Light (or optical) microscopy is an important tool used by biologists. It enables them to study specimens that are too small to see with the naked eye. Light (natural or artificial) is transmitted through, or reflected from, the specimen and then passed through a system of lenses that produce a magnified image.
The type of microscopy most commonly used by students is called bright field. Light (from an inbuilt source) is aimed at a lens system called a substage condenser. The condenser focuses the light onto an area the same size as the objective lens. The condenser has an iris diaphragm that is used to control the diameter of the beam of light passing through the condenser. The light then passes through the specimen and into the objective lens followed by the eyepiece lens to produce a magnified image. The objective lens is the one closest to the specimen. Most of the microscopes used at school and college have four objective lenses (usually with magnifications of x4, x10, x40 and x100).
Looking at the image
What can you see?
It doesn’t matter how big your image appears, it won’t be particularly useful if it is blurred, too dark or dazzlingly bright. Here are a few handy hints.
- Adjust the iris diaphragm to achieve the optimum balance between definition and glare. If the diaphragm is open, the image is brighter but the contrast is low. If the diaphragm is closed, the image is darker but the contrast greater. Make sure you re-adjust the iris diaphragm for each objective lens. Do not try to control light intensity by moving the substage condenser, the position of which should be to focus light on the specimen.
- Use the low power objective lens for focusing on and searching a large field of view BEFORE turning to the much smaller field of view of a higher objective lens (e.g. x40). Then, without altering the focus, turn to the high power lens and then gently re-focus with the fine focus.
How much bigger does it look?
The magnification is a measure of how much bigger an image appears than the actual size of the specimen.
To calculate the overall magnification use this simple equation:
Total magnification = magnification of objective lens x magnification of eyepiece lens
If the objective lens has a magnification of x40 and the eyepiece lens x10, the overall magnification would be x400.
But size isn’t everything – the level of detail that you can see is called the resolution. This is the ability to distinguish between two points.
Measuring the size of the specimen
It is possible to measure the actual size of your specimen. You will need a mini ruler called an eyepiece graticule that is placed in the eyepiece. This is then calibrated using a special microscope slide called a stage micrometer. An excellent guide to calibrating your microscope is given in the book Maths for Advanced Biology by Alan Cadogan and Malcolm Ingram. If you are given an image in an exam question, you can calculate the actual size of a specimen using this equation:
Actual size = observed size / magnification
Magnification = observed size /actual size
- Make sure you keep the units for size the same.
- Magnification has no units.