Using Your Microscope
Adjusting the Microscope
Written by Frank Prince-Iles
FishDoc
Part 2

Scheme for setting up a simple monocular microscope

1. make sure the 10x eyepiece is in place at the top of the draw tube
2. raise the body tube a few inches above the stage - by looking from the side and turning the course focus knob
3. rotate the nosepiece and click the lowest power objective into place above the stage (usually a 10x)
4. adjust the illumination if using a mirror, turning the flat side of the mirror towards the light source so that light is reflected up towards the condenser
5. rack the condenser up to within 2mm below the stage and adjust the iris diaphragm until it is half open
6. place the specimen on the stage making sure that the cover glass is uppermost and secure it with either the stage clips or the mechanical stage arms
7. adjust the angle of the mirror so that a spot of light appears on the slide directly below the objective lens
8. looking from the side and using the course control knob, lower the objective until it is just above the slide
9. look through the eyepiece. Adjust the mirror to give an even amount of illumination
10. use the course control knob to slowly rack the objective upwards and look through the eyepiece until the specimen is in focus. (Tip) it is sometimes easier to focus on the edge of the cover slip to start with as this gives a nice clean edge when in focus - whereas mucus can sometimes be difficult "to find"
11. use the fine focus to obtain the sharpest possible image
12. if the light is too bright either use a bulb with a lower wattage (if using a table lamp to illuminate the mirror) or adjust the iris diaphragm to reduce glare
13. focus the light source onto the slide by slowly racking down the condenser - watch that this does not affect the mirror angle. Adjust the condenser and iris diaphragm to give optimum illumination. Ideally, once the condenser is set in the optimum position, there shouldn't be any need to keep altering it.

While this long list may seem daunting, it is because I have tried to cover every step. You will also note that much of it revolves around optimizing the light source if it is mirror based. With a fixed light source many of these steps can be ignored. After you have set up the microscope a few times it should become second nature.

General microscopy

For general microscope work, a magnification between 40X and 400X is usually sufficient and will allow for relatively easy identification of most common ectoparasites.

Higher magnification

When talking about higher magnification it is important to understand the difference between simply magnifying the size and increasing the amount of visible detail. With high-power microscopy the most important consideration is the image resolution, which means being able to distinguish two objects that are close together as being separate, distinct entities.

For technical reasons there is a limit to the amount of resolution that can be obtained with an ordinary light microscope. Usually, beyond 1000X magnification there is no significant increase in resolution, and any extra magnification merely gives an increase in size without any extra detail.

As you might guess, increasing resolution comes at a price - and means investing in more expensive components such as oil-immersion objectives and condensers. For example, a fairly good oil-immersion objective lens can cost over £300! It is possible to increase resolution beyond 1000x, but the type of equipment required is prohibitively expensive.

The reasons for using oil-immersion objectives are technical - but basically the oil reduces light being reflected back from the slide as well as bending the light rays inwards to focus on the specimen - giving more light, less interference and therefore better detail

Using an oil-immersion objective

Focus on the specimen using a dry objective lens (40x), making sure that the specimen is central in the field of view.

Move the dry objective to one side and place a drop of oil on top of the specimen. Swing the oil-immersion lens into place, ensuring that it's front lens makes contact with the oil drop. If it doesn't, slowly lower the objective while watching from the side until it just touches the drop

If you are using modern parfocal lens, which means that all objectives are approximately in focus at the same setting, the specimen should already be roughly in position. Look down the microscope and make any necessary fine adjustments to the focus, iris diaphragm and condenser to obtain the best image. Be very careful not to lower the objective onto the slide as the working distance with high magnification lens is very narrow.

After use it is important to clean the oil from the lens using a soft tissue. It should be cleaned using a wiping movement, rather that a circular, scrubbing movement as this might scratch the lens. Any dried on oil can be removed using xylene, but be careful as this could dissolve the cement securing the lens!

So what can you see at 1000x mag?

You will see the internal structures of algae and parasites - if you can get them to stay still! However, at this magnification we would normally be looking at bacteria and other micro-organisms. In honesty, you may be initially disappointed with what you actually see. Most micro-organisms are transparent and even at this magnification are difficult to see with an ordinary bright-field microscope. With special stains they are visible, but still fairly small and insignificant (see photos).

However, as understanding develops it is possible to determine significant information and interest from high-power microscopy. Special staining techniques such as Gram and acid-fast staining enable us to determine the basic group of bacteria - for example most bacterial fish pathogens are Gram-negative and stain red with Gram staining.

It is also possible to determine approximate size and thereby differentiate between long Flexibacter. sp and the smaller ulcer-related bacteria such as Aeromonas sp. The shape of the bacterium can also be seen as either straight rods, bent rods or round (cocci).

While conclusive identification can only be determined by culturing the bugs and subjecting them to biochemical testing, high powered microscopy enables us to go part way to discovery this normally hidden, fascinating world

Setting up

The most common cause of disappointing results is poor setting up of the microscope before use. The performance of almost all microscopes can be improved if a little time is spent focusing before use. No matter what quality microscope you buy, it makes sense to get the best possible image it is capable of.

Neglected

An often neglected part of the microscope is the substage condenser, found on all but the most basic of instruments. The condenser focuses and concentrates the light uniformly onto the specimen. Most importantly, because it controls the size of the cone of light illuminating the stage. It also controls resolution - i.e. how sharp or fuzzy the image is.

Critical focusing

Ideal illumination is obtained by critical focusing which ensures that the specimen and light source are properly centred and focused, with just the right amount of light to give a clear, uniformly bright image.

Setting up for critical focusing

Put a prepared slide on the stage and bring it into focus with the 10x or 20x objective

Next focus the condenser. How this is done depends on whether light source has a field diaphragm such as found with Köhler-type illumination. Köhler lighting systems have an iris or field diaphragm which controls the aperture of light going into the condenser. Although this form of illumination is gaining it popularity, it is would not normally be found on the average hobbyist microscope.

Focusing the condenser on a microscope without an iris diaphragm is carried out by removing the slide from the stage and placing a piece of thin card half-way across the light source aperture. Adjust the condenser - not the stage or objective - by racking it up or down until the card is in sharp focus when viewed through the eyepiece lens. This usually occurs just as the light interference halo turns from blue to red.

At this point the condenser is properly focused and should not need to be adjusted again. With Köhler lighting systems focusing is carried out in exactly the same way - only focusing on the leaves of the diaphragm rather than a piece of card.

Centering the condenser

Next, it is important that the condenser is centred or it will focus the light at some point to the side of the specimen. To centre the condenser close down the iris diaphragm and remove the eyepiece. Looking down the viewing tube you will see a small point of light (see diagrams). Make sure that the light appears in the centre of the tube by adjusting the condenser positioning screws. This takes a bit of fiddling about - but is simple once you are used to it.

The iris diaphragm

The final step is to adjust the substage or iris diaphragm. This is done with a lever or screw found on the condenser which works in a similar fashion to that of the iris of a camera. As the iris is opened it allows more light into the condenser. The iris diaphragm is often used incorrectly to control light intensity. While this might seem logical, its proper use is to control the size of the cone of light entering the objective lens. The correct iris diaphragm setting varies with each objective. Consequently it needs to be re-adjusted every time you change magnifications

To set up the iris diaphragm look down the viewing tube without the eyepiece in place and slowly open up the iris diaphragm until the circle of light just about fills the viewing tube. Finally replace the eyepiece, re-focus and adjust the light intensity if your microscope has rheostat control.

Provided that your lens and slide are clean you should now get the best possible image.

Read further in Part 3