MICROSCOPY - use of microscopes in biology

Doc Brown's Biology Revision Notes

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.

Microscopes use a glass lens system to magnify images - with a bigger image you see more detail.

You can increase the resolution of an image by using more powerful and better quality lenses. Resolution means how good a microscope is at distinguishing between two points that are close together on an image. The higher the resolution the more clear is the image, especially when looking for fine details e.g. in a cell.

Microscopes enable you to see objects (like microorganisms) which you cannot see with the naked eye.

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.

Light microscopes using visible light and lenses to form a magnified image of the object under investigation e.g. cells of plant or animal tissue. With a light microscope you can see individual cells and large subcellular structures like the nucleus, but not internal cell structures such as ribosomes or plasmids. The best light microscopes can give a magnification of 2000 times of a specimen's length.

Changes in microscope technology have enabled us to see cells with more clarity and detail than in the past, including simple magnification calculations.

Over time the design and usefulness of microscopes has improved, particularly using new technology in the 20th century and into the 21st century.

In the 20th century, with advances in atomic physics, the electron microscope was invented in the 1930s which uses beams of electrons instead of visible light photons. Electron microscopes have a much greater magnifying power and resolving power than optical light microscopes.

So, using an electron microscope, using electrons instead of light photons, you can form images of very small subcellular structures such as ribosomes, plasmids and the internal structure of mitochondria and chloroplasts, because they have a much higher resolving power.

Electron microscope images have a higher resolution - a higher resolving power increases the distinction between points on an image, i.e. you get a much sharper image of the fine detail of cell structure.

Electron microscopes can produce magnified images of up to ten million (109) times the real length of the specimen under investigation.

So electron microscopes are superior to optical light microscopes in terms of both magnification and resolution.

This has enabled the magnification produced by a microscope to be considerably increased to the point where you can see even smaller structures such as the internal detailed structure of mitochondria, chloroplasts and plasmids (hoops of DNA) so as to give a better understand of their structure and how their role in cell behaviour - in other words a powerful tool for better understanding how a cell works and the function of sub-cellular structures.

You need to be able to use the formula: magnification = length of image / real length of object

and with a variety of units e.g. micro, nano etc. as well as expressing small numbers in standard form!

 

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.

1. The eyepiece, through which you look, is at the top of the tube that can move up and down when focussing the microscope.

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.

3. The prepared microscope slide (see below) is 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.

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.

 

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.

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

With a pipette you place a drop of pure water onto the middle of a clean slide which helps keep the specimen in place.

For example: Cut up an onion to separate it into layers and with tweezers peel off a 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.

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

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

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).

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.

 

(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 m (500 micrometres, means 10-6 in standard form).

The average size of an onion cell is ~200 m, and the average size of the onion cell nucleus is ~6 m.

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

Note: In calculations I've used the symbol to indicate equivalence.

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 m

Suppose the length of 5 onion cells = 1 mm, average length 0.2 mm.

1 mm 1000 m, so the average length of one onion cell = 1000/5 = 200 m

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 m scale bar on your drawing.

scale bar length (for 500 m) = 500 x drawing length of cell (m) actual length of cell (m)

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 m and the actual real average length is 200 m

scale bar length = 500 x 20,000 200 = 50,000 m

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.

The magnification of your drawing = average length of drawn cell (m) average real length of cell (m)

The drawn cell is 20 mm long 20 x 1000 = 20,000 m, real length of cell is 200 m

magnification of drawing = 20,000 200 = 100X

 

Other microscope drawings

e.g. on the left a typical plant cell

 

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:

total magnification = eyepiece lens magnifying power x objective lens magnifying power

e.g. if the eyepiece lens power is x 5 and the objective lens power is x 50, the total magnification is 5 x 50 = 250 x

 

Examples of calculations

Q1 A white blood cell has a diameter of 13.0 m

Express the diameter of the white blood cell in metres, centimetres, millimetres and nanometres, in standard form.

Using equivalents e.g.: 1 m 10-6 m, 1 nm 1.0 x 10-9 m, 1 mm 1.0 x 10-3 m, 1 cm 1.0 x 10-2 m,

1 m 1.0 x 106 m, 1 m 1.0 x 109 nm, 1 mm 1.0 x 103 m, 1 cm 1.0 x 104 m, 1 m 1.0 x 106 m (etc.!)

(use the above for scale conversions, you should know them all)

13.0 m 13 x 1.0 x 10-6 m = 1.3 x 10-5 m  (you get the same answer from 13/106)

1 cm 10 mm 10 x 1 x 103 = 1.0 x 104 m, so diameter = 13 104 = 1.3 x 10-3 cm

1 mm 1000 m, so 13 m 13 1000 = 0.013 mm = 1.3 x 10-2 mm

1 m 1000 nm, so 13.0 m 13  x 1000 = 13,000 = 1.3 x 104 nm

Just try working them out in your own way and see if you agree with my answers

 

Q2 A red blood cell has a diameter of 0.012 mm.

Give the red blood cell diameter in metres, millimetres, micrometres and nanometres all written in standard form.

0.012 1000 = 1.2 x 10-5 m  (1 m  ≡ 1000 mm)

0.012 mm = 1.2 x 10-2 mm  (just changed to standard form)

0.012 mm 0.012 x 1000 = 1.2 x 101 m  (1 mm  ≡ 1000 m)

0.012 mm 0.012 1000 = 1.2 x 10-5 m, 1.2 x 10-5 10-9 = 1.2 x 104 nm  (1 m  ≡ 1000 m, 1 m  ≡ 109 nm)

Again, just try working them out in your own way and see if you agree with my answers

 

Q3 Suppose the real length of a cell is 150 m.

What is the size of the cell image with a microscope magnification of X 50? (give you answer in m and mm)

image size = magnification x object size, 1 mm 1000 m

image size = 50 x 150 = 7500 m and 7500 1000 = 7.5 mm

 

Q4 The image of a red blood cell is 7.0 mm under a microscope magnification of 1000 X.

What is the real size of the red blood cell? (give your answer in micrometres and nanometres)

1 mm 1000 m, so 7.0 mm 7.0 x 1000 = 7000 m.

object size = image size magnification

real object size = 7000 1000 = 7.0 m

1 m 1 x 10-6 m, 1 nm 1 x 10-9 m, so 1 m 10-6 10-9 = 1000 nm

Therefore cell diameter = 7.0 x 1000 = 7.0 x 103 nm

 

Q5 What magnification is needed to produce a 10 mm image of a cell from a specimen cell of diameter 125 m.

magnification = image size actual size

1 mm 1000 m, so 10 mm ≡ 10,000 m

magnification needed = 10,000 125 = 80 X

 

Q6 If a cell has diameter of 0.000090 m what is its diameter in ...

(a) standard form

move the decimal place 5 place to the right and multiply the answer by x 10-5 giving 9.0 x 10-5 m

(b) micrometres (m)

1 m = 10-6 m, so move the decimal point forward 6 places to give 90 m

 

Q7 ?


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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