The complete microscope
The traditional layout of a complete ‘compound’ microscope is shown here. The objective forms a real, magnified and inverted image because the sample is further from the lens than its focus. The image is ‘real’ because it can be projected on a screen – a slide projector produces a real image in this way (and we therefore have to put slides in upside down). The eyepiece is placed quite close to this real image – too close to form a real image of it. Instead the rays which reach the eye appear to come from a magnified ‘virtual image’ (of the real image) located further away. The virtual image is not inverted relative to the real image, so in the end we always see an inverted image of the specimen.
Modern research microscopes modify this layout a bit. The problem with the simple arrangement is that the distance between objective and eyepiece must be absolutely fixed, since spherical aberration can only be corrected for one position of the image. If we want to add in components for fluorescence, polarization and so on we are in trouble. Modern objectives put the specimen at the focus of the lens, so they will form an image ‘at infinity’ – that is, the rays from any one point on the sample leave the lens parallel to each other. This won’t form an actual image, so an additional lens, the tube lens brings the rays to a focus just in front of the eyepiece, as before. The diagram below shows this layout, and indicates where each component is in the actual microscope.
The great advantage of this plan is that it doesn’t matter (within reason) what the distance is between the objective and the tube lens – the rays are parallel and so the SA correction is unaffected. There is a limit, of course, or rays from objects at the edge of the field of view will get cut off. Nevertheless the few centimetres of free space we gain are very valuable.
The other feature of a modern research microscope is that the illumination system is built in. Abbe showed that when we view an object with transmitted light diffraction at the sample, not just the objective, limits our resolution. We therefore need a condenser lens to illuminate the sample with an NA matching that of the objective. Since the illuminator has to be aligned with the condenser it makes sense to build this into the microscope as well.
Just because the condenser and illuminator are built on does not absolve the user from the need to adjust them correctly, and the next section explains how to optimise the system. Do not fall into the trap of assuming that if you are just doing fluorescence or confocal you don’t need this. You will almost always want to capture a phase or DIC image to match your fluorescence, and if you are going to use the (non-confocal) transmission detector built into most confocal microscopes the condenser and illuminator must be accurately set up or the image will be terrible.
Koehler illumination was first introduced by August Koehler in 1893 to provide optimal contrast and resolution in light microscopy that complement the numerical aperture of the objective lens. The process involves aligning and focusing the light path, and adjusting the apertures. Koehler illumination has to be performed every time objective lenses are changed. Below are a description of the microscope parts that are important for the process and a brief summary of the steps involved. For virtual Koehler illumination, please click on this link.
The light path and microscope
There are two sets of conjugate planes in Koehler Illumination. In the first set, the field diaphragm, objective front focal plane (specimen), the intermediate image plane and the retina are in conjugation with one another. In the second set, the filament, condenser focal plane, objective back focal plane and the iris of the eye are in conjugation (Fig 1). The latter is best viewed by removing the eyepice and inserting an eyepiece telescope or Bertrand lens.
The collector lens is located between the lamp and the field diaphragm. It gathers the light from the lamp, and magnifies and focuses an image of the filament at the front focal plane of the condenser (Set 2). This can be achieved by focusing the condenser using the condenser focus dial.
The field diaphragm is located in front of the condenser. It is used to adjust the illumination field reaching the specimen and should not exceed the capacity of the objective lens. Illuminating extraneous objects can cause light to scatter into the lens and cause glare. This will in turn, reduce contrast and resolution.
The condenser focuses light onto the specimen plane. This light then spreads from the specimen onto the objective lens (Set 1). The condenser also forms an image of the field diaphragm (Set 1).
The condenser diaphragm is adjusted so that the light achieves an angle that sufficiently fills the back focal plane of the objective lens (Set 2). This is important to achieve good resolution in the image. Removing the eyepiece allows you to view the back focal plane of the objective where an image of the condenser diaphragm appears. Adjusting the condenser levers focuses the image (Set 2).
The objective lens focuses the specimen image onto the intermediate focal plane (Set 1). This lens should also form an image of the filament at its back focal plane. (Set 2).
The ocular focuses the cone of light emerging from the image of the filament at the objective’s back focal plane onto the eye’s iris (Set 2).
The eye lens focuses the diverging rays of light from the ocular onto the retina where an image of the specimen is formed (Set 1). The filament, however, cannot be focused but is viewed as a field of light.
Performing koehler illumination
- Place specimen on stage and focus.
- Focus the field diaphragm by adjusting the condenser levers.
- Open the field diaphragm to the edge of the field of view and centre using the condenser centering controls.
- Adjust the condenser diaphragm until it is 2/3 open.
Figure 1. The positions of conjugate planes in light microscopy. These planes are located where the light rays crossover. Note that there are two sets of conjugate focal planes in a light microscope adjusted for Koehler illumination.