Microscopy

Question 1

Describe in principle, what a microscope does and name four different types of microscope.

Answer 1

A miscrope is an instrument which enables you to magnify an object a lot.

• compound light microscope
• laser scanning confocal microscope
• scanning electron microscope (SEM)
• transmission electron microscope (TEM)

Question 2

Give the functions of each component of a compound light microscope.

Answer 2

1) EYE PIECE: what you look through. It magnifies the object x10
2) SCANNING LENS: used in preliminary observation and magnifies x4
3) LOW POWER OBJECTIVE LENS: magnifies x10 and gives a moderate amount of detail
4) HIGH POWER OBJECTIVE LENS: magnifies x40. gives the most amount of detail and is used last
5) STAGE CLIPS: holds the glass slide in place
6) STAGE: a stand above the light source for the glass slide
7) ARM: microscope held by the arm when carried
8) CONDENSER: used to the vary the intensity of the light projected on to the object
9) COARSE KNOB: for rough focusing on low magnification
10) FINE ADJUSTMENT KNOB: for careful focusing on higher magnification
11) ILLUMINATION: projects light on to the slide
12) BASE: the support/ the base of microscope

Question 3

Outline how an SEM works.

Answer 3

A beam of electrons is fired at the surface of the specimen and the reflected electrons are collected to create a 3D image.

Question 4

Outline how a TEM works.

Answer 4

A beam of electrons is transmitted through the specimen and focused to produce an image

Question 5

What is fluorescence?

Answer 5

The absorption and the re-radiation of light. Substances can be treated with fluorescent chemicals.

Question 6

Outline how a laser scanning confocal microscrope works.

Answer 6

It moves a single spot of focused light (the illumination point) across a specimen. This causes fluorescence from the components labelled with a dye. The emitted light from the specimen is filtered through a pinhole aperture. Only light radiated from very close to the focal plane (the distance that gives the sharpest focus) is detected.

Question 7

State the features of images produced from compound light microscopes. (Identifying different micrographs)

Answer 7

• 2D images
• A wide range of colours can be seen due to staining specimens
• usually observing small organisms, parts of small organisms or whole cells
• can observe larger organelle like the nucleus, mitochondria or chloroplasts

Question 8

Know a bit about the history

Answer 8

First cells observed in 1665

Question 9

Explain how to use a light microscope to view a specimen at low and high powers.

Answer 9

When using low powers (the scanning lens and low power objective lens) use the coarse adjustment knob to bring the specimen into focus.
When using a high power, use the fine adjustment knob.

Question 10

Describe how to produce a temporary wet mount of living tissue.

Answer 10

Specimens are suspended in liquids such as water or an emmersion oil. A cover slip is placed on the specimen from an angle. Aquatic samples and other living organisms can be viewed this way.

Question 11

Describe and explain the characteristics of a good slide preparation.

Answer 11

• No air bubbles or artefacts
• Staining, if needed
• Glass transparent
• Cover slip (acts as protection)
• A thin specimen which is good quality and a good size
• Not too much liquid in sample
• Everything should be clean

Question 12

Explain why slide preparations need to be thin.

Answer 12

Transmitted light is going through it so it needs to be transparent or you will not be able to see anything. You want to look a single cell so it can be measured accurately. This cannot be done if the multiple cells overlap because the specimen is too thick.

Question 13

Explain how to use a stage micrometer to work out the distance represented by the small divisions in an eyepiece graticule under 3 different objective lenses.

Answer 13

1) Get the scale on the stage micrometer slide in clear focus.
2) Align the micrometer scale with the scale on the eye piece graticule and take a reading from both of them.
3) See what the equivalent in stage in units to eye piece units is (e.g. 12 stage = 42 eye piece units)
4) Work out what the stage units are in millimeters by dividing by 10.
5) Divide the stage units in millimeters by the number of eye pices units (e.g. 1.2 / 42)
6) Multiply the result by 1000 to get the value in micrometers.
7) This value is the calibration between the stage units and eye piece graticule under that particular magnification e.g. 1 epu = x micrometers. This is called the magnification factor

Question 14

Explain how to use a stage micrometer and eye-piece graticule to add a scale bar to a drawing. ask at school

Answer 14

1) Measure the specimen under the microscope with the eye piece graticule
2) Then mulitply the value by the magnification factor.
3) This will get the size of the specimen in micrometers
4) Draw specimen and draw a bar underneath it. Add its length in micrometers.

Question 15

Explain how to use a stage micrometer and eye-piece graticule to calculate the size of a specimen.

Answer 15

1) Measure the specimen under the microscope with the eye piece graticule
2) Then mulitply the value by the magnification factor.
3) This will get the size of the specimen in micrometers

Question 16

Describe how to choose an appropriate number of significant figures, or decimal places to present data.

Answer 16

Use the highest number of decimal places in the question for your answer.

Question 17

9. Explain how an adjustment to the “plane of focus” can alter what is viewed within a cell. (F)

Answer 17

If you change the plane of focus while looking through a microscope different parts of the specimen will come in and out of focus. This is because the plane of focus is the horizontal plane where what you are looking at is in focus, it is usually not very thick so it is hards to focus are things which are not flat. When you focus the microscope you move the plane of focus up and down. This is why different parts you are looking at go in and out of focus.

Question 18

Explain how a tissue slice might be misleading due to the very thin nature of the slice.

