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Cell Biology > Light microscopy > Flashcards

Light microscopy Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

What is diffraction?

A

Scattering or bending of path of incident light by the detailed substructure within the object / sample.

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2
Q

What is refraction?

A

Change in the path of light due to passing through the interface between 2 media with different densities.

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3
Q

What is resolution?

A

The smallest distance between two points on a specimen that can still be distinguished as two separate entities.

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4
Q

Why is there a theoretical limit to resolution?

A

Because lenses can only capture up to a certain angle of diffraction - miss some of the diffraction pattern. Limit to physical size of lenses.

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5
Q

What is the theoretical limit to resolution (value)?

A

200-300nm

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6
Q

What wave properties of light can we see?

A

Wavelength (colour), amplitude (intensity)

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7
Q

What wave properties of light can we not see?

A

Polarisation (without a polariser), phase

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8
Q

What is phase (of light)?

A

The fraction of the oscillation cycle covered up to a certain time. Phase shifting would be moving the wave forward or backward on a diagram.

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9
Q

What does image formation depend on?

A
  • Interactions of the light with matter (diffraction, refraction)
  • Interactions of the light with itself (interference)
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10
Q

What is wave interference?

A

The sum of 2 (or more) waves.

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11
Q

What is constructive interference?

A

The waves are in phase and add to increase amplitude (brighter light).

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12
Q

What is destructive interference?

A

The waves are out of phase and add to decrease amplitude (cancel each other) (dimmer light).

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12
Q

High to low energy colours

A

Violet (short wavelength) to red (long wavelength).

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13
Q

Snell’s law

A

Describes how light (and other waves) change direction when passing through different media. Formula defines relationship between angle of incidence and angle of refraction. The light takes the fastest path.

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14
Q

How does a prism refract light?

A

Different wavelengths are refracted to different extents, creating a rainbow.

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15
Q

How can a lens refract light?

A

Depending on the lens shape, it can converge or diverge light rays.

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16
Q

What is focal length (f)?

A

The distance from the lens at which incident parallel light rays converge (on the horizontal axis) after refraction (image is formed).

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17
Q

What does focal length of a lens depend on?

A
  • Lens curvature (more curve = shorter f)
  • Refractive index
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18
Q

Object > 2f from lens

A

Miniature image formed between f and 2f

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19
Q

Object at 2f from lens

A

Same size image formed at 2f from lens

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20
Q

2f > object > f

A

Magnified image at >2f

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21
Q

Object at f from lens

A

No image - rays emerge parallel

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22
Q

Object < f from lens

A

No image - rays diverge

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23
Q

What is a compound microscope?

A

A microscope with 2 lenses (objective and ocular/eyepiece) working together to focus an image onto the retina.

24
Q

Why do you move the stage (upright) or lens (inverted) in a compound microscope?

A

So the objective lens can focus the light into the designated part of the microscope for the image.

25
Q

What does the objective lens in a compound microscope do?

A

Creates image inside the microscope.

26
Q

What does the ocular / eyepiece lens in a compound microscope do?

A

Magnifies the image created by the objective lens. Diverges the rays so they can be converged by our eye onto our retina.

27
Q

Where does light come from in an upright microscope?

A

Underneath the sample.

28
Q

Where does light come from in an inverted microscope?

A

Above the sample.

29
Q

What does a point source object look like under a microscope?

A

An Airy (fuzzy) disk.

30
Q

What do we use inverted microscopes for?

A

Viewing cells in liquid culture - don’t want to dip lens in liquid.

31
Q

Huygen’s principle

A

Every point on a propagating wavefront serves as source of secondary wavelets.

32
Q

What does Huygen’s principle explain

A

Why waves are able to propagate behind a blockade.

33
Q

What did Young’s experiment illustrate?

A

The wave properties of light - interference of 2 waves.

34
Q

What did Young do?

A

Generate 2 in phase light waves from 2 point sources and record the banded diffraction pattern on a screen (constructive and destructive interference).

