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HISTOPATH LEC - TRANS > F1-TRANSMISSION ELECTRON MICROSCOPY > Flashcards

F1-TRANSMISSION ELECTRON MICROSCOPY Flashcards

(79 cards)

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1
Q

Which microscopy method uses a beam of electrons instead of light to create images

A

Electron microscopy

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2
Q

Which source of electrons is commonly used in electron microscopy

A

Heated tungsten filament

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3
Q

Which property of electron microscopy allows observation of ultrastructures

A

Higher magnification and greater resolving power

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4
Q

Which lenses are used to control the path of electrons in electron microscopy

A

Electromagnetic and/or electrostatic lenses

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5
Q

What is the basic design of an electromagnetic lens

A

Solenoid with coiled wire

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6
Q

How does the electron beam travel in an electron microscope

A

Through the center of the solenoid down the column toward the sample

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7
Q

Which type of electron microscope uses electron beams to illuminate the specimen

A

Transmission electron microscope

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8
Q

What is the resolution of a transmission electron microscope

A

0.2 nanometers

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9
Q

What is the magnification of a transmission electron microscope

A

2

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10
Q

What are the main parts of a transmission electron microscope

A

Electron source + Electromagnetic lens system + Sample holder + Imaging system

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11
Q

Which material is commonly used as the electron source

A

Tungsten filament

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12
Q

What does the imaging system in a transmission electron microscope include

A

Electromagnetic lens system + Screen with phosphorescent plate

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13
Q

When is transmission electron microscopy used for diagnostics

A

When it provides useful structural functional and compositional information

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14
Q

When is transmission electron microscopy used for minor or atypical abnormalities

A

When only atypical or minor abnormalities are visible by light microscopy

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15
Q

When is transmission electron microscopy used for equivocal affinity labelling results

A

When affinity labelling results are equivocal

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16
Q

When is transmission electron microscopy used as a last resort

A

When there is no realistic alternative or simple test available

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17
Q

When is transmission electron microscopy used for new diseases or microorganisms

A

For investigation of new diseases and microorganisms

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18
Q

When is transmission electron microscopy considered time and cost effective

A

When it is time and cost effective compared to alternative techniques

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19
Q

Why is electron microscopy not a routine technique

A

Because it is a special technique used only for specific cases

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20
Q

What is the first and most important step in processing tissue for electron microscopy

A

Primary fixation

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21
Q

What is the purpose of primary fixation

A

To cross-link cellular structures and preserve morphology volume and spatial relationships with minimal loss

