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Microbiology > 3.1: Microscopy > Flashcards

3.1: Microscopy Flashcards

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1
Q

What are the two units of measurement most commonly used by researchers?

A
  1. micrometer (µm)
    - one-millionth of a meter
    -10 to the power of -6 m
  2. nanometer (nm)
    - one-billionth of a meter
    - 10 to the power of-9 m
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2
Q

The micrometer (µm) is ___1___ of a meter and is commonly designated at ___2___.

A
  1. one-millionth
  2. 10 to the power of -6 m
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3
Q

The nanometer (nm) is ___1___ of a meter and is commonly designated at ___2___.

A
  1. one-billionth
  2. 10 to the power of -9 m
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4
Q

µm stands for what unit of measurement?

A

Micrometer

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5
Q

nm stands for what unit of measurement?

A

Nanometer

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6
Q

The unaided eye can resolve (see clearly) objects typically > ______.

A

200 µm.

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7
Q

Cellular components, organelles, etc. can be as small as ______.

A

0.2 µm in diameter.

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8
Q

What two critical factors influence our ability to see an object?

A
  1. Resolution
  2. Contrast
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9
Q

The ___1___ refers to the distance between two objects at which the objects still can be seen as separate. Thus, the closer two objects are to each other, the greater the ___1___ requirement will be to maintain viewing the two objects as separate.

A
  1. resolution
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10
Q

The resolution refers to the………

A

…… distance between two objects at which the objects still can be seen as separate. Thus, the closer two objects are to each other, the greater the resolution requirement will be to maintain viewing the two objects as separate.

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11
Q

The closer two objects are to each other, the greater the ______ requirement will be to maintain viewing the two objects as separate.

A

resolution

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12
Q

The ___1___ is the difference in light absorbance between two areas (objects). The lower the ___1___ between an object and its background, the harder it will be to see that object.

A
  1. contrast
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13
Q

The contrast is the……

A

…… difference in light absorbance between two areas (objects). The lower the contrast between an object and its background, the harder it will be to see that object.

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14
Q

The greater the ______ between two areas the easier it will be to visualize.

A

Contrast

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15
Q

The type of microscope largely influences the ___1___ and the ___2___.

A
  1. power of resolution
  2. degree of contrast
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16
Q

______ microscopes are the simplest form of light, or optical, microscopes. Light, most often emitted from a standard halogen bulb, enters the microscope from the base (bottom) and is reflected via mirrors towards the sample.

A

Brightfield

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17
Q

Brightfield microscopes are the simplest form of ___1___. Light, most often emitted from a standard halogen bulb, enters the microscope from the ___2___ and is ___3___ towards the sample.

A
  1. light, or optical, microscopes
  2. base (bottom)
  3. reflected via mirrors
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18
Q

BRIGHTFIELD MICROSCOPES: Before the light reaches the sample, it first passes through a _____ converging the light beams into a focused area on the sample.

A

condenser

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19
Q

BRIGHTFIELD MICROSCOPES: The ___1___ controls the amount of light that passes through the sample and into the ___2___.

A
  1. iris diaphragm
  2. objective lens
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20
Q

BRIGHTFIELD MICROSCOPES: The ______ is the lens closest to the sample and yields the greatest amount of magnification.

A

objective lens

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21
Q

BRIGHTFIELD MICROSCOPES: The objective lens is the lens ___1___ sample and yields the ___2___ magnification.

A
  1. closest to the
  2. greatest amount of
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22
Q

BRIGHTFIELD MICROSCOPES: The degree of magnification is directly proportional to the what?

A

amount of light needed

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23
Q

BRIGHTFIELD MICROSCOPES: To image samples clearly at higher magnifications, more what is required?

A

Light

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24
Q

BRIGHTFIELD MICROSCOPES: Once light passes through the sample and the objective lens, it is directed through the ______ to your eye.

A

ocular lens, or eyepiece,

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25
Q

BRIGHTFIELD MICROSCOPES: What is the most common power of an ocular lens?

A

10x

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26
Q

BRIGHTFIELD MICROSCOPES: For a microscope using two lenses (objective and ocular) the total magnification of a specimen is ___1___. Thus, a 40x objective and a 10x ocular result in a total magnification of ___2___.

A
  1. multiplicative
  2. 400x
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27
Q

BRIGHTFIELD MICROSCOPES: In order to visualize cells, samples are most often ___1___ with specific combinations of ___2___ that are taken up by the cell.

