpendicular to the diatom's long axis) are visible and just about captured on the CCD camera. The dots aren't resolved visually.
 
Amphipleura pellucida - only the very best optics will 'crack' this one
Approx. 80-140 mm long, dots ca. 0.27 mm apart.
Right: general view of this diatom with the 45x CTS objective under phase contrast. Fine lines perpendicular to the long axis are resolvable with the best 100x objectives. Resolving these lines to dots pushes the finest optics and light microscope to the limit.
This species is included in the diatom test set I used but neither the Biolam or CTS optics are good enough to resolve the lines or dots! A very good system should show the lines of unresolved dots. The dot separation is near the resolution limit of an optical microscope although the finest optics may resolve the dots. Have a try if you own a very high quality 95x -100x objective and equally important - a very well corrected substage system to achieve the maximum potential of the objective. Oiling the condenser to the slide as well as the objective to the slide is necessary (a messy business!) and probably a blue filter and a high mag. eyepiece e.g. 25x. If you succeed with A. pellucida or have a better image of F. rhomboides, why not share your skills and send Micscape an image and we will be pleased to include it here with credits.

As an aside and a humbling thought, apparently microscopists first resolved this species to dots in 1868 so illustrates how fine optics were in the late 19th century! (See link below).
 
 

Other comments

To achieve the best performance from an achromatic objective it can be used with a yellow-green filter which minimises the uncorrected aberrations. Some of the finest photographs I've ever seen of diatoms were taken with achromatic objectives with a plate camera in 1904.

Diatoms are a good subject for understanding the importance of the correct condenser iris setting (not to be confused with the field iris which may be present on the lamp). Inspecting the image as the iris is moved from fully open to fully closed will show the effect on resolution and image quality.

Image capture for 'diatom dotting' also gives a good 'workout' for the image capture system, whether photo' film or in this case digital capture with a security CCD camera. The cheap B&W camera used (>560 line resolution) coped well and is less than £100 new from electronic suppliers in the UK. Arguably colour isn't essential for high mag. or phase work and a cheaper route into image capture (stills or video) than colour.

All the above captures were taken with the camera fitted to the eyepiece tube without any eyepiece. So despite the fact that the CTS objectives require compensating eyepieces the camera captured the features quite well.

Comments to the author Dave Walker (an amateur microscopist) are welcomed.

Acknowledgements

In addition to my own observations the leaflet accompanying the NBS diatom set 6/AK prepared by Eric Marson of Northern Biological Supplies was valuable in preparing this article. Also the measurements quoted in the http://glinda.lrsm.upenn.edu/diatom.html link below.

Appendix

Related Micscape articles on test diatoms
 

'A second look at some well known test diatoms' by Frithjof Sterrenburg.

Test diatoms - what you can expect to see even with modest objectives by Martin Mach.

Image gallery, two test diatoms by Will Varnell.

For other illustrated Micscape articles on the diatoms and their many beautiful forms, type 'diatom' in the Micscape Library search index. A good introduction to diatoms by Wim van Egmond is here.
 


Further reading

1) The measurement of three light microscope test diatoms by scanning electron microscopy by J B Sanderson. Royal Microscopical Society Proceedings, Vol 25/3, May 1990, pages 195-203.
Many thanks to J B Sanderson for sending me a copy of this fascinating article, which includes historical aspects of using test diatoms and ruled test-plates for assessing microscope objective performance.


External links of interest

bio-microtech.com/info/articles/article1.htm - 'Test Slides of Diatoms to Divisions - What Are You Looking At?' by Tim Richardson. An interesting article looking at the history of test slides as well as suggesting a new design.
http://glinda.lrsm.upenn.edu/diatom.html - has an image of the general forms of common diatom test species and relevant measurements.

http://www.wfu.edu/~gholz/research/ - a technical paper (on a contrast enhancement technique) with some nice illustrations of diatoms.

http://www.technicalvideo.com/Resolution.html - a page describing a special light source, but illustrated with Amphipleura pellucida images to show how well the technique defines the markings.

Test slide suppliers
Northern Biological Supplies, UK sell test set 6/AK. This is the set the author used and includes prepared slides of six diatom species with an illustrated instruction leaflet describing the features that should be seen with different objectives. This slide set can be ordered by contacting OnView.

Carolina Biological Supplies, US offer set WW-29-5984 which is a diatom test plate showing eight species.

Klaus Kemp, Microlife Services, UK also offers a variety of diatom slides, and is one of the few people skilled in the art of arranging diatoms into patterns.

Glossary
N.A. - numerical aperture; marked on the objective and the higher the N.A. the better its resolving power.

achromatic - many microscopes will include as standard a set of achromatic objectives. Chomatic aberrations are corrected for two colours and spherical aberrations for one (yellow-green).

fluorite - a corrected objective intermediate between an 'achro' and 'apo'. The CTS 45x phase objective is a fluorite.

apochromatic - expensive objectives where chromatic aberrations are corrected for three colours and spherical aberrations for two.

plan - indicates the objective is flat field i.e. gives sharp focus across the field of view. Both achromatic and apochromatic objectives can come in plan forms.

aplanatic - a better corrected condenser than the commonest Abbe design. The most highly corrected condensers are aplanatic-achromatic and demanded by the very best objectives.

phase contrast - a contrast enhancing technique that uses special objectives (often marked Ph.) and a matched condenser. Phase systems are available for most modular microscope stands.

compensating eyepieces - some objectives are designed to be used with the makers special eyepieces to help correct the aberrations.

CCD - charge coupled detector. The image sensor of modern video cameras, camcorders and digital stills cameras.
 
 

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