2.1.1 Microscopy

Question 1

What are the different types of microscopy?

Answer 1

• Light microscopy
• Transmission Electron microscope
• Scanning Electron Microscope
• Laser Confocal microscope

Question 2

What are the pros and cons of using a light microscope?

Answer 2

Pros : Easy to see large cellular structures, eg cell walls, chloroplasts, nuclei. Samples can be alive or dead, Images in colour

Cons : Low magnification and resolution, ultra-cellular structure cannot be seen

Question 3

What is the radiation source and radiation used for a light microscope? How is this radiation focused?

Answer 3

The radiation source is light and radiation used is light. This radiation is focused using glass lenses.

Question 4

How are cell structures seen with light microscope images?

Answer 4

By using staining dyes

Question 5

What is the highest magnification and resolution in a light microscope

Answer 5

M : x1500
R : 200nm

Question 6

How does a TEM work

Answer 6

U

Question 7

How does a TEM work

Answer 7

A beam of electrons is fired through a thin section of a specimen. Denser areas of the cell absorb more electrons making them appear darker on the image

Question 8

Max Magnification and resolution of TEM?

Answer 8

M : x500,000
R : 0.2 nm

Question 9

Pros and cons of TEM?

Answer 9

Pros: highest magnification and resolution,

Cons: Always a 2D greyscale image, colour can be added digitally, specimen always needs to be dead due to the vacuum used

Question 10

How does a SEM work

Answer 10

A beam of electrons is fired at the surface of specimen, knocks electrons off and these reflected electrons are collected t form an image.

Question 11

Max Magnification and resolution of SEM?

Answer 11

M : x100,000

R : 10nm

Question 12

Pros and cons of SEM

Answer 12

Pros: Can produce a 3d image of high magnification and resolution

Cons: always a greyscale image although colour can be added digitally, Specimen has to be dead since a vacuum is used

Question 13

What is the radiation source, radiation used and method of focusing radiation for electron microscopes?

Answer 13

The radiation source is an electron gun, electrons are the radiation used and electromagnets are used to focus the radiation

Question 14

Why do you need a vacuum for electron microscopes?

Answer 14

Scattering of electrons by air molecules interferes with image production

Question 15

How does a Laser scanning confocal microscope work?

Answer 15

Cells are stained with fluorescent dye and a thick section of tissue is scanned with a laser beam,
Light is focused through a pinhole on a detector - the pinhole means any out of focus light is blocked so clearer images

Question 16

Pros and Cons of a Laser Confocal Microscope?

Answer 16

Pros : Clearer images than light microscopes, laser bean can be focused at a specific depth eliminating the blur in optical scopes caused by out of focus lenses so clearer images, 3D images can be reconstructed as images taken can be fed into a computer
Cons:

Question 17

When were cells first discovered and by who?

Answer 17

1660s by Robert Hooke

Question 18

What is magnification?

Answer 18

The ability of a lens to enlarge the size of any optical image

Question 19

Magnification equation

Answer 19

Image / Actual size

Question 20

What is resolution

Answer 20

The ability to distinguish between two points as separate to see detail

Question 21

Why are stains used?

Answer 21

Stains are taken up better by different parts of the cell, the contrast is what makes the different parts show up better

Question 22

Preparing samples for staining - light microscope

Answer 22

Sample left to air dry, heat fixed, can then take up stains

Question 23

Preparing samples for staining - Electron microscopes

Answer 23

Samples dipped in solution of heavy metals, metal ions scatter creating contrast and some areas show up darker.

Question 24

Preparing a sample

Answer 24

• fixation - prevent decay - formaldehyde added in a form of phosphate buffered solution
• embedment - infiltrated with paraffin wax
• sectioning - a single layer of cells cut 3-5micrometers with a microtome
• staining
• covered with coverslip

Question 25

What’s the differences between a wet and dry mount

Answer 25

Wet slides you add a drop of water and place the coverslip at an angle to avoid air bubbles unlike a dry mount which involves no water so the cover slide doesn’t need to be placed at an angle