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Microscopy: 2. The design, function and use of an optical light microscope and slide preparation to examine biological materials

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There are various sections to work through.

Sub-index of biology notes: investigations using microscopes

(2) The design, function and use of an optical light microscope and slide preparation

Although scientists use the more powerful electron microscopes in research projects, many day-to-day investigations are performed using light microscopes. Examples include forensic science laboratories (e.g. evidence samples), hospital laboratories (e.g. examining the state/count of blood cells or looking for cancer cells by a pathologist).

The basic design of a light microscope

The basic design of an optical light microscope is shown in the left diagram. The back arch acts as a handle to carry the microscope around.

1. The eyepiece, through which you look at the image, is at the top of the tube that can move up and down when focussing the microscope. The eyepiece lens also magnifies the image produced by the objective lens.

2. There are two knobs for focussing - the course focussing knob is turned to get the image 'roughly' in focus and 'perfect' focus is enabled by turning the fine adjustment knob - moves in smaller increments on turning. The focussing knobs either move the stage or the eyepiece tube up or down to bring the image into focus.

3. On the stage, the prepared microscope slide (see below) is supported and held in place on the stage by clips - the slide needs to static to get good focus!

4. The lower end of the tube is connected to (usually) several objective lenses with different magnifications e.g. they might be marked X5, X10 or X20 etc. When at least two lenses are used it is referred to as a compound microscope.

5. The iris diaphragm under the stage controls the light intensity.

6. A mirror under the stage and iris directs a beam of light upwards through the stage, iris, slide and lens and up the tube to the eyepiece where you observe the image. You need a good bright light source to see any magnified image clearly.

Microscope slide preparation

The biological specimen you want to examine must be transparent, so you need a thin slice of it to let the light through.

To examine a specimen like plant or animal cells under a microscope you need to prepare microscope slide.

Live cells can be mounted in a drop of water or dilute sodium chloride salt solution (NaCl(aq), saline).

Since most cells appear colourless you can apply a stain (dye) to give colour and enhance contrast.

You might use methylene blue to stain animal cells or iodine to stain plant cells (starch turns dark blue).

A microscope slide is a rectangular thin strip of glass or clear hard plastic onto which you mount the specimen.

You need a very thin slice of the material under examination, if it is too thick, the image is too complicated and likely to be blurred - so you may have thin out a thick specimen.

The specimen to be examined must be thin and transparent to visible light.

The most common specimen for first time use of a microscope are onion cells.

With a pipette you place a drop of pure water (or a special clear liquid called a mountant) onto the middle of a clean slide which helps keep the specimen in place.

Cut up an onion to separate it into layers and with tweezers peel off a thin strip of epidermal tissue and place it in the water on the slide.

Add a drop of dilute iodine solution to the water and onion cells.

The iodine solutions acts as a stain to highlight particular features of the specimen - the thin layers of onion cells.

If all of the sample is colourless you might not see much detail.

Different stains can be used to highlight different structures e.g.

Eosin dye is used to stain cytoplasm and methylene blue dye stains DNA.

Then place a cover slip over the specimen. A cover slip is a very thin square of glass or hard transparent plastic.

The cover slip should be placed on with great care and lowered into position so that no air bubbles are trapped underneath.

You can use a mounted needle to hold the cover slip up at an angle and slowly and carefully lower it onto the slide.

Press down gently making sure no air bubbles are trapped under the cover slip.

Air bubbles will either obscure or distort the image you are attempting to observe.

The microscope slide is now ready to be examined with the light microscope.

Using the microscope - how to use a microscope prior to section (3)

Carefully clip the prepared slide onto the stage and make sure the specimen and cover slide are directly underneath the objective lens.

Initially use the less powerful objective lens to focus on the specimen, but first use the course adjustment knob to raise the stage to just below the objective lens.

It might be difficult to locate individual cells if you start off with too high a power lens, but a low power lens gives you an overall picture of the layout of the cells e.g. in human or plant tissue. It can also be easier to a cell count.

The circular area you see through the lens of a microscope is called the field of view.

With a very high powered lens the field of view is very small, almost at the level of few individual cells.

With a low power lens the field of view is much wider e.g. multiple cells of a thin slice of tissue.

Once you are focussed and made the appropriate observations and sketches, you can then increase the lens power to look for more detail.

Adjust the mirror and light source so the image seems bright, though not necessarily in focus yet.

Looking down the eyepiece, use the course adjustment knob to move the stage downwards until the image is roughly in focus.

Then use the fine adjustment knob to fully focus on the object and get a clear sharp image.

You then have the option of swapping to a higher powered objective lens to produce a more magnified image.

For scale measurements you can position a transparent ruler to measure the diameter of your field of view (FOV).

If you change to a more powerful objective lens for greater magnification you will reduce the FOV.

e.g. if you FOV was 10 mm and you swap to a lens that is five times more powerful your FOV is reduced from 10 mm to 10 5 = 2 mm.

Keywords, phrases and learning objectives for this part on the structure, function and use of a microscope including slide preparation for investigating the structure of biological materials

Know the design and function of parts of an optical microscope.

Know how to use a microscope, making up a side ready for study,

Be able to describe how to use an optical light microscope and how to prepare slides for the examination and investigation of biological materials



INDEX of biology notes on microscope investigations


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