Chapter 2: 2.2 Imaging in Cell Biology Flashcards

1
Q

List:

3 considerations when it comes to microscopy

A
  1. Magnification
  2. Resolution
  3. Clarity
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

In microscopy:

Magnification means…

A

The image size produced by the microscope is much larger than the actual size of the object

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

In microscopy:

What does resolution mean?

A

Ability to distinguish between two adjacent objects

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

True or False:

Larger resolution means a more powerful microscope

A

False, smaller resolution means a more powerful microscope

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

How is resolution measured?

A

Measured as the distance resolved between two points

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

State:

The formula for resolution

A

d = 0.61λ / n(sinα)
* d is distance resolved between two points
* λ is wavelength of the light
* n is refractive index of the medium/solution that the sample is submerged in
* α is half the angle of light entering the objective lens

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

In microscopy:

What does clarity mean?

A

Ability to identify different structures

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What are the two types of microscopy used today?

A
  1. Light microscopy
  2. Electron microscopy
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

For light microscopy:

  1. Describe
  2. State resolution and magnification
A
  1. Uses light and lenses to magnify images
  2. 200 nm resolution and >2000x magnification
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

For electron microscopy:

  1. Describe
  2. State resolution and magnification
A
  1. Uses a beam of electrons as a source of illumination
  2. Up to 50 nm resolution and 10,000,000x magnification
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

State resolution required to view:

Tissues

A

1 mm - 100 μm

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

State resolution required to view:

Cells

A

100 μm - 10 μm

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

State resolution required to view:

Organelles

A

1 μm - 100 nm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

State resolution required to view:

(Macro)molecules

A

10 nm - 0.1 nm

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

What structures can light microscopy be used to view?

A
  • Tissues
  • Cells
  • Organelles
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What structures can electron tomography be used to view?

A
  • Cells
  • Organelles
  • (Macro)molecules
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What light wavelengths are used to contribute to the resolving power of each microscopy technique?

A
  • Light (400-700 nm)
  • Electron (<300 nm)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Define:

Transmission Light Microscopy

A

An external source of light passes through the specimen, and is observed by the viewer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

List:

Types of Transmission Light Microscopy

A
  1. Bright field
  2. Dark field
  3. Methods to increase contrast (Phase contrast and Differential interference contrast/DIC)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Describe:

Bright field Transmission Light Microscopy

A

Light passes directly through the specimen (simplest)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Describe:

Dark field Transmission Light Microscopy

A

Light source is aligned such that directly-transmitted (unscattered) light that passes through the specimen is minimized; only scattered light is observed

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Describe:

Phase contrast

A

Specimens surrounded by a halo of light

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Describe:

Differential interference contrast (DIC)

A

Images appear 3-D

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

Describe:

Compound Light Microscope

A

Objective lens magnifies by 100x and ocular lens by 10x for a total of 1000x magnification

