2.3 More microscopy

Question 1

Put these in size order:
animal cell, bacterium, plant cell, virus

Answer 1

Smallest: virus
bacterium
animal cell
Largest: plant cell

Question 2

Define fluorescence

Answer 2

absorption then re-radiation of light

Question 3

How a light microscope works?

Answer 3

the objective lens magnifies the image and the eyepiece lens magnifies that image to increase the magnification by more
illuminated from below

Question 4

Advantages of a light microscope

Answer 4

inexpensive to buy and operate
small +portable
simple sample prep
specimens can be living or dead

Question 5

Disadvantages of a light microscope

Answer 5

Only up to x2000 magnification
resolving power of 200nm

Question 6

How a TEM works?

Answer 6

a beam of electrons is transmitted through a specimen and focussed to produce and image

Question 7

Advantages of a TEM

Answer 7

Over x500000 magnification
Resolving power of 0.5 nm (best)

Question 8

Disadvantages of a TEM/SEM

Answer 8

Expensive to buy and operate
Vacuum is needed
Complex sample prep
Specimens must be dead
Large and needs installation
Requires trained professionals

Question 9

How an SEM works?

Answer 9

a beam of electrons is sent across the surface of a specimen and the reflected electrons are collected

Question 10

Advantages of an SEM

Answer 10

3-10nm resolving power
Up to 500000 magnification
Produce 3D images

Question 11

How a laser scanning confocal microscope works?

Answer 11

move a single spot of focussed light across a specimen which causes fluorescence from the components
emitted light from specimen is filtered through a pinhole aperture
only light radiated from very close to the focal plane is detected

The laser scans a layer of the specimen which prevents blurring from too many layers merging together

Question 12

Stages of sample prep for an electron microscope

Answer 12

Double fixation - sterilise it and prevent decomposition
Dehydration - water could boil in a vacuum and damage the sample
Epoxy resin embedding - to make it rigid/ hold it firmly
Ultrathin slicing - to allow electrons to pass through the sample
Staining with heavy metals - heavy metals will attach to different structures and create contrast

Question 13

What is an artefact?

Answer 13

Something that is on the slide that shouldn’t be
e.g. hair, fingerprint etc

Question 14

Do samples need to be thin for a TEM/SEM?

Answer 14

TEM = must be thin
SEM = doesn’t need to be thin

Question 15

Why are lasers used in confocal microscopy?

Answer 15

Increase light intensity

Question 16

What is the pinhole aperture used for in confocal microscopy?

Answer 16

Scattered light
Reduces blurring and increases resolution

Question 17

Can CSM be used for deep tissue imaging?

Answer 17

No as the sample would be too thick for the laser to penetrate through as the light penetration is limited