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BIO1001 - Cell Biology > Methods of Celluar Biology > Flashcards

Methods of Celluar Biology Flashcards

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1
Q

What is the resolution of the human eye?

A

100-200μm

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2
Q

When were microscopes invented?

A

16th century

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3
Q

What are the 7 different types of optical microscopy?

A
  1. Transmitted light microscopy
  2. Bright-field microscopy
  3. Dark-field microscopy
  4. Phase contrast microscopy
  5. Differential interference contrast(polarised light microscopy)
  6. Fluorescence microscopy
  7. Confocal microscopy
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4
Q

What are the 2 different types of electron microscopy?

A
  1. Transmission electron microscopy (TEM)

2. Scanning electron microscopy (SEM)

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5
Q

Define ‘resolution’

A

He ability to perceive close objects as separate entities

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6
Q

What is the resolution of a light microscope?

A

0.2 μm (200nm)

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7
Q

What is the resolution of SEM?

A

20nm

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8
Q

What is the resolution of TEM?

A

0.2nm

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9
Q

Define ‘contrast’

A

The ability to see an object against its background

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10
Q

What 2 lenses are there in optical microscopy?

A

Ocular (x10) and objective (x10, x40, and x100)

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11
Q

What must be used when using objective lens x100 and why?

A

Immersion oil, because it increases the numerical aperture

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12
Q

What is transmitted light microscopy (TEM)?

A

A general term used for any type of microscopy where the light is transmitted from a source in the opposite side of the specimen to the objective lens

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13
Q

How does transmitted light microscopy work?

A

Light is focused on the specimen to get maximum illumination after crossing a light condenser (producing the Köhler illumination effect)

Light passes through the specimen, crosses the objective lens to magnify the specimen which is further magnified by the ocular lens

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14
Q

What techniques require a transmitted light path?

A
  • bright field
  • dark field
  • phase contrast
  • polarisation
  • differential interference contrast
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15
Q

When is bright field microscopy used?

A

When there is no optical contrast technique is employed

Works well on specimen that already contain contrast/colour

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16
Q

How does bright field microscopy work?

A
  • uses double diaphragm illumination because it employs both a field and an aperture iris diaphragm to set up the illumination
  • the condenser focuses parallel rays of light on the specimen, giving an evenly illuminated field so contrast/colour of specimen is visible
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17
Q

How does dark field microscopy work?

A

Illumination of the specimen is done obliquely (in an angle)

No direct light enters the objective lens to produce a dark field

Features in the specimen plane scatter light and can be clearly seen against a dark back ground

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18
Q

What types of illumination are used in dark field microscopy?

A

A simple patch stop

A dark-field element in a phase contrast condenser

A purpose built dark-field condenser

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19
Q

What is phase contrast microscopy?

A

A technique that exploits the fact that light slows down slightly when passing through biological samples

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20
Q

How does phase contrast microscopes work?

A
  • uses phase contrast objectives which have a corresponding phase plate
  • the specimen is illuminated by a hollow cone of light coming through a phase annulus in the condenser, light rays are differentially retarded by the specimen structures
  • inference between rays that cross the specimen creates a light and dark image of the specimen
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21
Q

How is phase contrast microscopy useful?

A

For observing transparent and colourless samples

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22
Q

How does differential interference contrast (polarised light microscopy) work?

A
  • it uses plane-polarised light to analyse structures that are birefringent
  • image contrast arises from the interaction of plane polarised light with a birefringent specimen to produce 2 individual wave components that are polarised to produce an image
23
Q

Define ‘birefringent’

A

The double refraction of light in a transparent, molecularly ordered material which is manifested by the existence of orientation-dependent difference in refractive index

24
Q

How can differential interference contrast (polarised light microscopy) be useful?

A

To have information about the molecular structure of objects (e.g orientation)

25
Q

How does fluorescence microscopy work?

A
  • based in fluorescence:property of emitting light
  • many biological substances emit light : they aren’t fluorescent but are labelled with a fluoresce probe(fluorochromme)
  • fluorochromme emits light at a certain wavelength when excited by incidence of a shorter wavelength of light
  • e.g. GFP emir green light when illuminated with blue light
26
Q

What is confocal microscopy?

A

All out of focus structures are suppressed at image formation via single plan focused light

27
Q

How does confocal microscopy work?

A

He confocal effect is obtained by diaphragms which act as a point light source and as a long detector respectively. The detection pinhole suppresses rays of light from out-of-focus points. The conjugated effects of light wavelength determines the depth of focus. To obtain full image the point of light is moved across the specimen by scanning mirrors

28
Q

Why is electron microscopy better than light microscopy?

