Setting up the light microscope

• Does it matter?
• Introduction
• Step-by-step instructions for achieving Köhler ilumination
• Critical illumination
• What if the image is still bad after I've followed all the rules?

Introduction

Experience shows that most casual users of the microscope, and also many experienced researchers, do not use their microscopes optimally. The usual problem is that clarity (resolution) is sacrificed in the interests of contrast, usually through faulty positioning of the condenser and/or inappropriate setting of the condenser diaphragm (closing it down too far). Setting up the light microscope is in fact very easy, except in some of the more basic instruments lacking a field diaphragm (an iris diaphragm placed near the lamp or near the collector lens of the illumination system). The appropriate illumination system for digital or traditional photomicroscopy is the Köhler system. This is described in detail on a separate page and but instructions on how to achieve it practically are given below.

Step-by-step to Köhler illumination

• 1. Place microscope slide preparation on the stage, choose an appropriate objective (low power first), select a specimen and focus upon it.
• 2. Identify the field diaphragm (It's not the one on the condenser; it'll be somewhere at the base of the microscope). Close it down almost fully.
• 3. Focus the field diaphragm using the condenser. You should see an area of light bounded by the sharply focused image of the field diaphragm (which is an iris diaphragm and will therefore have a polygonal outline, because the iris is formed by numerous thin strips of metal). Often, the image of the diaphragm will be surrounded by a slightly bluish halo.
• 4. Centre the image of the field diaphragm using the condenser centring screws.
• 5. Open up the field diaphragm until the light just fills the field of view.
  • If you are using a low magnification objective (e.g. ×10) and you cannot fill the field of view by opening up the field diaphragm, then you may find that your microscope condenser has an auxiliary lens that needs to be inserted or swung in at low magnifications, to spread the light from the condenser. If there isn't then you can either (1) live with it (at ×10, you're not going to see much of the diatoms anyway), or (2) put in a diffusing screen, e.g. ground glass to spread the light.
• 6. Open the condenser diaphragm fully. Then, while looking at the specimen, close the diaphragm slowly. At first, there will be almost or no detectable difference in the image as you close the diaphragm. Later, there will be a marked decrease in light intensity and at the same time a marked increase in contrast. Put the diaphragm at the threshold between these two regions, where the light just begins to dim and the contrast begins to increase. It is tempting to close down the diaphragm further. If you're using the microscope for observation only, then slightly suboptimal Köhler illumination can be tolerated: it's easier on the eyes. But it's best to try to get used to working with relatively low contrast image and for photomicroscopy, you must return the microscope to the proper settings (see Does it matter?). Indeed, for digital photomicroscopy, you could leave the condenser diaphragm wide open and it wouldn't really matter, because the low contrast produced by extra glare (superfluous light) can be removed by subsequent digital enhancement.
• 7. The microscope should now be properly and evenly illuminated. If it isn't, check you haven't forgotten something, then think about getting your microscope serviced, because the lenses may be out of alignment.
• 8. When you change to another objective, to increase or decrease magnification, you must repeat steps 2-6. You should find that the condenser position (focus) does not need much alteration. If it does, then check whether you have taken out or put in any auxiliary lens your microscope needs at low magnification.
• 9. Never use an oil immersion objective without oil. Never use a dry objective with oil. High resolution dry objectives (×25 or ×40) often have very short working distances, so that if a slide still has oil on it from previous use of an oil immersion lens, it is all too easy to transfer oil where it should not be. Wipe off the oil from the slide, if you need to go back to a low-power lens after using oil immersion obectives, or go straight back to ×10, where the presence of oil doesn't make an appreciable difference.

Nikon have produced an excellent on-line tutorial to demonstrate the procedures outlined above.

Critical illumination

A second system ('source focus' or 'critical' illumination) exists, in which the light source is focused into the same plane as the specimen, so that images of the specimen and light source are coincident (in conjugate planes) at the primary image plane viewed by the eyepiece, and on the retina of the observer. 'Critical illumination' can be useful in special circumstances (e.g. for detecting extremely faint striations, with enhancement of contrast by oblique illumination), but is suboptimal for photomicroscopy, because any unevenness in the light source (e.g. the lamp filament) is faithfully reproduced in the final image.

What if the image is still bad after I've followed all the rules?

Besides faults in how the microscope has been set up, poor images may be caused in several other ways, including:

• dirt on the specimen - clean it!
• debris on the surface of your eye or within the eye: seek advice from an optician.
• dirt (or oil) on the microscope lenses or on any graticule placed in the optical path.
  • in particular, check the front surface of the objective lens and, if necessary, clean it carefully with lens tissue (not standard medical wipes or toiletry tissues, which often have abrasive additives); wetting the tissue with ethanol may help, but then wipe also with dry tissue.
  • rotate the eyepieces: if the dirt rotates, you'll know where it is
  • get the microscope serviced!
• scratched lenses - you'll have to live with them or buy new ones!
• non-aligned lenses, producing oblique or uneven illumination: check that the condenser is inserted correctly and centres and/or get the microscope serviced
• (with oil immersion lenses) bubbles in the oil. This is not infrequent. Bubbles can sometimes be displaced by rotating the objective to one side and back. otherwise, wipe off the oil and replace.
• intrinsically low contrast between object and its surroundings - live with it, or use contrast enhancement methods (such as differential interference contrast, phase contrast, dark-field illumination), or stain the specimen, or use mountants that differ significantly in refractive index from the specimen.
• a poor quality microscope: this may be the problem, but exclude all other possibilities before you believe it. Manufacture of good microscope lenses has been routine for a century. That said, the more you pay, the better the lenses are likely to be, with better correction of well-known faults, such as chromatic aberration (failure to focus different wavelengths of light into the same plane), spherical aberration (differences in focal length across the objective lens, from centre to periphery), astigmatism (different focal lengths for different light paths, so that a point object is imaged as a line or ellipse).