A
  1. stained
  2. dyes
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28
Q

BRIGHTFIELD MICROSCOPES: Staining is often required due to the limitation of ______ on unstained cells because the flat and transparent regions of a cell may appear invisible under bright field conditions

A

resolution

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29
Q

BRIGHTFIELD MICROSCOPES: ______ is often required due to the limitation of resolution on unstained cells because the flat and transparent regions of a cell may appear invisible under bright field conditions

A

Staining

30
Q

BRIGHTFIELD MICROSCOPES: Staining typically requires ______ the cells by heat or chemical methods before adding the dye.

A

fixing

31
Q

BRIGHTFIELD MICROSCOPES: Staining typically requires fixing the cells by ___1___ or ___2___ before adding the dye.

A
  1. heat
  2. chemical methods
32
Q

BRIGHTFIELD MICROSCOPES: The cell fixation process produces its own challenges as it ___1___ and may even ___2___ the sample.

A
  1. kills
  2. distort
33
Q

______ have a distinct advantage over bright field microscopy in that they are often able to visualize certain structures that would otherwise be invisible

A

Phase contrast microscopes

34
Q

______ can provide detailed images of live cells without staining.

A

phase contrast microscope

35
Q

By using specialized condensers and objectives, a ______ amplifies the slight differences between cells and the surrounding medium (background) to make the cells highly distinguishable.

A

phase contrast microscope

36
Q

By using ___1___ and ___2___, a phase contrast microscope amplifies the slight differences between cells and the surrounding medium (background) to make the cells highly distinguishable.

A
  1. specialized condensers
  2. objectives
37
Q

Phase Contrast Microscope NOTE

A

Although phase contrast microscopy can image live cells in great detail, often scientists will still use organelle specific dyes to highlight a particular region of the cell. For instance, although the cell membrane, nucleus, and other organelles are readily apparent, a researcher interested in the trans-Golgi (TGN) region may still add a dye that specifically binds within the TGN just to make it more prominent in the final acquired image.

38
Q

______ can be used to visualize cell movements (motility), such as swimming or gliding, without altering the cell morphology commonly brought about from treating the cells with a fixing agent.

A

phase contrast microscopy

39
Q

Phase Contrast Microscopy can be used to visualize cell ______ without altering the cell morphology commonly brought about from treating the cells with a fixing agent.

A

movements (motility), such as swimming or gliding,

40
Q

______ can be used to greatly increase the contrast between a specimen and background, resulting in a dark background with bright objects in it

A

Dark field microscopes

41
Q

Dark field microscopes can be used to greatly increase the ______ between a specimen and background, resulting in a dark background with bright objects in it

A

contrast

42
Q

Unlike brightfield or phase contrast microscopy where light passes directly through the sample, dark field microscopy……

A

…… reflects light off of the specimen at an angle.

43
Q

The reflective approach of Dark field microscopes does not ______.

A

permit the visualization of intracellular structures.

44
Q

______ take advantage of fluorescent molecules called fluorophores to visualize cells on a dark background.

A

Fluorescence microscopes

45
Q

Fluorescence microscopes take advantage of fluorescent molecules called ______ to visualize cells on a dark background.

A

fluorophores

46
Q

FLUORESCENCE MICROSCOPES: Unlike brightfield, the energy of the incoming light is in the form of the ______.

A

ultraviolet (UV) spectrum

47
Q

FLUORESCENCE MICROSCOPES: UV light ___1___ different fluorophores at ___2___, enabling scientists to use a wide array of colors during imaging.

A
  1. excites
  2. varying wavelengths
48
Q

FLUORESCENCE MICROSCOPES: What are the 3 fluorescent colors used to highlight proteins?

A

Red(RFP)
Green(GFP)
Yellow(YFP)

49
Q

FLUORESCENCE MICROSCOPES: These fluorescent proteins alone can be expressed in a cell:

A
  1. nonspecifically illuminating the cell as a whole
  2. linked (coupled) to a normal cellular protein of interest whereby the fluorescent color is indicative of protein movement and localization
  3. or used as tags on molecules or antibodies used to designate the presence (fluorescence detected) or absence (no fluorescence) of a specific protein target.
50
Q

______ combine the usefulness of fluorescence microscopy with the ability to visualize cells in 3-D.

A

Confocal (laser scanning) microscopes

51
Q

Unlike light or fluorescence microscopy where light is focused a single plane (2-D), ______ can capture images in either 2-D or 3-D.

