2.3 More microscopy

Question 1

What is the limiting factor in light microscopy?

Answer 1

Resolution

Question 2

Define electron microscopy.

Answer 2

Microscopy using a microscopy that employs a beam of electrons to illuminate the specimen.
Since electrons have a much smaller wavelength than light, they produce images with higher resolutions than light microscopes.

Question 3

Electron microscopy - what is used to illuminate the specimen?

Answer 3

A beam of electrons with a wavelength of less than 1 nm.

Question 4

Electron microscopy - how can more detail of cell ultra-structure be seen?

Answer 4

Electrons have a much smaller wavelength than light waves - they can magnify x500 000 with clear resolution

Question 5

What are the disadvantages of electron microscopy?

Answer 5

• Expensive
• Only usable in controlled environment
• Specimens can be damaged by the electron beam
• Artefacts often made due to complex preparation process

Question 6

What are the two types of electron microscope?

Answer 6

• Transmission electron microscope (TEM)

- Scanning electron microscope (SEM)

Question 7

How does a transmission electron microscope (TEM) work? What is its resolving power?

Answer 7

A beam of electrons is transmitted through a specimen and focused to produce an image
Produces the best resolution with resolving power of 0.5nm

Question 8

How does a scanning electron microscope (SEM) work? What is its resolving power?
What’s the good thing about SEM?

Answer 8

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

• Resolving power is 3-10 nm
• 3D images can be produced, providing us info about the appearance of organisms

Question 9

Compare light microscopy with electron microscopy

Answer 9

LIGHT vs ELECTRON
- Cheap - Expensive
- Small/portable - Large, requires installation
- Simple sample prep - Complex sample prep
- Sample prep doesn’t - Sample prep often causes
cause distortion distortion
- Vacuum not required - Vacuum required
- Natural colour of sample - Black and white images
- x2000 magnification - x500 000 magnification
- 200nm resolving power - RP of: TEM = 0.5nm
SEM - 3-10nm
-Specimens can be live/dead -Specimens are dead

Question 10

In terms of microscopy, what is an artefact?

Answer 10

A visual structural detail caused by processing the specimen - not a feature of the specimen
(appears in both electron and light microscopy)

E.g. bubbles trapped under cover slip during prep for electron microscopy are artefacts

Question 11

How does a laser scanning confocal (LSC) microscope work?

Answer 11

-Moves a single spot of focused light across a specimen, causing fluorescence from components labelled with a dye
(Fluorescence is the absorption and re-radiation of light)
-Light emitted from the specimen is filtered through the pinhole aperture
-Only light radiated from close to the focal plane is detected

Question 12

Why are high resolution images obtained using a laser scanning confocal (LSC) microscope?

Answer 12

• Thin sections of specimen are examined

- Light from elsewhere is removed

Question 13

Define resolution

Answer 13

Shortest distance between 2 objects that are still seen as separate objects.

Question 14

Define fluorescence

Answer 14

Absorption and re-radiation of light

Question 15

How do fluorescent microscopes work?

Answer 15

Higher light intensity used to illuminate a specimen treated with a fluorescent dye

Question 16

What is a laser scanning confocal microscope?

Answer 16

Microscope that uses a laser beam and pin-hole aperture to produce an image with very high resolution

Question 17

Why is blurring reduced in laser scanning confocal microscopy?

Answer 17

Light emitted from other parts of the specimen doesn’t pass through the pinhole aperture.
So high resolution images are obtained.

Question 18

What is the benefit of using a laser instead of light in laser scanning confocal microscopy?

Answer 18

To get higher illumination

Question 19

How are 2D images produced with LSCM?

Answer 19

Spot illuminating the specimen is moved across the specimen

Question 20

How are 3D images produced with LSCM?

Answer 20

By creating images at different focal planes

Question 21

What is the beamsplitter in LSCM?

Answer 21

A dichroic mirror which only reflects one wavelength, from the laser, but allows other wavelengths, from the sample, to pass through

Question 22

What does the position of the 2 pinholes in LSCM mean?

Answer 22

Light waves from the laser illuminating the sample follow the same path as light waves radiated when the sample fluoresces.
This means they have the same focal plane, hence the name confocal