Rec Reading Mol Bio Cell Chapter 9 Visualising Cells

Question 1

Schleiden and Schwann: the cell doctrine 1838

Answer 1

all plant and animal tissues are aggregates of individual cells

Question 2

The light microscope can resolve details 0.2um apart

Answer 2

bacteria and mitochondria are the smallest objects that can be clearly discerned by a light microscope

Question 3

The wavelike nature of light causes optical diffraction effects

Answer 3

Two trains of waves can either be in phase (inc brightness) or out of phase (decrease clarity)

Question 4

The limit of resolution

Answer 4

The limiting separation at which two objects appear distinct

depends on both wavelength and numerical aperture of the lens

Question 5

Numerical aperture

Answer 5

A measure of the width of the entry pupil of the microscope scaled to the distance from the object

the wider a microscope opens it’s eye, the more sharply it can see

under ideal conditions:
UV light wavelength = 0.4um
Numerical aperture: 1.4
Resolution: 0.2um

Question 6

Resolution vs. Detection

Answer 6

Objects are still detectable under 0.2um even though they may not be able to be resolved - e.g. fluorescently labelled microtubules (dia. 0.025um) can still be seen, just blurred and at least 0.2um thick

Question 7

Lenses

Answer 7

• the objective lens collects a cone of light rays to create an image
• the condenser lens focuses a cone of light rays onto each point of the specimen

Question 8

Resolution

Answer 8

• the resolving power of the microscope depends on the width of the cone of illumination and therefore on both the condenser and the objective lens.
• calculation: Resolution = lambda/n sin theta

lambda= wavelength of light used
n= refractive index of medium (air or oil)
theta= half the angular width of the cone of the objective lens

Question 9

Numerical aperture

Answer 9

n sin theta = numerical aperture (NA) of the lens

• means light collecting ability
• the higher the numerical aperture, the greater the resolution and the brighter the image
• high NA means a short working distance and a small depth of field

Question 10

Living cells - phase contrast or Differential Interference Contrast microscope

Answer 10

• exploit interference effects produced when 2 sets of waves recombine, creating an image of cell structure

Question 11

Refractive Index

Answer 11

amount of retardation of light passing through a specimen (e.g dense nucleus has a high RI)

Question 12

Living cells - dark field and bright field

Answer 12

• DF: rays of light are directed from the side so ONLY SCATTERED light enters the lens (cell appears light against a dark background)
• BF: rays of light passing through specimen forms a direct image