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My Packs: (WIZEPREP) (BIO 2382) > Chapter 2: 2.2 Imaging in Cell Biology > Flashcards

Chapter 2: 2.2 Imaging in Cell Biology Flashcards

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Biology 101 flashcards
1
Q

List:

3 considerations when it comes to microscopy

A
  1. Magnification
  2. Resolution
  3. Clarity
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2
Q

In microscopy:

Magnification means…

A

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

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3
Q

In microscopy:

What does resolution mean?

A

Ability to distinguish between two adjacent objects

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4
Q

True or False:

Larger resolution means a more powerful microscope

A

False, smaller resolution means a more powerful microscope

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5
Q

How is resolution measured?

A

Measured as the distance resolved between two points

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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

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7
Q

In microscopy:

What does clarity mean?

A

Ability to identify different structures

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8
Q

What are the two types of microscopy used today?

A
  1. Light microscopy
  2. Electron microscopy
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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
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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
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11
Q

State resolution required to view:

Tissues

A

1 mm - 100 μm

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12
Q

State resolution required to view:

Cells

A

100 μm - 10 μm

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13
Q

State resolution required to view:

Organelles

A

1 μm - 100 nm

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14
Q

State resolution required to view:

(Macro)molecules

A

10 nm - 0.1 nm

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15
Q

What structures can light microscopy be used to view?

A
  • Tissues
  • Cells
  • Organelles
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16
Q

What structures can electron tomography be used to view?

A
  • Cells
  • Organelles
  • (Macro)molecules
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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)
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18
Q

Define:

Transmission Light Microscopy

A

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

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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)
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20
Q

Describe:

Bright field Transmission Light Microscopy

A

Light passes directly through the specimen (simplest)

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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

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22
Q

Describe:

Phase contrast

A

Specimens surrounded by a halo of light

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23
Q

Describe:

Differential interference contrast (DIC)

A

Images appear 3-D

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24
Q

Describe:

Compound Light Microscope

A

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

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25
Q

List:

Components of a compound microscope (13)

A
  • Eyepiece lens
  • Barrel
  • Objective lenses
  • Stage
  • Diaphragm
  • Lamp
  • Arm
  • Coarse and fine knobs focus
  • Base
  • Stage clips
  • Stage control
  • Power button
  • Dimmer
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26
Q

How are Phase Contrast and DIC Microscopy similiar?

A

Both use the different refractive indexes in the cell to highlight key structures

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27
Q

Describe:

Phase Contrast Microscopy, how it highlights key structures

A

Uses varying wavelengths: creates well defined edges where the refractive index contrasts a lot

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28
Q

Describe:

DIC Microscopy, how it highlights key structures

A

Uses polarized light: Softens the edges but resolutes the 3D structure more for better detail

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29
Q

Define:

Emission Light Microscopy

A

An external source of light is used to excite fluorescent molecules present in the specimen, causing emissino of light from those molecules

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30
Q

List:

Types of Emission Light Microscopy

A
  1. Epifluorescence
  2. Confocal
31
Q

Describe:

Epifluorescence Emission Light Microscopy

A

Fluorescent molecules throughout the entire specimen are illuminated, and they all emit light

32
Q

Describe:

Epifluorescence Emission Light Microscopy samples

A

Sample may look blurry; poor resolution of fluorescent molecules

33
Q

Describe:

Confocal Emission Light Microscopy samples

A

Samples will have good resolution of fluorescent molecules

34
Q

What is the illuminated plane in confocal emission light microscopy known as?

A

Optical slice or Z stacks

35
Q

List:

Staining and fluorescent dye techniques (3)

A
  1. Direct staining
  2. Indirect staining (immunofluorescence)
  3. GFP tagging
36
Q

Describe:

The physics/optics of a compound microscope

A
  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
Q

Define:

Direct staining

A

Dyes known as flurochromes bind directly to molecule/region of interest

38
Q

What does direct staining work with? (4)

A
  • Membranes
  • Nucleus (DNA)
  • Mitochondria
  • Cytoskeleton
39
Q

Describe:

Fluorochromes

(How they work)

A

Have electrons that when excited then relaxed, release energy at a wavelength that is visible

40
Q

How are fluorochromes excited?