A

confocal microscopy

52
Q

Rather than using halogen (brightfield) or UV (fluorescence) light, confocal microscopes use ______ to focus on a single plane within an object and with a higher degree of accuracy.

A

lasers

53
Q

Rather than using halogen (brightfield) or UV (fluorescence) light, ______ use lasers to focus on a single plane within an object and with a higher degree of accuracy

A

confocal microscopes

54
Q

CONFOCAL MICROSCOPE: Rendering a three-dimensional image is a ______ whereby once an image is taken, the laser then moves to an adjacent plane, captures an image, then repeats this process until the desired depth of the sample has been covered.

A

sequential process

55
Q

______ is used to visualize incredibly small specimens.

A

Electron microscope (TEM and SEM)

56
Q

As light microscopy (brightfield, phase contrast, fluorescence, and confocal) is limited to a resolution of about 0.2 µM, it cannot efficiently visualize viruses or even some subcellular compartments. To circumvent this restriction, electron microscopes use beams of ___1___, which have significantly shorter wavelengths than light, to increases its resolution capacity to less than ___2___!

A
  1. electrons (rather than light)
  2. 1nm—that’s 200x better
57
Q

Tue or False: Electron Microscopes use beams of electrons, which have significantly longer wavelengths than light, to increases its resolution capacity to less than 1nm—that’s 200x better!

A

False. Electrons have a significantly shorter wavelength than light.

58
Q

Electron microscopes requires samples to be ___1___, and the process may alter the ___2___.

A
  1. fixed (killed)
  2. cell structure
59
Q

What are the 3 types of Electron microscopes?

A
  1. Transmission electron microscopes (TEM)
  2. Scanning electron microscopes (SEM)
  3. Scanning transmission electron holography microscopes (STEHM)
60
Q

______ use thin slices of a sample, heavily treated and coated in preservatives, and placed between the electron beam source and the detector. An image is formed from the interactions of the electrons as they pass through the thin sectioning of the sample. This process can be used to visualize subcellular organelles, substructures, and viral particles

A

Transmission electron microscopes (TEM)

61
Q

______ also use a beam of electrons, but the image is obtained as the electrons reflect off (not through) the surface of the specimen. Samples are coated with either gold or palladium to enhance electron reflection. Thus, it can only be used to generate a detailed three-dimensional shell model of the surface of a specimen. Live samples cannot be viewed.

A

Scanning electron microscopes (SEM)

62
Q

______also use an electron beam but coupled with a holography technique to study surfaces of proteins and subcellular structures. It has the capacity to magnify subatomic structures up to 20 million times larger than what can be viewed with the naked eye. While EM can resolve 1 nm (10-9), this has the capacity to resolve 35 pm (a picometer is one-trillionth of a meter, or 10-12) and possibly even smaller.

A

Scanning transmission electron holography microscopes (STEHM)

63
Q

What microscope was used here?

A

Brightfield Microscope:
Adherent cells are imaged using a bright field microscope. As the cells are unstained, they largely appear invisible. Upon closer examination you can discern the nucleus (dark areas) as well as the plasma membrane (thin lines) outlining the shape of the cell.

64
Q

What microscope was used here?

A

Phase Contrast Microscope:
Noticeable detail and depth is achieved to what would normally be a nearly invisible cell. Note the defined cell periphery (plasma membrane) and internal organelles.

65
Q

What microscope was used here?

A

Dark Field Microscope:
The spirochete borrelia (the causative agent of Lyme disease) is often imaged using dark field microscopy. Unlike bright field or phase contrast, the background is dark, while the sample appears illuminated.

66
Q

What microscope was used here?

A

Fluorescence Microscope:
Adherent cells expressing GFP (A) and mRFP (B) allow researchers to monitor the subcellular localization of specific proteins. Protein expression levels, protein localization, and trafficking can then be studied globally, as shown in the merged image (C).

67
Q

What microscope was used here?

A

Confocal (Laser Scanning) Microscope:
Fluorescent staining of adherent cells counterstained against the nucleus (blue), actin (green), and mitochondria (red). Notice the increased resolution and contrast compared to standard fluorescence microscopy. Individual actin filaments are clearly visible (right inset; green).

68
Q

What microscope was used here?

A

Electron Microscope (TEM):
(A) Image from a TEM of a nanoparticle shows the 2-D rendering

69
Q

What microscope was used here?

A

Electron Microscope (TEM):
(B) SEM image is able to capture 3-D architecture of a nanoparticle.

70
Q

What microscope was used here?

A

Microbiology (39 decks)

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