35
Q

How did Young maximise interference?

A

Had light coming from the point sources be in phase.

36
Q

How do you reduce the angle between diffraction spots and the optical axis (associated interference)?

A

Increase the distance between point sources of light.

37
Q

How do you increase the angle between diffraction spots and the optical axis (associated interference)?

A

Reduce the distance between point sources of light.

38
Q

Where is information about close points on the diffraction pattern?

A

Further from the origin / optical axis.

39
Q

Where is high resolution information of an object in a diffraction pattern?

A

The perimeter.

40
Q

Abbé’s theory of image formation

A

Light scattered by an object is captured by a lens and refracted so rays reconverge and form an image.

41
Q

Why is an Airy disk formed?

A

There is incomplete interference of diffracted light. The highest angle diffracted light does not enter the optical system therefore can’t produce the final image.

42
Q

What does resolution of a microscope depend on?

A
  • Angle of light that the lens can collect from the sample (depends on lens diameter and distance from sample). Immersion oil improves angle of collection too. (Higher angle of collection = greater resolution). Contributes to numerical aperture (NA value).
    And the light’s wavelength.
  • Wavelength of the light (lower wavelength = greater resolution).
43
Q

What is the maximal numerical aperture (NA) that we are able to achieve with a microscope?

A

1.5

44
Q

What is a useful structure size range for light microscopy?

A

300µm - 300nm (works well for cells, not viruses).

45
Q

Why does shorter wavelength result in higher resolution?

A

The associated interference of 2 spots occurs at a smaller angle to the origin, so can be collected by the microscope.

46
Q

In what situation can wavelength have improve resolution?

A

When it is much smaller than the spacing between the objects.

47
Q

What changes were tested in light microscopes to improve resolution that failed?

A

Use of UV light instead of visible light (shorter wavelength).
Scrapped due to being mutagenic.

48
Q

How can we view dead (transparent) cells?

A

Chemical staining.

49
Q

How can we view live (transparent) cells?

A

Phase contrast microscopy.

50
Q

Where do many blood cell names come from?

A

The dyes used to stain them!

51
Q

What is the theory behind staining objects to visualise them?

A

Objects absorb light of a specific wavelength, and it comes out the other side with a decreased amplitude, dependent on the density of the object. ‘Amplitude objects’.

52
Q

What is the theory behind phase contrast microscopy?

A

Objects refract light and slow it down dependent on their density. This changes the phase of the light so it peaks earlier in comparison to light that did not pass through the object, or passed through a different density object.
Objects also diffract light (change its direction) which is utilised in the microscope design.

53
Q

Who invented phase contrast microscopy?

A

Fritz Zernike

54
Q

How does phase contrast microscopy work?

A
  1. Light diffracted by object is found all over the diffraction plane (between the object and the image plane) and is considered to be phase-shifted by ~wavelength/4 relative to non-diffracted light.
  2. Light not diffracted by object (sometimes called “zeroth order” light) is at the centre of the diffraction pattern.
  3. A special piece of glass (“phase plate”) at diffraction plane can alter phase of most of the diffracted light by an extra wavelength/4 without changing phase of the non-diffracted light.
  4. The diffracted light has its phase shifted by wavelength/2, which means there is destructive interference between the diffracted and non-diffracted light.
  5. Destructive interference is visualised as darkness - denser areas are darker in image.
55
Q

What is a phase plate?

A

A circular piece of glass placed between the object and image plane. It has a thinner ring within it where the diffracted light passes through - phase is changed. The rest of circle is thicker and maintains the phase of the non-diffracted light.

56
Q

Why is the ring of the phase plate darkened?

A

Less of non-diffracted light contributes to the final image. Therefore, interference of diffracted and non-diffracted waves at image plane is not “swamped out” by huge excess of non-diffracted light.

57
Q

What type of microscopy gives a 3D-like image?

A

Differential interference contrast. Also utilises phase differences of diffracted light, but relative differences instead of absolute.

Cell Biology (8 decks)

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