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.