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22
Q

What is the preferred primary fixative for electron microscopy

A

2.5 percent glutaraldehyde in 100 millimolar phosphate buffer at pH 7.0

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23
Q

How long is primary fixation typically performed

A

2–24 hours with 2–3 hours preferred

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24
Q

At what temperature is primary fixation started

A

At room or physiological temperature for 15–30 minutes

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25
At what temperature is primary fixation continued
At 4 degrees Celsius
26
Why is fixation continued at 4 degrees Celsius
To slow autolysis and reduce tissue shrinkage
27
What is the largest recommended tissue size for electron microscopy
1 cubic millimeter
28
What is done after primary fixation
Rinsing in 200 millimolar phosphate buffer
29
What is the purpose of rinsing
To remove traces of aldehyde and prevent contamination
30
What buffer is used for rinsing
Sodium cacodylate
31
What is the effective buffering range of sodium cacodylate
5.1–7.4
32
What can be added to the primary fixative
Calcium chloride and/or magnesium chloride
33
Which buffers should not be used with aldehyde fixatives
Veronal buffers containing barbitals
34
What is the purpose of secondary fixation
To increase stability and contrast of fine structure
35
What is used for secondary fixation
1 percent osmium tetroxide in 100 millimolar phosphate buffer for 1–2 hours at 4 degrees Celsius
36
How many times is the specimen washed after secondary fixation
At least 5 times in distilled water
37
What is used for dehydration in tissue processing
Series of ethanols or acetones and propylene oxides
38
Which is preferred for dehydration
Acetone because it causes less lipid loss than ethanol
39
What are the steps and durations for dehydration with acetone or ethanol
30 percent 10 min + 50 percent 20 min + 70 percent 20 min + 90 percent 20 min + 100 percent 3 × 20 min + 100 percent 20 min
40
What is the purpose of infiltration
To fill cell spaces with hard plastic material that tolerates cutting pressure
41
Which resin is used for infiltration
Epoxy resin
42
What is the purpose of embedding
To provide a stable hard matrix for thin sectioning
43
What are the three principal types of resins for embedding
Epoxy resin + Polyester resin + Methacrylate resin
44
Which step follows tissue infiltration with resin
Polymerization
45
How are tissues prepared before polymerization
Wrapped in aluminum foil and placed in appropriate molds
46
Which molds are commonly used for specimen embedding
Beem capsules
47
How is polymerization initiated
Allowed to set overnight at room temperature
48
What is the next step after setting at room temperature
Placed in an oven at 60 degrees Celsius for 2–3 days
49
How is correct polymerization tested
Denting one of the side ridges of the capsule tip with a fingernail
50
Which fixative mixture is used for initial tissue fixation
2.5 percent glutaraldehyde + 2 percent paraformaldehyde in 100 millimolar cacodylate buffer with 2 millimolar calcium chloride and 0.2 percent picric acid
51
How long is the initial fixation
30 minutes
52
What size are specimens cut into after initial fixation
1 cubic millimeter
53
How long is continued fixation performed
16–24 hours at 4 degrees Celsius
54
Which buffer is used for washing after fixation
200 millimolar cacodylate buffer
55
What is used for post-fixation
1 percent osmium tetroxide in 100 millimolar cacodylate buffer for 2 hours at 4 degrees Celsius
56
What is used to remove free cacodylate and/or phosphate ions
Excess distilled water
57
What is used for en bloc staining
2 percent aqueous uranyl acetate for 2 hours at 4 degrees Celsius in the dark
58
Which solvents are used for dehydration
Acetone or ethanol + Propylene oxide
59
Which step follows dehydration
Embedding in resin
60
Which instrument is used for trimming
Dissecting microscope
61
Which tools are used for trimming
Old glass knives or knives not suitable for sectioning
62
What is the goal of trimming
To expose the tip of the tissue
63
What shape is the capsule trimmed to
Pyramid
64
What is the angle of the pyramid sides
About 45 degrees
65
What thickness should sections be for electron microscopy
30–60 nanometers
66
How are sections collected after sectioning
Float onto a surface of liquid and collected on grids
67
How are sections prepared for light microscopy
Placed on microscope slide + Dried + Lightly heat fixed + Stained with toluidine blue
68
Which type of stain deposits electron dense material on the area of interest
Positive stain
69
Which stains are positive stains
Uranyl acetate + Lead citrate
70
Which type of stain penetrates and darkens the interstices between areas of interest
Negative stain
71
Which stains are negative stains
Ammonium molybdate + Uranyl acetate + Uranyl formate + Phosphotungstic acid + Osmium tetroxide + Osmium ferricyanide
72
Which stain is considered the best available
Lead citrate
73
At what pH can lead citrate be used
High pH
74
Which cellular components does lead citrate stain
Nuclear components + Ribosomes + Membranes + Microfilaments + Glycogen
75
How can uranyl acetate be used during dehydration
By making the 50 percent acetone up to 2 percent with the stain
76
How can uranyl acetate be used for sections on grids
As a 2 percent aqueous solution to float or dip sections
77
Which stain is used as a negative stain and does not bind well to cellular material
Phosphotungstic acid
78
What concentration of phosphotungstic acid is used
1–2 percent aqueous solution
79
How long is phosphotungstic acid used during dehydration
About 30 minutes

HISTOPATH LEC - TRANS (26 decks)

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