Since Antonie van Leeuwenhoek first saw mysterious ‘animalcules’ (bacteria) through his simple glass lens in the late 1600s, scientists have wanted to understand more about the strange and wonderful things they were discovering by using microscopes. Being able to look more closely (that is, at higher magnification and resolution) has always been a major goal, but scientists also have other things on their wish lists. Some want to look at a surface of an object, while others want to see its inner workings; some want to see processes happening in real time in living things; for some, being able to label specific molecules in a sample is important.

Over time, specialised light microscopes have been developed such as the confocal laser scanning fluorescence microscope and the polarised light microscope. Specialised microscopes can provide different kinds of information about a microscope sample so that scientists can choose the microscope that is most likely to answer their questions about their sample.

What is light microscopy?

Light microscopes (also known as optical microscopes) are the original microscopes. Light microscopes are the ones you’re most likely to find in the classroom or school science lab. They use visible (white) light to illuminate (light up) the object being looked at and focus the light using one or more glass lenses. Two kinds of light microscope are common in the classroom: the stereomicroscope (which looks at the surface of a sample) and the compound microscope (which looks at a thin cross-section).

The maximum resolution (and therefore magnification) of light microscopes is quite limited compared to electron microscopes – at best, they can magnify up to approximately 2000 times. The advantage of light microscopes (and stereomicroscopes in particular) is that objects can be looked at with little or no preparation. This makes them very useful for looking at living things, such as flower parts, insects, earthworms and human skin.

Preparing biological samples

Biological samples for the light microscope (particularly compound microscopes) often need to be ‘stained’ (coloured) in some way to make it easier for users to understand what they’re seeing. This is important because these samples often lack contrast, which makes it hard to distinguish between parts of the sample.

Stains interact with a specific part of the sample, turning it a different colour from its surroundings. For example, iodine is often used to stain plant cells because it colours the starch stored within the cells a blue colour and other structures a pale brown. Other common stains include H&E (haematoxylin and eosin), which stains the cell nucleus purple and other tissue components pink. In fluorescence microscopy, fluorescent stains are used to highlight specific parts of the cell or tissue.

Stains can be used on living or non-living biological material. Some stains require the sample to be treated beforehand, and in this case, the tissue is no longer living. Other stains are used on living tissue, which is important for observing biological processes under the microscope.

Specialised forms of light microscopy

Specialised types of light microscope have been developed to help investigate different aspects of a sample.

Confocal laser scanning fluorescence microscopy (or ‘confocal’ for short) makes it possible to look at different cross-sections of a thick sample without actually cutting the sample up. This is especially useful for looking at biological processes in living tissue, because it can continue to function throughout the microscope experiment. The movement of mitochondria around cells, mitosis and the change in length of primary cilia are all processes that are well suited to being viewed on the confocal.

Confocal is also good for identifying specific components (such as organelles or proteins) in the cell or tissue being studied. The components are labelled with fluorescent tags, then laser light is shone at the sample. The fluorescent molecules glow brightly (and everything else stays dark), so they’re easy to see.

 

Polarised light microscopy helps us to learn about minerals (such as those in rocks) in a sample. Different minerals show up as different colours under polarised light (light waves that vibrate in only one direction), so polarising microscopes use special filters to polarise the light that shines on the sample. Looking at minerals through a polarising microscope can provide information about their shape, size and orientation.

Useful links

Learn more about polarised light microscopy.
www.microscopyu.com/articles/polarized/polarizedintro.html 

    Published 29 February 2012