2. Tools in Cell Biology

Question 1

Units

Answer 1

pico - 10 (-12)
nano - 10 (-9)
micro - 10 (-6)
milli - 10 (-3)

kilo - 10 (3)
mega - 10 (6)
giga - 10 (9)
tera - 10 (12)

Question 2

Resolution

Answer 2

measure of clarity of image or the minimum distance of two distinguishable points

Question 3

Contrast

Answer 3

visible differences in parts of sample.

… or making sample visible against background

Question 4

Light microscopy

Answer 4

400 nm - 700 nm

Question 5

Compound microscope

Answer 5

The condenser:
Focuses light onto specimen

The objective:
in turret, light is collected from image + focuses to form image within barrel of microscope

The Eyepiece:
allows image to be viewed as if it were a projection in plane of section

Question 6

Objectives

Answer 6

Labelling of objective:
objective class, special designations used

Magnifiction / Numerical aperture:
additional details on immersion medium (oil) , adjustable cover glss correction, contrast method

Tube length / cover glss thickness in mm:
standard cover glass - 0.17
without cover glass - 0

Mechanical correction:
cover glass thickness correction, different immersion, temperature, adjusting iris diaphragm

Question 7

Lenses

Answer 7

objective lens:
collects cone of light rays - create image

condenser lens:
focuses cone of light rays to each point of specimen

Question 8

Resolution

Answer 8

resolving power of microscope depends on width of cone of illumination + therefore on both condenser + objective lens.

o.61 sigma / n sin 0

0 = half of angular width of cone of rays collected by objective lens from typical point in specimen.
maximum width 180 degrees / sin 0 has maximum width of 1

n = refractive index of medium (air or oil) separating specimen from objective + condenser lens

sigma = wavelength of light (white light figure of 0.53 micro metres commonly assumed)

Question 9

Numerical aperture

Answer 9

n sin 0 in equation called numerical aperture of lens - function of its light-collecting ability. Dry lenses don’t have one of more than 1, but for oil immersion lenses, it can be higher than 1.4. The resolution is bigger if the numerical aperture is higher, meaning the image appears brighter which is important to the fluorescence of microscopy. This advantage is obtained at the expense of very short working substances + very small depth of field.

Question 10

Dry lens

Answer 10

slide -> cover slip -> air -> objective lens

Question 11

Oil-immersion lens

Answer 11

slide -> cover slip -> oil -> objective lens

Question 12

Light microscope contd

Answer 12

• images enhanced + analyzed by digital techniques

- intact tissues usually fixed + sectioned before microscopy

Question 13

Principles of microscopy

Answer 13

Visualising stained samples:
A) incident light (white) -> only one wave light coming through

Phase contrast:
B) incident light (green) -> both wave lights coming through
-> described in 1934 by Fritz Zernike
-> high contrast image - transparent specimens -> living cells (usually in culture), microorganisms + subcellular particles (nuclei + other organelles)
=> 2 waves in phase - bright - higher amplitude
=> 2 waves overlapping out of phase - Dim - lower amplitude