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
# List: Components of a compound microscope (13)
* Eyepiece lens * Barrel * Objective lenses * Stage * Diaphragm * Lamp * Arm * Coarse and fine knobs focus * Base * Stage clips * Stage control * Power button * Dimmer
26
How are Phase Contrast and DIC Microscopy similiar?
Both use the different refractive indexes in the cell to highlight key structures
27
# Describe: Phase Contrast Microscopy, how it highlights key structures
Uses varying wavelengths: creates well defined edges where the refractive index contrasts a lot
28
# Describe: DIC Microscopy, how it highlights key structures
Uses polarized light: Softens the edges but resolutes the 3D structure more for better detail
29
# Define: Emission Light Microscopy
An external source of light is used to excite fluorescent molecules present in the specimen, causing emissino of light from those molecules
30
# List: Types of Emission Light Microscopy
1. Epifluorescence 2. Confocal
31
# Describe: Epifluorescence Emission Light Microscopy
Fluorescent molecules throughout the entire specimen are illuminated, and they all emit light
32
# Describe: Epifluorescence Emission Light Microscopy samples
Sample may look blurry; poor resolution of fluorescent molecules
33
# Describe: Confocal Emission Light Microscopy samples
Samples will have good resolution of fluorescent molecules
34
What is the illuminated plane in confocal emission light microscopy known as?
**Optical slice** or **Z stacks**
35
# List: Staining and fluorescent dye techniques (3)
1. Direct staining 2. Indirect staining (immunofluorescence) 3. GFP tagging
36
# Describe: The physics/optics of a compound microscope
1. Image 1 is created from the object through the objective lens 2. Image 2 (the one we see) is created from Image 1 through the eyepiece lens
37
# Define: Direct staining
Dyes known as flurochromes bind directly to molecule/region of interest
38
What does direct staining work with? (4)
* Membranes * Nucleus (DNA) * Mitochondria * Cytoskeleton
39
# Describe: Fluorochromes | (How they work)
Have electrons that when excited then relaxed, release energy at a wavelength that is visible
40
How are fluorochromes excited?
By using UV light which is a shorter wavelength
41
What is the released energy at a visible wavelength in direct staining known as?
Emission
42
# Describe: The basic process of direct staining
1. Light is reflected to the specimen 2. The light excites the fluorochromes on the specimen 3. The emission is shown through the lens
43
# Define: Indirect staining
A primary antibody binds to the molecule of interest; a secondary antibody, with attached fluorescent marker, binds the primary antibody
44
What is indirect staining also known as?
Immunofluorescence
45
# For excitation and emission, describe: 1. Wavelength 2. Energy
Excitation * Shorter wavelength * Higher energy Emission * Longer wavelength * Less energy
46
# Define: GFP tagging
A gene encoding green fluorescence protein is attached to the gene of interest
47
What is GFP tagging used for?
Used to observe proteins inside a cell
48
# True or False: In GFP tagging, the GFP gene is transcribed along with the gene of interest
True
49
Is the GFP gene transcribed? Is the transcription also translated?
1. Yes, the GFP gene is transcribed 2. Yes, the GFP gene is translated, too
50
# Define: FRET
Fluorescence Resonance Energy Transfer (FRET) * A technique used to look at protein-protein interactions
51
# In FRET: Explain the setup of FRET
1. One protein is cloned (like GFP) so that it contains a CFP 2. The other protein which is suspected to interaction with the first protein is labelled with YFP
52
# In FRET: Define and explain CFP
Cyan fluorescent protein * Emits blue visible light * Can also stimulate fluorescence of other molecules
53
# In FRET: Define and explain YFP
Yellow fluorescent protein * Requires excitation energy to emit yellow light
54
# In FRET: Explain the results and its purpose
* When the two proteins interact and come together, yellow fluorescence is observed (CFP transfers energy to YFP) * If no yellow is seen, one can assume the proteins do not interact
55
# Explain: Electron microscopy
Uses electrons to illuminate specimens; can be used to view smaller structures than it is possible with a light microscope
56
# List: The types of Electron Microscopy
1. Transmission electron microscopy (TEM) 2. Scanning electron microscopy (SEM)
57
# Describe: Transmission electron microscopy (TEM)
Specimen is cut into extremely thin sections and stained with a heavy metal to increase electron density * An electron beam passes through the section (similar to transmission light microscopy)
58
# Describe: Scanning electron microscopy (SEM)
An electron beam is scanned across the surface of a specimen coated with a very thin layer of a heavy metal * The metal emits electrons, and a 3D image of the surface can be generated
59
# True or False: Electron micrographs can only be shown in black-and-white
False, they are often shown in black-and-white but may be coloured during processing
60
What is the resolution of TEM?
* Resolution of about 0.005-0.1 nm (2000 times more powerful than light microscopy)
61
# True or False: Resolution of TEM does not depend on the refractive index of the medium | (Explain why)
True, as it uses electrons and it passes through without change
62
# State: The formula for resolution of TEM
d = 0.61λ / α
63
# Overview: Types of Microscopy
Light Microscopy * Transmission (Bright/Dark field, Phase contrast, DIC) * Emission (Epifluorescence, confocal) Electron Microscopy * TEM * SEM
64
# For Light Microscopy, state: 1. Resolution limit 2. Advantages 3. Disadvantages
1. 200 nm 2. Can image live cells, can use coloured stains, can image whole tissues 3. Low resolution
65
# In TEM, state: 1. Resolution limit 2. Advantages 3. Disadvantages
1. 0.2 nm 2. High resolution and detail 3. Sample prep kills specimen, difficult sample prep, colourless, no 3D information
66
# In SEM, state: 1. Resolution limit 2. Advantages 3. Disadvantages
1. 0.2 nm 2. High resolution and detail, can view 3D surface 3. Sample prep kills cells, colourless, cannot see inside of structures
67
# State the relative size of cellular structure (approximate): Cell
10-100 μm
68
# State the relative size of cellular structure (approximate): Nucleus
3-10 μm
69
# State the relative size of cellular structure (approximate): Mitochondrion
0.5-3.0 μm
70
# State the relative size of cellular structure (approximate): Chloroplast
5 μm
71
# State the relative size of cellular structure (approximate): Endoplasmic reticulum
10-20 μm
72
# State the relative size of cellular structure (approximate): Golgi apparatus
20-25 μm
73
# State the relative size of cellular structure (approximate): Lysosome
0.2-1 μm
74
# State the relative size of cellular structure (approximate): Peroxisome
0.2-1 μm