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Module 06: Microscopy Flashcards

(39 cards)

0
Q

What do you during when you fix cells?

A

Covalently crosslink macromolecules to each other

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1
Q

What is the purpose of dehydration when you fix cells?

A

Prevention of microbial growth and hydrolysis

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2
Q

What is the purpose of permeabilizing cells during fixation?

A

Degrades the membrane, allows access of antibodies and stains

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3
Q

What does a microtome do?

A

It cuts tissue into sections thin enough that light can transmit rough

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4
Q

What are the steps of preparing a thick specimen?

A

Fixing the tissue, dehydration, embedding in block of wax, microtoming

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5
Q

What classes of chemicals are used in chemical fixation?

A

Acids (acetic acid) and aldehydes (formaldehyde)

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6
Q

What processing is required to use phase contrast microscopy?

A

None; cells are unprocessed

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7
Q

How does phase contrast microscopy work?

A

Uses the different in phases between direct (unaltered) and diffracted (altered by specimen) light

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8
Q

What does DIC microscopy stand for?

A

Differential interference microscopy

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9
Q

What is another name for DIC microscopy?

A

Normanski microscopy

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10
Q

Describe the basics of fluorescence.

A

A photon causes an electron to move to an excited state, which then drops to a lower, more stable state. This energy is released as an emitted photon (of a longer wavelength)

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11
Q

What is it called when a photon causes an electron to move to a higher energy state?

A

Excitation

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12
Q

What is it called when an electron drops to a more stable energy state and releases a photon?

A

Emission

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13
Q

excitation (n.)

A

the act of a photon exciting an electron into a higher energy state

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14
Q

How does immunofluoresence work?

A

Fluorescently labeled antibodies bind directly to antigen

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15
Q

How does indirect immunofluoresence work?

A

An unlabeled primary antibody binds to the antigen; secondary antibodies labeled with a fluorescent dye binds to the primary antibodies

16
Q

What does DAPI stain?

17
Q

What does GFP stand for and where does it come from?

A

Green fluorescent protein, and it comes from jellyfish

18
Q

What is special about GFP?

A

It allows us to image and record live cells

19
Q

How is GFP added onto tubulin?

A

DNA coding for GFP is added on to the sequence that encodes for tubulin; the end result is a fusion protein of tubulin and GFP

20
Q

What does TEM stand for?

A

Transmission electron microscopy

21
Q

What are two heavy metals used in TEM?

A

OsO4 and gold

22
Q

What does SEM stand for?

A

Scanning Electron Microscopy

23
Q

What is the preparation process for SEM?

A

Fixation, dehydration, soaked in OsO4, then sprayed with a fine coat of gold

24
What is OsO4?
Osmium tetroxide
25
Does OsO4 bind to polar or non-polar molecules?
Polar molecules
26
How does negative staining work?
Puddle an electron dense solution around a 3D structure
27
What kind of microscopy do you use to look at the result of the "shadowing" process?
TEM
28
What is freeze fracture used for?
Used for looking inside membranes
29
What type of microscopy is used after freeze fracturing?
TEM
30
How big is the average eukaryotic cell?
20 µm
31
How big is the average protein?
2 nm
32
What type of energy is light?
Electromagnetic energy
33
What property of light changes to make the different colors of visible light?
The wavelength
34
How does an electron get to a higher orbital?
Absorb energy
35
How does an electrons get to a lower orbital?
Release energy
36
What is a photon?
A "packet" of light
37
How are frequency and wavelength related?
The higher the frequency, the shorter the wavelength
38
How are electromagnetic energy and wavelength related?
The shorter the wavelength, the higher the energy