A

By using UV light which is a shorter wavelength

41
Q

What is the released energy at a visible wavelength in direct staining known as?

A

Emission

42
Q

Describe:

The basic process of direct staining

A
  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
Q

Define:

Indirect staining

A

A primary antibody binds to the molecule of interest; a secondary antibody, with attached fluorescent marker, binds the primary antibody

44
Q

What is indirect staining also known as?

A

Immunofluorescence

45
Q

For excitation and emission, describe:

  1. Wavelength
  2. Energy
A

Excitation
* Shorter wavelength
* Higher energy

Emission
* Longer wavelength
* Less energy

46
Q

Define:

GFP tagging

A

A gene encoding green fluorescence protein is attached to the gene of interest

47
Q

What is GFP tagging used for?

A

Used to observe proteins inside a cell

48
Q

True or False:

In GFP tagging, the GFP gene is transcribed along with the gene of interest

A

True

49
Q

Is the GFP gene transcribed? Is the transcription also translated?

A
  1. Yes, the GFP gene is transcribed
  2. Yes, the GFP gene is translated, too
50
Q

Define:

FRET

A

Fluorescence Resonance Energy Transfer (FRET)
* A technique used to look at protein-protein interactions

51
Q

In FRET:

Explain the setup of FRET

A
  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
Q

In FRET:

Define and explain CFP

A

Cyan fluorescent protein
* Emits blue visible light
* Can also stimulate fluorescence of other molecules

53
Q

In FRET:

Define and explain YFP

A

Yellow fluorescent protein
* Requires excitation energy to emit yellow light

54
Q

In FRET:

Explain the results and its purpose

A
  • 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
Q

Explain:

Electron microscopy

A

Uses electrons to illuminate specimens; can be used to view smaller structures than it is possible with a light microscope

56
Q

List:

The types of Electron Microscopy

A
  1. Transmission electron microscopy (TEM)
  2. Scanning electron microscopy (SEM)
57
Q

Describe:

Transmission electron microscopy (TEM)

A

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
Q

Describe:

Scanning electron microscopy (SEM)

A

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
Q

True or False:

Electron micrographs can only be shown in black-and-white

A

False, they are often shown in black-and-white but may be coloured during processing

60
Q

What is the resolution of TEM?

A
  • Resolution of about 0.005-0.1 nm (2000 times more powerful than light microscopy)
61
Q

True or False:

Resolution of TEM does not depend on the refractive index of the medium

(Explain why)

A

True, as it uses electrons and it passes through without change

62
Q

State:

The formula for resolution of TEM

A

d = 0.61λ / α

63
Q

Overview:

Types of Microscopy

A

Light Microscopy
* Transmission (Bright/Dark field, Phase contrast, DIC)
* Emission (Epifluorescence, confocal)

Electron Microscopy
* TEM
* SEM

64
Q

For Light Microscopy, state:

  1. Resolution limit
  2. Advantages
  3. Disadvantages
A
  1. 200 nm
  2. Can image live cells, can use coloured stains, can image whole tissues
  3. Low resolution
65
Q

In TEM, state:

  1. Resolution limit
  2. Advantages
  3. Disadvantages
A
  1. 0.2 nm
  2. High resolution and detail
  3. Sample prep kills specimen, difficult sample prep, colourless, no 3D information
66
Q

In SEM, state:

  1. Resolution limit
  2. Advantages
  3. Disadvantages
A
  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
Q

State the relative size of cellular structure (approximate):

Cell

A

10-100 μm

68
Q

State the relative size of cellular structure (approximate):

Nucleus

A

3-10 μm

69
Q

State the relative size of cellular structure (approximate):

Mitochondrion

A

0.5-3.0 μm

70
Q

State the relative size of cellular structure (approximate):

Chloroplast

A

5 μm

71
Q

State the relative size of cellular structure (approximate):

Endoplasmic reticulum

A

10-20 μm

72
Q

State the relative size of cellular structure (approximate):

Golgi apparatus

A

20-25 μm

73
Q

State the relative size of cellular structure (approximate):

Lysosome

A

0.2-1 μm

74
Q

State the relative size of cellular structure (approximate):

Peroxisome

A

0.2-1 μm

My Packs: (WIZEPREP) (BIO 2382) (20 decks)

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