Answer 18

• You can only see the cross section of a cell, not its overall shape.
• There are different organelles in different parts of the cell so you might miss structures which are inside the cell.
• You may not measure the actual diameter of the cell, just the diameter of the cross section you took.

Question 19

Explain why staining is useful for light microscopy.

Answer 19

A lot of specimens are transparent and colourless and so difficult to see. Staining provides colour and contrast with the background.
You can also differential staining. This means you can distinguish between organism and different organelles.

Question 20

Describe the properties a stain needs to have to be useful for light microscopy.

Answer 20

• Needs to be able to bind to other molecules.
• In differential staining, it needs to bind to different parts of the cell.
• Must be liquid and able to get through cell membrane.
• Shouldn’t damage the cell
• Needs to have a very intense colour so it is able to colour something in very small amounts

Question 21

Describe how to prepare a stained specimen for viewing under a light microscope.

Answer 21

1) place sample on a slide and allow it to air dry
2) Then it is heat fixed by passing through a flame
3) The specimen will adhere to the microscope slide and will take up stains.

Question 22

Name some common stains and the molecules they bind to.

Answer 22

• Methylene blue used to stain DNA
• Giemsa stain is commonly used to differentiate bewteen different types of blood cells
• Eosin dyes the cytoplasm pink

Question 23

State the rules of biological drawings.

Answer 23

• Inlcude a title
• state magnification
• use a sharp pencil for drawings and labels
• use white unlined paper
• the bigger the better
• draw smooth continous lines
• do not shade
• draw clearly defined structures
• ensure proportions are correct
• labelled lines should not cross and should not have arrow heads
• Label lines should be parellel to the top of page
• include a scale

Question 24

Produce a labelled and annotated tissue plan using a light microscope.

Answer 24

You do not draw individual cells, instead it is drawn out in blocks in proportion (see book for an example).

Question 25

State the magnification formula and represent the magnification formula in triangle diagram.

Answer 25

Magnification = Size of image/ actual size of object

the image is a biological drawing

Question 26

Explain how to calculate the magnification of an image using the magnification formula.

Answer 26

1) measure image in mm
2) convert to micrometers by mulitplying by 1000
3) the divide by the actual size given to get magnification

Question 27

State the symbols used for millimetres, micrometres and nanometres.

Answer 27

Millimeters - mm
micrometers - um
nano meters - nm

Question 28

Explain how to convert measurements from one unit into another. Try writing them out.

Answer 28

mm —> um: x1000
mm —> nm: x 1,000,000

um —> mm: /1000
um —> nm: x1000

nm—> mm: /1,000,000
nm —> um: /1000

Question 29

Define the term resolution

Answer 29

The shortest distance between the two objects that are still seen as two separate objects.

Question 30

Define the term magnification.

Answer 30

How much bigger an image from a microscope is compared to the specimen.

Question 31

State the resolution and useful maximum magnification of light microscopes.

Answer 31

Resolution: 200nm
Magnification: x1500 - x2000

Question 32

State the resolution and useful maximum magnification of SEMs.

Answer 32

Resolution: 3- 10nm
Magnification: x100,000 - x500,000

Question 33

State the resolution and useful maximum magnification of TEMS.

Answer 33

Resolution: 0.2 - 0.5 nm
Magnification: up to 500,000

Question 34

State the features of images produced by laser scanning confocal microscopes.
(Comparing micrographs)

Answer 34

• light collected is from fluorescent stains so image is mostly black
• limited colour variation as it is restricted by the number of fluorescent dyes used
• image first appears 2D but a 3D image can be virtually constructed
• usually observing whole cells or stained structures with in the cells

Question 35

State the features of images produced by a TEM microscope. (Comparing micrographs)

Answer 35

• only grey scale images but ‘false colour’ can be virtually added
• always a 2D image
• very high magnification
• high resolution
• cells may appear grainy
• often observing organelles or small cell
• very thin specimen used

Question 36

State the features of images produced by an SEM.

Comparing micrographs

Answer 36

• only grey scale images though colour can be virtually added
• always a 3D image
• high resolution
• freeze fracture allows 3D images of inside the cell
• outside cell surface specimens can be seen in great detail
• usually observing organelles or surface specimens

Question 37

Draw out a table that gives the image type, resolution, colour, background and type of specimen for:

• SEM
• TEM
• Compound light
• laser scanning confocal

Answer 37

Cross reference with pre drawn table in folder

Question 38

What are the advantages of electron microscopy?

Answer 38

• Magnification of up to x500,000
• SEMs can give a 3D image
• Very high resolution possible: TEM has a resolving power of 0.2 - 0.5nm, SEM has a resolving power of 3- 10nm
• the high magnification and resolution provides crucial information about structures in and outside the cell

Question 39

What are the disadvantages of electron microscopy?

Answer 39

• expensive to buy and operate
• complex sample preparation
• creation of artefacts likely due to the complex sample preparation process
• specimen must be dead
• vacuum is required

Question 40

What are the advantages of light microscopy?

Answer 40

• inexpensive to buy and operate
• small and portable
• natural colour of sample is seen
• specimen can be alive or dead
• sample preparation is relatively simple
• vacuum is not required

Question 41

What are the disadvantages of light microscopy?

Answer 41

• Resolving power is quite low (200nm)

- The highest useful magnification is x2000