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

electron miscopy (lecture 3) Flashcards

(45 cards)

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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

Study These Flashcards
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

Study These Flashcards
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?

Study These Flashcards
A

small particles such as viruses, DNA, RNA

22
Q

When are replicas relevant (direct examination TEM)

Study These Flashcards
A

fragile samples

23
Q

Fixatives function?

Study These Flashcards
A

crosslink molecules

24
Q

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

Study These Flashcards
A

Preserved during sample preparation

25
3 Examples of fixatives?
1- Glutaraldehyde (protein amino groups) 2- Osmium tetroxide (proteins & lipids) 3- Potassium permanganate KMnO4 (membranes)
26
Why do we use small pieces of samples (sections from tissues TEM)??
0.5mm^3 to allow rapid infiltration of fixative
27
Dehydration method
10%> 100% ethanol serves in 10% steps with very gentle agitation (mixing)
28
Embedding method, dehydration, Sections from tissues TEM
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
Sectioning method?
Ultra-microtome (estimate section thickness by interference colours). Sections (50-90 nm thick) are picked up on formvar coated grids.
30
Katherine Esau, time period & research
1960's | information about phloem.
31
Fixatives vary in their
rate of penetration in tissues and ability to fix different molecules
32
Fixatives can cause what to occur in tissues?
Shrinkage or swelling of tissues
33
3 Methods of localisation of cell components by EM
1) Antibody linked to colloidal gold. 2) Enzyme localisation by linking product to heavy metal 3) Enzyme localisation (if products are electron dense)
34
Structure of complex which is used when an antibody is lined to colloidal gold?
Antigen, antibody, protein A, Fc domain, gold (attached to more protein a's)
35
Use of antibodies being linked to colloidal gold??
localisation of proteins within a cell
36
Scanning Electron Microscopy works by
Electron beam scans specimen causes it to emit electrons giving detailed image.
37
Magnification Scanning Electron Microscopy range
Wide range of magnification x10 to x100,000
38
Depth of focus Scanning Electron Microscopy??
Great depth of focus
39
Resolution Scanning Electron Microscopy
resolution <10nm
40
Specimen can be?? - Scanning Electron Microscopy
rotated and tilted
41
What types of specimens can be viewed- scanning electron microscopy
Large irregular specimens can be viewed
42
Structures of TEM and SEM
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
Preparations of SEM
Delicate specimens need to be dehydrated
44
critical point drying SEM
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
Problems with SEM and how to overcome
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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