MICROSCOPY - use of microscopes in biology
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Suitable for GCSE/IGCSE/O level Biology/Science courses or equivalent
This page will answer many questions e.g.
What is a microscope? How is it constructed?
How does a microscope work? How can we measure the size of a cell?
What is the advantage of studying structures with a microscope?
What do we mean by the resolution/resolving power of a microscope?
What is the formula for magnification? How do you do magnification calculations?
What is the difference between a light microscope and an electron microscope? Which is the most powerful?
(a) MICROSCOPES - use and development
What do we use a microscope for in biology? and why is it such a useful investigative tool?
A microscope is an important instrument for studying cells e.g. the type of cell and the structure of cells.
Microscopes enable to see structures that we cannot see with the unaided naked eye.
Plant and animal cells can be studied in greater detail with a light microscope by magnifying the image.
Microscopes using the visible part of the electromagnetic spectrum (visible light) were invented in the late 16th century and the optical lens systems have been improved through the following centuries even until today. With these microscopes, by passing light through a specimen up into a lens system, you can see individual cells and smaller details such as nuclei and mitochondria in all cells, and chloroplasts in plant cells.
Changes in microscope technology have enabled us to see cells with more clarity and detail than in the past, including simple magnification calculations.
You need to be able to use the formula: magnification = length of image / real length of object
See also Introduction to plant and animal cell structure and function gcse biology revision notes
(b) The design, function and use of an optical microscope and slide preparation
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. It 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 to bring the image into focus.
3. 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.
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.
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 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.
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.
The microscope slide is now ready to be examined with the light microscope.
Using the microscope
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.
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).
(c) Drawing your observations - scale drawings
Give your drawing a title e.g. "Onion Cells" and quote the microscope magnification e.g. X50
You should record your observations as neatly and accurately as you can with a sharp pointed black pencil on white paper.
Make sure your sketch is a good size and cell walls with unbroken lines!
Don't use colouring or shading, just show the structures with neat thin lines.
Do you best to get the features in proportion, either the cells of the layer or any subcellular structures you can see like the nucleus.
Carefully label the features of your drawing e.g. the cell features such as cell wall, nucleus, cytoplasm or chloroplasts, but not their very fine detailed structure.
Make sure your straight labelling lines match the label exactly!
Sketch - diagram of onion cells as seen under a microscope.
I've just labelled the cell wall, nucleus and cytoplasm.
I've added a scale showing a length of 500 µm (500 micrometres, µ means 10-6 in standard form).
The average size of an onion cell is ~200 µm, and the average size of the onion cell nucleus is ~6 µm.
The size of each cell and its nucleus do vary a bit from cell to cell.
How to work out a scale bar for your drawing by measuring the average real size of a cell
You fix a clear plastic ruler on the microscope slide and clip both of them onto the stage.
Select a magnification of at least X100 and refocus the microscope to obtain another clear image of the cells.
Adjust the slide so that the cells line up with the scale and count the number of cells along a line of 1 mm.
1 mm ≡ 10-3 m (1/1000th of a metre).
A micrometre is 1 millionth of a metre, 1.0 x 10-6 m (in standard form).
Therefore 1 mm ≡ 10-3/10-6 = 1000 µm
Suppose the length of 5 onion cells = 1 mm, average length 0.2 mm.
1 mm ≡ 1000 µm, so the average length of one onion cell = 1000/5 = 200 µm
Once you know the average length of one cell you can use it to calculate the length of the scale bar on you diagram using the formula below. Suppose you want a 500 µm scale bar on your drawing.
scale bar length (for 500 µm) = 500 x drawing length of cell (µm) ÷ actual length of cell (µm)
e.g suppose each cell you have drawn was on average 2.0 cm long on paper.
This is 20 mm, which equals 20 x 1000 = 20,000 µm and the actual real average length is 200 µm
scale bar length = 500 x 20,000 ÷ 200 = 50,000 µm
50,000 ÷ 1000 = 50, so the scale bar on my drawing would be 50 mm or 5 cm long.
AND you can calculate the actual magnification of your drawing compared to the specimen's real size.
Other microscope drawings
e.g. on the left a typical plant cell x 200 to x 500 magnification.
Note that is a simple black and white line drawing.
See also Introduction to plant and animal cell structure and function gcse biology revision notes
(d) Examples of numerical calculations in microscopy
Calculations involving scale drawings and magnification I've dealt with above.
Note: In these exemplar calculations I've used the symbol ≡ to indicate equivalence.
You need to be able to use the prefixes centi (10-2), milli (10-3), micro (10-6) and nano (10-9) and express answers in standard form when carrying out calculations involving magnification, real size and image size using the magnification formula (below).
The reason for this is that that the real size of the objects under investigation with a microscope are very small!
The formula for magnification
e.g. solving magnification problems and the relative sizes of object e.g. a cell and its observed image in a microscope.
The formula 'triangle' for magnification is shown on the right (size = length).
When using the formula: magnification = image size ÷ object size
make sure the image size and object size are in the same length units!
Rearrangements: image size = magnification x object (specimen) size
and the real object size = image size ÷ magnification
If you know (and you should!) know the magnifying power (x...) of both the eyepiece lens and the objective lens, it is quite easy to calculate the total magnification from the formula:
Examples of calculations
Q1 A white blood cell has a diameter of 13.0 µm (13 micrometres)
Q2 A red blood cell has a diameter of 0.012 mm.
Q3 Suppose the real length of a cell is 150 µm.
Q4 The image of a red blood cell is 7.0 mm under a microscope magnification of 1000 X.
Q5 What magnification is needed to produce a 10 mm image of a cell from a specimen cell of diameter 125 µm?
Q6 If a cell has diameter of 0.000090 m what is its diameter in ...
Q7 A specimen is 60 µm wide.
aqa gcse 9-1 biology: You should be able to: understand how microscopy techniques have developed over time, explain how electron microscopy has increased understanding of sub-cellular structures, and appreciate the differences in magnification and resolution that have both increased with technological development in microscopy. Know and understand that an electron microscope has much higher magnification and resolving power than a light microscope. This means that it can be used to study cells in much finer detail. This has enabled biologists to see and understand many more sub-cellular structures. You should appreciate the differences in magnification and resolution between a light microscope and an electron microscope. Be able to explain how electron microscopy has increased understanding of subcellular structures. Be able to use prefixes centi (10-2), milli (10-3), micro (10-6) and nano (10-9) (expressing answers in standard form) and carry out calculations involving magnification, real size and image size using the formula: magnification = size of image / size of real object . With light microscopes you can see individual cells and large subcellular structures like the nucleus. With electron microscopes, using a beam of electrons instead of a beam of light, you gain a much higher resolution seeing much smaller objects e.g. the structures of mitochondria, chloroplasts, ribosomes and plasmids.
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