- Living things are composed of Cells. Cells are very small (ususally between 1 and 100 μm) and can only be seen by magnification with a microscope. A distinction is made between Magnification and Resolution: Magnification is how large the image is compared to real life, whereas Resolution is the amount of information that can be seen in the image - defined as the smallest distance below which two discrete objects will be seen as one.
- To work out the size of an object viewed with a microscope, a Graticule is used. It is a small transparent ruler that becomes superimposed over the image. As the same sample may look to be different sizes under different magnifications, the Graticule must be calibrated.
- Actual Size, Image Size and Magnification are related by the formula:
Image Size = Actual Size × Magnification
The Light Microscope
- Light Microscopes, or Optical Microscopes, as they are more correctly termed, use light and several lenses in order to magnify a sample. Light from the Condenser Lens, and then through the Specimen where certain wavelengths are filtered to produce an image. The light then passes through the Objective Lens, which focuses it and can be changed in order to alter the magnification. Finally, the light passes through the Eyepiece Lens, which can also be changed to alter the magnification, and into the eye.
- The maximum magnification of light microscopes is usually ×1500, and their maximum resolution is 200nm, due to the wavelength of light. An advantage of the light microscope is that it can be used to view a variety of samples, including whole living organisms or sections of larger plants and animals. It is also relatively inexpensive.
- There are two types of light microscope. Compound Microscopes contain several lenses and magnify a sample several hundred times. Dissecting Microscopes on the other hand have a low final magnification but are useful when a large working distance between the objectives and the stage is required (e.g. during dissection). They have two eyepieces to produce a 3D stereoscopic view.
- Many specimens require preperation before being viewed by a light microscope, as some may not be coloured or might distort when cut. Samples are Stained with coloured stains that bind to certain chemicals or cell structures. For example, Acetic Orcein stains DNA dark red. Samples may also be Sectioned - embedded in wax; this helps with preserving structure while cutting.
The Electron Microscope
- Light microscopes are great and all, but sometimes their (relatively) low magnification and resolution are insatisfactory for viewing very small things, like Organelles within cells. In these circumstances, and Electron Microscope may be used. Electorns have a much lower wavelength than light (100000 times shorter in fact, at 0.004nm) which means that they can be used to produce an image with resolution as great as 0.1nm. Electron Microscopes can have magnifications of ×500000.
- There are different types of Electron Microscope. A Transmission Electron Microscope (TEM) produces a 2D image of a thin sample, and has a maximum resolution of ×500000.
- A Scanning Electron Microscope (SEM) produces a 3D image of a sample by 'bouncing' electons off and dectecting them at multiple detectors. It has a maximum magnification of about ×100000.
- The preparation of a sample for electron microscopy is a complex process. It may involve
- Chemical Fixation: Stabilising an organism/sample's mobile macrostructure
- Cryofixation: Freezing the sample very rapidly to preserve its state
- Dehydration: Removing the water form a specimen, for example, by replacing it with ethanol
- Embedding: Embedding in resin, ready to be sectioned
- Sectioning: Cutting the sample into thin strips that are semitransparent to electrons, for example with a diamond knife
- Staining: Using heavy metals to scatter electrons and produce contrast
- Freeze Fracturing: Freezing the sample rapidly, and then fracturing it, for example, when viewing cell membranes
- Mounting: Placing the sample on a copper grid
- It is advantageous to use an Electron Microscope in many situations because they offer a much higher resolution that Light Microscopes, so they can be used to image very small objects in detail, and also because of the 3D images that SEMs offer. However, samples must be placed in a vacuum as electrons are deflected by particles in the air, they are very expensive to buy and maintain, and preparing the samples requires a lot of skill to do.