Module 06: Microscopy

Question 0

What do you during when you fix cells?

Answer 0

Covalently crosslink macromolecules to each other

Question 1

What is the purpose of dehydration when you fix cells?

Answer 1

Prevention of microbial growth and hydrolysis

Question 2

What is the purpose of permeabilizing cells during fixation?

Answer 2

Degrades the membrane, allows access of antibodies and stains

Question 3

What does a microtome do?

Answer 3

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

Question 4

What are the steps of preparing a thick specimen?

Answer 4

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

Question 5

What classes of chemicals are used in chemical fixation?

Answer 5

Acids (acetic acid) and aldehydes (formaldehyde)

Question 6

What processing is required to use phase contrast microscopy?

Answer 6

None; cells are unprocessed

Question 7

How does phase contrast microscopy work?

Answer 7

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

Question 8

What does DIC microscopy stand for?

Answer 8

Differential interference microscopy

Question 9

What is another name for DIC microscopy?

Answer 9

Normanski microscopy

Question 10

Describe the basics of fluorescence.

Answer 10

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)

Question 11

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

Answer 11

Excitation

Question 12

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

Answer 12

Emission

Question 13

excitation (n.)

Answer 13

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

Question 14

How does immunofluoresence work?

Answer 14

Fluorescently labeled antibodies bind directly to antigen

Question 15

How does indirect immunofluoresence work?

Answer 15

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

Question 16

What does DAPI stain?

Answer 16

DNA

Question 17

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

Answer 17

Green fluorescent protein, and it comes from jellyfish

Question 18

What is special about GFP?

Answer 18

It allows us to image and record live cells

Question 19

How is GFP added onto tubulin?

Answer 19

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

Question 20

What does TEM stand for?

Answer 20

Transmission electron microscopy

Question 21

What are two heavy metals used in TEM?

Answer 21

OsO4 and gold

Question 22

What does SEM stand for?

Answer 22

Scanning Electron Microscopy

Question 23

What is the preparation process for SEM?

Answer 23

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

Question 24

What is OsO4?

Answer 24

Osmium tetroxide

Question 25

Does OsO4 bind to polar or non-polar molecules?

Answer 25

Polar molecules

Question 26

How does negative staining work?

Answer 26

Puddle an electron dense solution around a 3D structure

Question 27

What kind of microscopy do you use to look at the result of the "shadowing" process?

Answer 27

TEM

Question 28

What is freeze fracture used for?

Answer 28

Used for looking inside membranes

Question 29

What type of microscopy is used after freeze fracturing?

Answer 29

TEM

Question 30

How big is the average eukaryotic cell?

Answer 30

20 µm

Question 31

How big is the average protein?

Answer 31

2 nm

Question 32

What type of energy is light?

Answer 32

Electromagnetic energy

Question 33

What property of light changes to make the different colors of visible light?

Answer 33

The wavelength

Question 34

How does an electron get to a higher orbital?

Answer 34

Absorb energy

Question 35

How does an electrons get to a lower orbital?

Answer 35

Release energy

Question 36

What is a photon?

Answer 36

A "packet" of light

Question 37

How are frequency and wavelength related?

Answer 37

The higher the frequency, the shorter the wavelength

Question 38

How are electromagnetic energy and wavelength related?

Answer 38

The shorter the wavelength, the higher the energy