or why knocking the mirror or condenser out of alignment sometimes helps!
by Dave Walker
A brief survey of the benefits of using off-axis lighting to view some subjects, and how it can be easily accomplished without cost for most microscopes. Also a look at some rather bizarre 19th century microscopes which took the use of oblique illumination a bit too far!
If, like the author, you own a microscope with a mirror for external lighting, you may well have accidentally discovered the benefit of oblique illumination for studying some subjects. Microscope mirrors are occasionally knocked out of alignment at some stage during viewing, with the result that the subject is lit with an off-axis light source. This oblique illumination is in fact a useful way of improving the visibility of some low contrast subjects like protozoa and the details on diatoms.
Essentially oblique illumination works by accentuating any phase gradients within a transparent specimen. It's an easy form of lighting to achieve up to medium powers (e.g. 40x objectives) with most microscopes with or without a mirror, and is a 'cheap and cheerful' technique the amateur can use to improve visibility when studying low contrast subjects, especially if you're not fortunate enough to possess phase contrast or high power dark field illumination.
illustrate its effect, the image right shows part of the classic diatom
Pleurosigma angulatum using a 40x achromatic objective (Russian,
NA 0.65 dry). This is one of the cheapest 40x objectives you can buy, but
is perfectly capable of resolving the dots i.e. the fine detail on the
silica shell, but in normal brightfield this detail is quite difficult
to see. Only the central 'raphe' (on the right of the image) and an edge
of the diatom frustule at the left can be made out.
not resolution that's the problem, it's contrast. If the diatom is studied
with an oblique patch stop in the filter tray (see below) the image right
is seen. The dots which are fine markings on the diatom frustule are now
clearly seen. (Incidentally I have to push my ancient image capture
card to the limit to capture these sort of images, so they are a lot less
convincing than the visual image down the microscope).
It's also possible to resolve the dots under dark-field with this objective, but I have never found using home-made patch stops for a 40X dry objective very easy and have achieved less than successful dark-field, so oblique illumination is an easier alternative to improve contrast.
In a second example, the image left and right shows the classic epithelial cells taken by scraping the inside of the cheek lightly with the finger nail and mounting the saliva under a cover slip (again using the 40x dry objective). The image left is in brightfield with the iris stopped down more than normal to improve contrast - it's an acceptable image but rather flat. The same view with the oblique patch stop gives a very pleasing depth to the cell especially the nucleus, cell granules and folding of the cell (again, the visual image is a lot clearer than in the captured image).
Oblique illumination also works well on single cells such as protozoa. One book I referred to (ref. 1) illustrates that the internal vacuoles are particularly distinctive in this sort of illumination.
If you haven't tried the technique, how can it be achieved? A few methods are given below.
Knock the mirror!
As commented above, if you have a microscope with a mirror, moving the mirror (plane side) so that light is shone off-axis can achieve the effect. This one of the few techniques for improving contrast that the owner of a simple microscope without a condenser can achieve i.e. unlike dark-field or phase contrast. I first spotted the effect myself as a ten year old with a no-frills toy microscope, and I got into the habit of rocking the mirror on its axis when studying transparent live pond-life to see if more details were revealed.
Simple stop for the condenser filter tray
If you have a condenser and filter tray, a simple stop can be made as shown left. Just cut out a piece of opaque card to fit the filter tray and experiment with various sizes of sector cut out of the disc for a given objective to allow light from an angle to reach the subject. Normal axial illumination should be setup first. As for dark field, the condenser iris should be fully open and the condenser focus adjusted to get the best effect.
You may need to experiment with the size of the notch for a given objective. The orientation of the notch with respect to the subject should also be changed to see what achieves the best effect. If you have a rotating stage this is easier than rotating the patch stop in the filter tray. If the filter tray is a swing out type, also experiment with the extent the filter is swung in, as well as varying the orientation of the notch in the filter tray.
Condenser with oblique illumination
Buying a condenser specifically for oblique illumination is probably not justified, but this is a feature found in some condensers. My Russian Biolam microscope is supplied with a modest Abbe condenser, but for critical work I use the better quality Russian aplanatic condenser (N.A. 1.4). As a bonus this also has an oblique illumination feature, see image right. (Supplementary low power lens N.A. 0.3 also shown).
The iris can be moved up to 10mm off-axis in opposite directions, which also rotates in the mount to change the orientation of the light to the subject. The latter feature, as mentioned above for the oblique patch stop, is important as the off-axis lighting often works best in a specific orientation to a subject e.g. for non-symmetric diatoms like Pleurosigma.
In the late19th century but before the advent of high numerical aperture objectives, oblique illumination was quite popular and some makers developed microscopes especially to exploit the benefits of the technique. One of the first models was shown by Zentmayer of Philadelphia in 1876 at an Exposition in this city. It had a swinging substage, i.e. the whole substage assembly swung about an axis in the same plane as the stage. An image of this microscope is in the Moody Medical Library On-line (item 1.028 in catalogue). (This site illustrates a wonderful selection of historical microscopes from the famous Moody collection, most makers are represented).
Some makers designed microscopes that took oblique illumination to rather extreme limits. The classic example is the Ross Radial microscope built by the famous English maker Ross to a design by Wenham. This is a superb example of quality engineering and optics by a respected maker, but a rather bizarre microscope nevertheless. Almost every conceivable plane and axis of the microscope limb, body and substage has the facility to be tilted or rotated (see ref. 2 or similar history of microscopes where this model is often illustrated). An image of this microscope is also in the Moody Medical Library On-line (item 1.031 in catalogue).
The technique fell out of favour towards the end of the 19th century as high numerical aperture objectives became available with the ability to resolve the fine detail of diatoms etc. with good axial illumination. A firmer understanding of the requirements for a critical image with axial illumination were also realised.
I wasn't sure how well the technique is used nowadays given the many other ways of improving contrast. However, a web search for 'oblique illumination' shows that some of the mainstream microscope makers mention this form of lighting as a feature in some of their models (particularly metallurgical), so presumably it does still have a worthwhile role. The oblique patch stop is certainly a cheap and cheerful way for the amateur to improve contrast for some subjects, especially at medium powers where patch stops for dark-field get trickier to make.
I was also intrigued to see that at least one manufacturer, 'Edge', market a microscope with 'patented Multiple Obliquetm Illumination' which apparently gives 3D images. Visit the Edge web site for details and read the theory behind the technique for 3D imaging.
Comments on the article to the author Dave Walker welcomed.
1) 'Under the Microscope' by A. Curry, R. F. Grayson and G. R. Hosey, (Blandford Press, UK, 1982), Chapter 8. Now out of print, but worth looking out for in second-hand bookshops as it gives clear explanations of many techniques and principles, especially the use of polarised light.
2) 'The Microscope Past and Present' by S. Bradbury. Pergamon Press, UK, 1968, pp. 180-183. A compact but thorough overview of the history of the microscope and it's development. Illustrates and discusses the Ross Radial and Ross / Zentmayer microscope and the fad for oblique illumination.
Text and images © David Walker.