2.2 - Magnification and calibration (2.1.1)

Question 1

What is magnification?

Answer 1

Magnification is how many times larger the image is than the actual size of the object being viewed.

Question 2

What allows the user to adjust the magnification?

Answer 2

Interchangeable objective lenses on a compound light microscope

Question 3

What is resolution?

Answer 3

• The resolution of a microscope determines the amount of detail that can be seen - the higher the resolution, the more details are visible.
• Resolution is the ability to see individual objects as separate entities.

Question 4

Describe how resolution is limited

Answer 4

Resolution is limited by the diffraction of light as it passes through samples (and lenses). Diffraction is the tendency of light waves to spread as they pass close to physical structures such as those present in the specimens being studied. The structures present in the specimens are very close to each other and the light reflected from individual structures can overlap due to diffraction. This means the structures are no longer seen as separate entities and detail is lost.

Question 5

In optical microscopy, structures that are closer than ……. cannot be seen separately (resolved).

Answer 5

half the wavelength of light

Question 6

How can resolution be increased?

Answer 6

Resolution can be increased by using beams of electrons which have a wavelength thousands of times shorter than light. Electron beams are still diffracted but the shorter wavelength means that individual beams can be much closer before they overlap. This means objects which are much smaller and closer together can be seen separately without diffraction blurring the image.

Question 7

What is the equation for magnification?

Answer 7

Magnification= size of image/actual size of image

Question 8

What is the equation to find the actual size of the object?

Answer 8

Actual size of object = size of image/magnification

Question 9

What is the equation to find out the size of the image?

Answer 9

Size of image = magnification x actual size of object

Question 10

How piece of equipment would you use to measure the size of a sample under a microscope?

Answer 10

Eyepiece graticule

Question 11

Why is an eyepiece graticule used?

Answer 11

As the true magnification of the different lenses of a microscope can vary slightly from the magnification stated so every microscope, and every lens, has to be calibrated individually using an eyepiece graticule and a slide micrometer.

Question 12

What is an eyepiece graticule?

Answer 12

An eyepiece graticule is a glass disc marked with a fine scale of 1 to 100. The scale has no units and remains unchanged whichever objective lens is in place. The relative size of the divisions, however, increases with each increase in magnification.

Question 13

What do you need to know about eyepiece graticules?

Answer 13

You need to know what the divisions represent at the different magnifications so you can measure specimens.

Question 14

The scale on the graticule at each magnification is ______________ using a ______ ____________.

Answer 14

calibrated, stage micrometer

Question 15

What is a stage micrometer?

Answer 15

A stage micrometer is a microscope slide with a very accurate scale in micrometers engraved on it.

Question 16

Using the 4x objective lens, 1 division on the stage micrometer scale = ___ ___________ on the eyepiece graticule.

Answer 16

2 divisions

Question 17

How many millimetres equal 100 micrometer divisions?

Answer 17

1

Question 18

What is the equation to calculate 1 graticule division?

Answer 18

1 graticule division = number of micrometres / number of graticule divisions

Question 19

How would you use the magnification factor and eyepiece graticule to find the actual size of the object?

Answer 19

To use the magnification factor remove the stage micrometer and place a prepared slide on the stage. Measure the size of an object in graticule units. To find the actual size multiply the number of graticule units measured by the magnification factor to give you the length in micrometers.

Question 20

What is the equation to find the measurement of the object using a graticule and the magnification factor?

Answer 20

Graticule divisions x magnification factor = measurement (micrometers)

Question 21

A student was asked to calibrate the x10 lens of a light microscope and then to determine the diameter of a pollen grain from a sample slide provided. Describe their method. (3)

Answer 21

1) The student placed a scale on the stage of the microscope and focused on it with the x10 lens. 100 small divisions of the scale are 1mm long, so 1 division is 10 micrometers.
2) The student then aligned the scale in the eyepiece with the scale on the microscope stage and calibrated it.
3) The student replaced the micrometer slide with pollen sample slides and used the calibrated scale in the eyepiece to measure the diameter of the pollen grains.

Question 22

Using the x40 objective lens, 20 divisions on the eyepiece graticule = ___ __________ on the stage micrometer

Answer 22

1 division

Question 23

Describe how you would use the x4 objective lens to measure and calculate the size of a pollen grain which is 50 micrometers

Answer 23

The diameter of the pollen grain seen using the x4 objective lens measures 10 graticule divisions. The magnification factor for the x4 lens is 5. The diameter of the pollen grain = graticule units x magnification factor = 10 x 5.0 = 50 micrometers.

Question 24

Describe how you would use the x40 objective lens to measure and calculate the size of a pollen grain which is 50 micrometers

Answer 24

1) 20 divisions on the eyepiece graticule = 1 division on the stage micrometer. Each division of the micrometre is 1/100mm = 10 micrometres.
2) Using our observations 20 graticule divisions = 1.0 micrometre division.
1.0 micrometre division = 1 x 10 micrometres = 10 micrometres.
20 graticule divisions = 10 micrometres.
1 graticule division = 10/20 = 0.5 micrometres.
The magnification factor is 0.5
3) The stage micrometer is removed and the prepared slide placed on the stage. In this example you measure the same pollen grain: Calibrate the eyepiece graticule using the x40 eyepiece lens.
4) The diameter of the pollen grain seen using the x40 objective lens measures 100 graticule divisions. The pollen grain diameter = graticule units x magnification factor = 100 x 0.5 = 50 micrometers.

Question 25

What are 4 advantages of optical microscopes?

Answer 25

• Relatively cheap
• Easy to use
• Portable and able to be used in the field as well as in laboratories
• Able to be used to study whole living specimens

Question 26

What are the 5 steps you should follow when using an optical microscope?

Answer 26

1) The specimen on a slide is placed on the stage and clipped into place.
2) By rotating the nosepiece, the lowest power (smallest) objective lens is placed over the specimen.
3) Adjust the coarse focus knob while looking into the eyepiece, until the image you see is clear and in focus.
4) Whilst viewing the image adjust the iris diaphragm for optimum light.
5) Make sure that the object you wish to view is directly over the hole in the stage. Mow rotate the nosepiece and bring the x10 objective into place over the specimen. Look down the ocular tube and use the fine focus knob to focus the image.

Question 27

A photograph of the image seen using an optical microscope is called a _______________.

Answer 27

photomicrograph