Setting up the microscope

Work in a darkened room if you can, in order to keep the intensity of the light to a minumum

Start with a low magnification objective

Place a slide preparation with a coverslip on the stage, and focus on something, such as the edge of the coverslip

Adjust (interocular) distance between the eyepieces so you can see easily through both oculars. Make sure both are focussing at the same level.

Köhler illumination

For Köhler illumination, close the iris in the lamp housing, move the condenser up and down with the appropriate knob , until the edge of the iris is in sharp focus; use centring knobs on the condenser to center the iris in the field of view, and remove an eyepiece and looking down the tube open up the iris to a point where it stops just inside the maximum field of view (this last step is required for each change in magnification).

Contrast enhancement (examples )

Köhler illumination achieves good resolution, but most usually in with protists resolution is not the biggest concern, and the emphasis tend to be on making a strong and contrasty image. The can be done in several ways.

Close the iris in the condenser (a similar effect is achieved by lowering the condenser). Considerable resolution can be lost, but the technique is simple and cheap

Phase contrast microscopy . This technique is suitable for small objects. This can only be done if you have the right kind of objectives. The condenser should be set for Köhler illumination, then all irises opened completely. There should be phase rings in the condenser, and you will need to select the ring appropriate to the objective, and then center it using (probably 2) adjustment devices. You can check on centering either by removing an eyepiece and looking down the tube - when you should also see a bright and a dark ring, the second being the phase ring in the objective - and the two will need to be aligned, or simply assess the quality of the image when moving the phase-ring alignment knobs.

Differential interference contrast optics . The condenser should be set for Köhler illumination. DIC requires four additional components in the light path. The first is a polarizer that is usually located below the condenser, the second is another polariser (referred to as an analyser) and this is located above the objective. These should be 'crossed' allowing minimal transmission to light. You can check this without the other components for Nomarksi or Phase contrast being in the light path by the darkness of the image. The third component is a prism that is located immediately above the objective, and the final element is a prism that is located in the condenser - typically selected by rotating a ring in the condenser. The prisms for the objhectives and condenser are matched to the the objective, the polariseer and analyser are the same for all magnifications. With the polarise and analyzer you can also create polarised light for imaging items that have a regular or crystalline substructure. You will find that the image quality is dependent on the setting of the

Dark ground optics. This is achieved when specimens are illuminated obliques, and no light passes directly from the condenser to the objective. This can be achieved at lower magnifications by using the higher magnification contrast rings and can be achieved crudely by misaligning the condenser.

In order to develop familiarity

Begin with abundant and diverse material. We find the mud preparations to be ideal because of the diversity of organisms that are produced

Work initially only with non-oil immersion objectives. This is because the objectives are at high risk of damage if they are pushed into the slide. Until you feel relaxed with the machine, stop with the X40 objectives

Begin with high contrast optics, i.e. phase contrast

Move to X 100 phase

Finally, extend to DIC

Take images as you develop your skills, share them with experienced colleagues, who can advise if the technique is satisfactory.