2.3 MORE MICROSCOPY

Question 1

What is the main disadvantage for light microscopy?

Answer 1

Increased resolution is not easily achieved (limiting factor)

Question 2

What are the main components using beams of electrons?

Answer 2

• Wave Particle duality
• Beams of electrons wavelengths are less than 1nm
• Resolution between 0.5nm to 10nm
• show cell ultrastructure

Question 3

What are some components of a Transmission Electron Microscope (TEM)?

Answer 3

• vacuum
• ultra thin specimens (70/350nm)
• The specimen is embedded in Resin then stained with a heavy metal compound, i.e. OsO4.
• The specimen is cut into thin slices on a Microtome
• 3D Black and white image of inside the cell

Question 4

What are some components of a Scanning Electron Microscope (SEM)?

Answer 4

• vacuum
• uses a secondary electron detector
• specimen needs to be prepared to increase the electrical conductivity
• specimen is coated with a thin layer of electrically conducting materials i.e. gold
• specimen needs to be freeze-fractured to reveal the inside
• shows the surface of a cell (3D black and white)

Question 5

What is an artefact?

Answer 5

Visual artefacts are anomalies apparent during visual representation - not actually there.

Question 6

What is a Mesosome?

Answer 6

Folded invaginations in the plasma membrane of bacteria that are produced by the chemical fixation techniques used to prepare samples for electron microscopy.

Question 7

Compare light microscopes to electron microscopes.

Answer 7

LM = simple sample preparation, EM = complex sample preparation
LM = does not lead to distortion, EM = often distorts material
LM = 200nm resolution, EM = TEM - 0.5nm SEM - 3-10nm
LM = specimens can be living or dead, EM = specimens are dead

Question 8

What are the main components of a Fluorescence Microscope?

Answer 8

Optical microscope, uses fluorescence to create an image of a specimen
Emits light of longer wavelengths (FLUOROPHORES)

Question 9

What is the definition of Fluorescence?

Answer 9

absorption of colour and radiation of different colours of light.

Question 10

What are the main components of Laser Scanning Confocal Microscope?

Answer 10

Optical microscope, refined fluorescence microscope, single spot focused light from a laser is moved across specimen.
The illumination light/radiated fluorescent light follow the same path - hence confocal

Question 11

What are the main components of a Super Resolved Fluorescent Microscope?

Answer 11

Resolution greater than the theoretical limit of 0.2 micrometers for optical microscopes.
There are two methods of optical microscopy.

Question 12

What are the two main methods of optical microscopy?

Answer 12

1. Creating a higher resolution image by digitally combining many smaller and overlapping images of a specimen.
2. Digitally superimposing many images on top of each other to create a higher resolution image.

Question 13

What are the main components of an Atomic Force Microscope?

Answer 13

High resolution type of Scanning Probe Microscope.
Works by scanning a small probe over the surface of a sample, and then changes the amount of laser light reflected by into the Photodiode, producing an image.