A

The penetrating power of electrons allows higher magnification and better resolution

29
Q

Who created the first real prototype of an electron microscope in 1933 which could resolve to 50nm?

A

Ruska

30
Q

How does electron microscopy work?

A
  • beam of electrons produced by a tungsten filament accelerated at 100kv
  • whole microscope in a vaccum(electrons don’t move through air)
  • electrons focused by a series of electro-magnetic lenses
  • the image is projected onto a fluorescent screen or CDD camera at the bottom of the column - human eye not sensitive to electrons
31
Q

How does transmission electron microscopy (TEM) work?

A
  • a high voltage beam is transmitted Brough a very thin (semi-transparent for electrons) specimen
  • the variation of the specimens result Ina n image which is magnified by magnetic lenses
32
Q

How does scanning microscopy (SEM) work?

A
  • in SEM detection of secondary electrons emitted from the surface of the specimen after extraction by the primary electron beam produce an image of the sample
  • the electron beam is scanned across the surface of the sample to build its image
33
Q

What does a scanning electron microscope (SEM) work best on?

A

SEM works best on surfaces

34
Q

In sample preparation for microscopy, how is a sample preserved as close as possible to living state?

A
  • fixation of the sample
35
Q

Name the different ways of sample fixation in microscopy

A
  • cryofixation
  • chemical fixation
  • dehydration
  • embedding
  • sectioning
  • staining
36
Q

When preparing a sample what is ‘cryofixation’?

A

-freezing a sample rapidly in liquid nitrogen

37
Q

When preparing a sample what is ‘chemical fixation’?

A

Glutaraldehyde is often used to cross link proteins and osmium tetroxide to preserve lipids

38
Q

When preparing a sample what is ‘dehydration’?

A

Removing water from the samples and replacing it with organic solvents such a ethanol amid acetone for subsequent embedding for TEM specimens

39
Q

When preparing sample what is ‘embedding’?

A

Infiltration of the tissue with wax for light microscopy or a resin for electron microscopy

40
Q

When preparing a sample what is ‘sectioning’?

A

The production of thin slices of the specimen using ultramicrotome

41
Q

When preparing a sample what is ‘staining’?

A

Use heavy metals such as lead plans uranium to give contrast by scattering electrons

42
Q

What are the different techniques of staining and labelling?

A
  • dye staining
  • fluorescent labelling
  • immunostaining
43
Q

What is ‘dye staining’?

A

Use of dyes that bind specifically to different compartments of the cell and increase their contrast

e.g. Methylene blue dye to the nucleus

44
Q

What is ‘fluorescent labelling’?

A

There are fluorescent dyes and proteins

Dyes = fluorscein and rhodamine 
Proteins = green fluorescent protein
45
Q

What is ‘immunostaining’?

A

Uses fluorescently labelled antibody specific to a target protein in a sample.

A fluorescent secondary antibody might be used to detect the primary antibody for indirect detection of the target - sample then observed

46
Q

What does ‘FACS’ stand for?

A

Fluorescent Activated Cell Sorters

47
Q

What do FACS do?

A

Separate cells in solution from each other by detecting scattered, reflected and fluorescent light emitted

48
Q

How does FACS work?

A

Cells travel in a thin capillary and are separated base on light characteristics

  1. cells are forced to enter a small vibrating nozzle one at a time
  2. cells are scanned by a laser beam (cell counting and cell size)
  3. By measuring scattered reflected and fluorescent light the computer determines which cells to separate and collect
49
Q

What are the applications of flow cytometry?

A
  • sorting out cells in a mixed sample
  • sorting out living cells from dead
  • measuring DNA and RNA in vivo (living)
  • sorting out cells by functional types
  • separating cellular organelles
  • sorting out chromosomes
50
Q

What is cell fractionation used for?

A
  • separation of intact cells from a mixed sample in to factions
  • separation of cellular organelles
  • separation of membranes
51
Q

What is ‘homogenisation’?

A

Tissue disruption in a stabilising buffer

Only once homogenised can the cell/s be fractionated

52
Q

What are the physical means of homogenisation?

A
  • grinding the tissue using a pestle and mortar or mixer
  • compression and/or expansion
  • ultra sonication
53
Q

What are the non-physical means of homogenisation?

A
  • hydrologic enzymes e.g. Cellulases, lysozymes

- osmotic force

BIO1001 - Cell Biology (5 decks)

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