Microscopy

Question 1

What is resolution?

Answer 1

Shortest distance between two points on a specimen that can still be distinguished clearly. Higher the resolution the greater the detail you can see.

Question 2

What are microscopes used for?

Answer 2

To visualise or photograph specimens

Question 3

What are the characteristics of a light microscope?

Answer 3

• max magnification is 1500x
• resolution is 200nm ( objects closer than that will blur together and be seen as one object
• wide range of specimens can be viewed in colour (including living organisms eg daphnia and euglena)
• can view thin sections of larger plants and animals
• cannot be used for ultra structure
• Can be used for lab analysis of tissues, research and education

Question 4

What is ultrastructure?

Answer 4

Small components in the internal structure that cannot be viewed by a light microscope

Question 5

Why will objects closer than 200nm blur together when using a light microscope?

Answer 5

The wavelengths of visible light are from 400-750nm.
As light rays of these wavelengths pass through the specimen and the microscope lenses, they are diffracted and will overlap each other if they come from pointing the specimen that are closer than 200nm

Question 6

What are the 2 stages of preparing specimens for light microscopes?

Answer 6

1. Sectioning - specimens are embedded in wax and then thin sections are cut without distorting the structure of the specimen
2. Staining - light passes through the specimen and many structures in the cell are transparent so cannot be seen. Coloured stains are chemicals that bind to chemicals in or on the specimen. Some structures take up the stain more than others do, this allows you to see contrast between components

Question 7

What are the 2 types of electron microscopes?

Answer 7

1) transmission electron microscopes (TEM)

2) scanning electron microscopes (SEM)

Question 8

What are the characteristics of TEMs?

Answer 8

• electron beam passes through very thin section of sample
• sample prepared by being dipped in heavy metals such as lead: some structures absorb more than others therefore appear dark. Other parts appear bright
• produces 2D black and white images on a screen (colour can be added to image by computer)
• magnification = x500 000
• resolution = 0.1nm

Question 9

What are the characteristics of a SEM

Answer 9

• electron beam directed onto surface of sample (not through)
• electrons bounce off sample and are detected by several sensors
• final computed image is 3D and black and white
• image is of the surface of specimen
• magnification = x100 000
• resolution = 20nm

Question 10

What are limitations of electron microscopes?

Answer 10

• whole system must be in a vacuum therefore living specimens cannot be observed
• complex staining process
• specimen must be extremely thin (for TEM)
• image may contain artefacts (things that result from specimen being prepared eg air bubble)

Question 11

State the resolution and magnification for all 3 microscopes

Answer 11

Question 12

What is an eyepiece graticule?

Answer 12

Glass disc with a scale etched on. - It is inserted into the barrel of the eyepiece
- needs to be calibrated for the objective lens you are using

Question 13

What is a stage micrometer?

Answer 13

A slide that has a ruler etched onto it

Question 14

How do you calculate the length of each division on the eyepiece graticule?

Answer 14