Lecture 2

Question 1

Is structure related to function in cell biology?

Answer 1

Structure is always related to function in cell biology.

Question 2

What happened in 1665?

Answer 2

Robert Hooke published a collection of assays under ‘Micrographica’.
Described seeing a honeycomb of chambers or cells.

Question 3

What happened in 1675?

Answer 3

Van Leeuwenhoek improved the art of polishing lenses, able to resolve to 1.5µm. Could see protozoa. Then went on later to discover bacteria.

Question 4

What happened in the 19th century regarding light microscopes?

Answer 4

Maximum theoretical resolution was obtained (0.25µm).

Question 5

What was developed in the 1930s? And what term was given to the new level of detail observable?

Answer 5

Electron microscope.

Ultrastructure.

Question 6

Best resolution of LM?

Answer 6

0.2µm.

Question 7

6 types of light microscopy?

Answer 7

Brightfield (unstained), 
Brightfield (stain),
Fluorescence,
Phase-contrast,
Differential-interference-contrast,
Confocal.

Question 8

Seven steps in light microscope sample preparation?

Answer 8

Whole mounts - small, translucent specimens can be mounted directly.
Tissue sections - most tissues need sectioning first.
Fixation - prevents cell autolysis, preserves structure.
Dehydration and clearing - Removes water from tissue to prepare for wax impregnation.
Embedding - specimen infiltrated with molten wax.
Sectioning - sections 5 microns thick cut on microtome, collected onto glass slides.
Staining - wax removed, tissue stained with coloured dye.

Question 9

What does Eosin stain?

Answer 9

Cytoplasm.

Question 10

What does haematoxylin stain?

Answer 10

Nuclei.

Question 11

What light microscopy method can be used to generate 3D images of living cells? What other benefits does this method confer?

Answer 11

Confocal.

Can also remove out of focus images, and look inside thick specimens.

Question 12

2 light microscope techniques that break the resolution limit? How do they work?

Answer 12

Deconvolution microscopy - algorithms remove out of focus light. Sharpens image and improves resolution.
Super resolution microscopy - gathers light from individual fluorescent molecules and records their position. Combining information from these individual molecules breaks limit.

Question 13

Who were electron microscopes developed by?

Answer 13

Ruska and Knoll.

Question 14

What is the theoretical resolution limit for EMs? And why?

Answer 14

0.002nm. Electrons have very short wavelength.

Question 15

What is the currently achieved maximum resolution of the EM?

Answer 15

0.1nm.

Question 16

As electrons have poor penetrating power, what state is the EM kept under?

Answer 16

Vacuum.

Question 17

How are electrons focussed?

Answer 17

Magnetic fields.

Question 18

Two types of EM?

Answer 18

Transmission electron microscope.

Scanning electron microscope.

Question 19

How does a TEM work?

Answer 19

Electron gun, usually heated tungsten, produces electrons by thermionic emission.
Electron beam passes through specimen, having been focussed and magnified by magnetic objective and projector lenses.
Electron image then converted into visible image by fluorescent screen, viewed through a glass window.

Question 20

How is a TEM sample prepared?

Answer 20

Whole mounts - bacteria and viruses can be examined directly.
Fixation - Usually glutaraldehyde (protein cross linking), followed by second fixation in Osmium Tetroxide (lipid cross linking).
Dehydration - in ethanol series.
Embedding - specimens embedded in plastic resins, e.g. epoxy.
Sectioning - 50nm thick sections cut using ultramicrotome.
Staining - Heavy metal stains e.g. lead used to improve contrast under electron beam.

Question 21

Why is an SEM called an SEM?

Answer 21

Because electron beam scanned across surface of the specimen.

Question 22

How does an SEM work?

Answer 22

Electron beam scanned across surface of the specimen, reflected from its surface, before being collected by an electron detector and converted into an electronic signal displayed on TV screen.

Question 23

What gives SEM images a 3D appearance?

Answer 23

Depth of focus of SEM.

Question 24

How are SEM samples prepared?

Answer 24

Fixation - Glutaraldehyde (protein cross linking), then Osmium Tetroxide (lipid cross linking).
Dehydration - water replaced with ethanol.
Critical point drying - enables all ethanol to be removed in a way that minimises shrinking.
Coating - Coated in a thin layer of gold, to protect from electron beam damage.

Question 25

How many g’s are required for how long to create a pellet rich in nuclei and cellular debris?

Answer 25

1000g for 10 minutes.

Question 26

How many g’s are required for how long to create a pellet rich in mitochondria (and chloroplasts if plant cells)?

Answer 26

20,000g for 20 minutes.

Question 27

How many g’s are required for how long to create a pellet rich in microsomes?

Answer 27

80,000g for 60 minutes.

Question 28

How many g’s are required for how long to create a pellet rich in ribosomes?

Answer 28

150,000g for 3 hours.