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BB10005- Cell Biology > electron miscopy (lecture 3) > Flashcards

electron miscopy (lecture 3) Flashcards

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1
Q

Wavelength of electrons

A

-2.5 pm

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2
Q

Resolution of electrons

A

-0.1nm

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3
Q

Electron microscopy relies on?

A

High voltage electron beam in order to create an image (200keV)

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4
Q

Resolution is determined by?

A

Resolution determined by wavelength of radiation source used for illumination

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5
Q

What can we view using Electron microscopy

A 
eu cells 
nucleus 
most bacteria 
mycoplasma 
viruses 
ribosomes 
proteins 
lipids 
small molecules 
atoms
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6
Q

How do light microscopy and Transmission electron microscopy differ in their stucture

A

light m- eyepiece lens and light source

transmission -Projection lens and electron source

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7
Q

Structure of scanning electron microscope

A 
Electron source 
contender lens 
scanning coil 
condenser lens 
Specimen 
image viewed on a monitor
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8
Q

Where is the image viewed scanning electron microscope ?

A

Monitor

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9
Q

Structure of light microscopy and Transmission electron microscopy?

A

1) light source vs electron source
2) condenser lens
3) Objective lens
4) eye piece lens vs projection lens
5) Image viewed directly (light) vs image viewed on fluorescent screen

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10
Q

Where are electrons emitted from in electron microscopy?

A

Filament

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11
Q

Where are electrons accelerated in electron microscopy?

A

Electric filed

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12
Q

Purpose of condenser lens (in Electron microscopy)

A

Condenser lens focuses the electron beam

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13
Q

In TEM electrons either ________ or _______

A

electrons either scatter or hit a fluorescent screen at the bottom of the microscope

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14
Q

In SEM where are electrons focused?

A

SEM: electrons are focused on a metal coted specimen, electrons from the metal are collected by a detector

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15
Q

Column must be maintained at ______

A

very high vacuum

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16
Q

Transmission EM Techniques for direct examination?

A

GRIDS
FORMVAR
CONTRAST
REPLICATION

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17
Q

Transmission EM Techniques for sections from tissues

A 
FIXATION
DEHYDRATION
EMBEDDING
SECTIONING
STAINING
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18
Q

in direct examination- tem, samples are placed on?

A

FORMVAR 3mm diameter

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19
Q

How can we increase the contrast of direct examination TEM

A

negative staining

two types of ‘dyes’

biological elements composed of heavy metals such as lead uranium and gold- high electron scattering power

biological materials composed of light elements such as C, H, O N.- low electron scattering power (CREATES CONTRAST)

20
Q

How else other than negative staining can we increase the contrast of a direct examination of a sample using TEM

A

Shadowing

  • Heavy metal is evaporated from a wire in a vacuum chamber casting a shadow on the adjacent sample.
21
Q

Shadowing often used to visualise?

A

small particles such as viruses, DNA, RNA

22
Q

When are replicas relevant (direct examination TEM)

A

fragile samples

23
Q

Fixatives function?

A

crosslink molecules

24
Q

biological fine structure need to be (sections from tissues TEM)??

A

Preserved during sample preparation

25
Q

3 Examples of fixatives?

A

1- Glutaraldehyde (protein amino groups)
2- Osmium tetroxide (proteins & lipids)
3- Potassium permanganate KMnO4 (membranes)

26
Q

Why do we use small pieces of samples (sections from tissues TEM)??

A

0.5mm^3 to allow rapid infiltration of fixative

27
Q

Dehydration method

A

10%> 100% ethanol serves in 10% steps with very gentle agitation (mixing)

28
Q

Embedding method, dehydration, Sections from tissues TEM

A

1) Place sample in BEEM capsule
2) Infiltration with un-polymerised epoxy resin, Epon or Araldite 3) Polymerisation of resin in BEEM capsule
4) Remove resin block

29
Q

Sectioning method?

A

Ultra-microtome (estimate section thickness by interference colours).
Sections (50-90 nm thick) are picked up on formvar coated grids.

30
Q

Katherine Esau, time period & research

A

1960’s

information about phloem.

31
Q

Fixatives vary in their

A

rate of penetration in tissues and ability to fix different molecules

32
Q

Fixatives can cause what to occur in tissues?

A

Shrinkage or swelling of tissues

33
Q

3 Methods of localisation of cell components by EM

A

1) Antibody linked to colloidal gold.
2) Enzyme localisation by linking product to heavy metal
3) Enzyme localisation (if products are electron dense)

34
Q

Structure of complex which is used when an antibody is lined to colloidal gold?

A

Antigen, antibody, protein A, Fc domain, gold (attached to more protein a’s)

35
Q

Use of antibodies being linked to colloidal gold??

A

localisation of proteins within a cell

36
Q

Scanning Electron Microscopy works by

A

Electron beam scans specimen causes it to emit electrons giving detailed image.

37
Q

Magnification Scanning Electron Microscopy range

A

Wide range of magnification x10 to x100,000

38
Q

Depth of focus Scanning Electron Microscopy??

A

Great depth of focus

39
Q

Resolution Scanning Electron Microscopy

A

resolution <10nm

40
Q

Specimen can be?? - Scanning Electron Microscopy

A

rotated and tilted

41
Q

What types of specimens can be viewed- scanning electron microscopy

A

Large irregular specimens can be viewed

42
Q

Structures of TEM and SEM

A

1) Tungsten filament (cathode)
2) condenser lens
3) specimen (TEM), Scanning coils (SEM)
4) Electromagnetic objective lens
5) TEM: Projector lens
6) detecter (tem), specimen and then detector (SEM)

43
Q

Preparations of SEM

A

Delicate specimens need to be dehydrated

44
Q

critical point drying SEM

A

Dehydrate in ethanol-water series
Replace with amyl acetate
Subject to liquid CO2 under pressure
Raise temperature, liquid CO2 converts to gas leaving dry specimen

45
Q

Problems with SEM and how to overcome

A

Shrinkage, removal of substances soluble in organic solvents e.g. leaf wax.
Cryo-SEM overcomes these limitations

BB10005- Cell Biology (6 decks)

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