Microscopy

Question 1

What is diffraction?

Answer 1

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

Question 2

What is refraction?

Answer 2

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

Question 3

What is resolution?

Answer 3

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

Question 4

Why is there a theoretical limit to resolution?

Answer 4

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

Question 5

What is the theoretical limit to resolution?

Answer 5

200-300nm

Question 6

What wave properties of light can we see?

Answer 6

Wavelength (colour), amplitude (intensity)

Question 7

What wave properties of light can we not see?

Answer 7

Polarisation (without a polariser), phase

Question 8

What is phase (of light)?

Answer 8

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.

Question 9

What does image formation depend on?

Answer 9

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

Question 10

What is wave interference?

Answer 10

The sum of 2 (or more) waves.

Question 11

What is constructive interference?

Answer 11

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

Question 12

What is destructive interference?

Answer 12

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

Question 12

High to low energy colours

Answer 12

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

Question 13

Snell’s law

Answer 13

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.

Question 14

How does a prism refract light?

Answer 14

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

Question 15

How does a lens refract light?

Answer 15

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

Question 16

What is focal length (f)?

Answer 16

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

Question 17

What does focal length of a lens depend on?

Answer 17

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

Question 18

Object > 2f from lens

Answer 18

Miniature image formed between f and 2f

Question 19

Object at 2f from lens

Answer 19

Same size image formed at 2f from lens

Question 20

2f > object > f

Answer 20

Magnified image at >2f

Question 21

Object at f from lens

Answer 21

No image - rays emerge parallel

Question 22

Object < f from lens

Answer 22

No image - rays diverge

Question 23

What is a compound microscope?

Answer 23

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

Question 24

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

Answer 24

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

Question 25

What does the objective lens in a compound microscope do?

Answer 25

Creates image inside the microscope.

Question 26

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

Answer 26

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

Question 27

Where does light come from in an upright microscope?

Answer 27

Underneath the sample.

Question 28

Where does light come from in an inverted microscope?

Answer 28

Above the sample.

Question 29

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

Answer 29

An Airy (fuzzy) disk.

Question 30

What do we use inverted microscopes for?

Answer 30

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

Question 31

Huygen’s principle

Answer 31

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

Question 32

What does Huygen’s principle explain

Answer 32

Why waves are able to propagate behind a blockade.

Question 33

What did Young’s experiment illustrate?

Answer 33

The wave properties of light - interference of 2 waves.

Question 34

What did Young do?

Answer 34

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

Question 35

How did Young maximise interference?

Answer 35

Had light coming from the point sources be in phase.

Question 36

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

Answer 36

Increase the distance between point sources of light.

Question 37

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

Answer 37

Reduce the distance between point sources of light.

Question 38

Where is information about close points on the diffraction pattern?

Answer 38

Further from the origin / optical axis.

Question 39

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

Answer 39

The perimeter.

Question 40

Abbé’s theory of image formation

Answer 40

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

Question 41

Why is an Airy disk formed?

Answer 41

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.

Question 42

What does resolution depend on?

Answer 42

The 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.
And the light’s wavelength.