Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BB10005 COPY > Electron microscopy > Flashcards

Electron microscopy Flashcards

1
Q

How does the resolution and wavelength in electron microscopy compare to light microscopy?

A

Light: resolution 200nm, Wavelengths: 400 - 710 nm
electron: Resolution 0.1nm, wavelengths: 2.5pm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is the basis of the electron microscope?

A
  1. Electrons are emitted from a filament and accelerated in an electric field.
  2. A condenser lens focuses the electron beam.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

How do TEM and SEM differ?

A
  1. In TEM, electrons scatter or hit a fluorescent screen at the bottom of the microscope. Electrons pass through the specimen.
  2. In SEM electrons are focused on a metal coated Specimen at the bottom of microscope. Electrons collide with specimen this excites electrons which are caught by detector.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Why can specimens not be alive in electron microscopy?

A

Due to the high pressure vacuum that needs to be present.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What are two methods used to view samples in TEM?

A
  1. Direct examination

2. Sections from tissues

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Why is a support film needed in TEM?

A

To enhance stability and conductivity when exposed to the electrons.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

How can sample contrast be increased in TEM?

A
  1. Negative staining using heavy metals such as lead, uranium, tungsten and gold.
  2. Shadowing can be used where heavy metal is evaporated from a wire in a vacuum chamber to cast a shadow on the adjacent sample.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

How does shadowing work?

A
  1. The sample is spread on a mica surface and then dried.

3. Spluttered particles of gold hit specimen and cause contrast

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How can protein amino groups be preserved during sample preparation?

A

Using glutaraldehyde.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

How can proteins and lipids be preserved during sample preparation?

A

Using osmium tetroxide.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How can membranes be preserved during sample preparation?

A

Using potassium permanganate.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Why do samples need to be preserved in sample preparation?

A

As samples are usually around 70% water and they would boil in the EM vacuum.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

How can the samples be dehydrated?

A
  1. 10% to 100% ethanol series in 10% steps with very gentle agitation. 10% then 20% etc
  2. Water is gradually replaced by ethanol
  3. Ensure artefacts aren’t introduced
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What is the purpose of embedding in TEM?

A

To produce blocks that are suitable for ultra-thin sectioning and still preserve the fine structure of the specimen.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

How can samples be embedded?

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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How can tissues be sectioned?

A
  1. Using an ultra-microtome to produce ultra-thin sections (50-90nm thick).
  2. These are then picked up on formvar coated grids.
  3. Judge thickness by colour
17
Q

How do fixatives differ?

A

They can vary in their rate of penetration, their ability to fix different molecules and can cause shrinkage or swelling of tissues.

18
Q

How can cell components be localized in light microscopy?

A
  1. Histochemical dyes
  2. Antibodies linked to fluorescein isothiocyanate,
  3. GFP
  4. chromogenic enzyme substrate.
19
Q

How can cell components be localized in electron microscopy?

A

An antibody linked to colloidal gold, enzyme localization by linking product to heavy metal e.g lipases with Pb and enzyme localization if products are electron dense e.g. peroxidase.

20
Q

How can proteins be detected in electron microscopy?

A
  1. Antibody linked to protein-A gold. Different sizes can differentiate two different compounds simultaneously.
  2. Enzyme localisation by linking product to heavy metal e.g. lipases with Pb.
  3. Enzyme localisation if products are electron dense e.g. peroxidase.
21
Q

How does protein A-gold work to localise cell components?

A
  1. Protein A-gold will bind to the antibodies added and can locate the molecule of interest.
  2. The different sizes of collodial gold can be used to identify different molecules simultaneously.- double labelling
22
Q

What can be used to identify phenols?

A

Peroxidase - it converts phenols to electron dense products.

23
Q

How does SEM work?

A

Electron beams scan the specimen and cause it to emit electrons to give a detailed image.

24
Q

What is the magnification range of SEM?

A

x10 to x100000

25
Q

What is the resolution of SEM?

A

<10nm

26
Q

What are some other key features of SEM?

A
  1. There is a great depth of focus,

2. the specimen can be rotated and tilted and large- irregular specimens can be viewed.

27
Q

What kind of images are produced with SEM?

A

3D images that can be artificially coloured.

28
Q

What is the preparation for samples in SEM?

A

Similar to TEM, but delicate specimens need to be dehydrated.

29
Q

What is the process for critical point drying?

A
  1. The species needs to be dehydrated in an ethanol-water series
  2. replaced with amyl acetate and subjected to liquid CO2 under pressure and then raise the temperature as liquid CO2 will convert to a gas to leave a dry specimen.
30
Q

What are the problems with critical point drying in SEM?

A

There may be shrinkage or the removal of substances in organic solvents such as leaf wax.

31
Q

How can the problems with critical point drying in SEM be overcome?

A

Cryo-SEM.

32
Q

What does Cryo-SEM mean?

A

Low temperature scanning electron microscopy.

33
Q

Why is cryo-SEM favoured over the normal SEM?

A

The sample retains water as it is flash freezed in liquid nitrogen. There is no exposure to fixatives or solvents, and the preparation time is short.

34
Q

What are the steps in cryo-SEM?

A

Mount the specimen on a holder, flash freeze in liquid nitrogen, transfer to a vacuum chamber of SEM (-100C), warm the stage above -96C to sublimeaway any surface ice crystals and then coat with metal and observe the specimen.

35
Q

How is a sample prepared using direct examination

A
  1. Samples are placed on a copper grid coated with formvar this is in water
  2. Formvar is floated off a glass slide onto the surface of water in a Petri dish.
  3. Grids on a sheet of wire gauze at the bottom of the Petri dish are lifted out of the water and the formvar film is deposited on them.
36
Q

What can polyoma virus be stained with

A

tungsten

37
Q

How are replicas prepared

A
  1. Replicas are made of fragile samples.

2. Sample surface coated with thin film of evaporated carbon or plastic- like a cast

BB10005 COPY (23 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions