Types of Microscopy
A Compound Microscope
What is Microscopy?
Microscopy is the scientific field where microscopes are used to observe things that cannot be seen with the naked eye.
Look at your hand. It seems quite solid? Indivisible? One large structure with four fingers, a thumb and a palm. Look more closely. You may be able to see your fingerprints, or tiny hairs on the back of your hands. But no matter how closely you look it still seems to be one solid structure. What you cannot see is that your hand is actually made up of billions of cells.
Cells are absolutely tiny - there are more than two billion in your hand alone. If we scaled each tiny cell up to the size of a grain of sand, your hand would be the size of a bus; scaled up to the size of a grain of rice and that same hand would be the size of a football stadium. Much of our knowledge of cells comes from the use of microscopes. In order to investigate cells, we need our microscopes to produce images that are both large and detailed...a big blurry picture is no good to anyone!
Magnification is the number of times greater an image is than the object being observed. It is usually expressed as a multiple e.g. x100, x250. If you know the magnification of an image, and the size of the image, you can calculate the actual size of the object. For example, if you are using a microscope at x1200 magnification, and can see a cell that is 50mm wide (50,000μm)*, you simply divide the image size by the magnification to calculate the actual width (41.6μm if you are interested)
Magnification = Image Size ÷ Actual Size
Actual Size = Image Size ÷ Magnification
Image Size = Actual Size × Magnification
Magnification is actually quite easy to achieve - most light microscopes are capable of x1500 magnification. However, magnification does not increase the detail you see.
*μm = micrometers; a more useful scale of measurement in cell biology. There are 1000mm in a meter, and there are 1000 micrometers in a millimeter.
What is Resolution?
At any reasonable distance, the light from a car's headlamps will appear to be a single beam of light. You can take a photo of that light, enlarge it, and it would still only appear as a single light source.The more you enlarge the photo, the blurrier the image becomes. You may have been able to magnify the image, but without detail, the photo is useless.
Resolution is the ability to distinguish between two different points that are very close together. As the car gets closer to you, the image resolves and you can clearly see light coming from two headlamps. In any image, the higher the resolution, the greater the detail you can see.
Resolution is all about detail.
Microscope Magnification Equation
Light and Electron Microscopes
There are many different types of microscope, but they can be broken down into two main categories:
- Light Microscopes
- Electron Microscopes
Light microscopes use a series of lenses to produce an image that can be viewed directly down the eyepiece. The light passes from a bulb (or a mirror in low power microscopes) under the stage, through a condenser lens and then through the specimen. This light is then focussed through the objective lens and then through the eyepiece. The magnification you achieve with a light microscope is the sum of the eyepiece magnification and the objective lens magnification. Using an objective lens of x40 and an eyepiece lens of x10, you get a total magnification of x400.
Light microscopes can magnify up to x1500, but can only resolve objects greater than 200nm apart. This is because a beam of light cannot fit between objects closer together than 200nm. If two objects are closer together than 200nm, you see a single object down the microscope.
Electron Microscopes use an electron beam as their light source, and need to use computer software to generate an image for us - there is no objective lens to look down in this case. Electron microscopes have a resolution of 0.1nm - 2000 times better than a light microscope. This allows them to see inside cells in great detail. Fhe electron beam has a much smaller wavelength than visible light, allowing the beam to move between objects that are very close together and providing a much better resolution. Electron microscopes come in two varieties:
- Scanning Electron Microscopes 'bounce' electrons off an object creating a 3-D image of the surface in stunning detail. The maximum effective magnification is x100,000
- Transmission Electron Microscopes beam electrons through an sample. This produces a 2-D image at a maximum effective magnification of x500,000. This allows us to see the organelles inside a cell
The final image from an Electron microscope is always black, white and grey. Computer software can be used afterwards to create 'false-colour' electron micrographs, such as those shown below.
Light and Electron Microscopes
x100,000 (SEM) x500,000 (TEM)
Visible Light (bulb or mirror)
Wide range of specimens can be viewed, including living samples.
High resolution allows for superb detail of structures within cells. SEM can produce 3D images
Poor resolution means it cannot tell us much about internal cell structure
Samples must be dead as EM uses a vacuum. Preparing samples and operating the EM requires a high degree of skill and training
Methylene blue, acetic orcein (stains DNA red);Gentian Violet (stains bacterial cell walls)
Heavy metal salts (e.g. Lead chloride) are used to scatter electrons and provide contrast. SEM requires samples to be coated in heavy metals such as gold.