Lecture 4: Microscopic techniques

Question 1

Wavelength

Answer 1

The distance from a point in the cycle to the corresponding point in the next cycle

Question 2

Frequency

Answer 2

The number of vibrations of a given wavelength in one second

Question 3

How does wavelength relate to frquency?

Answer 3

Longer wavelengths vibrate fewer times so the longer the wavelength, the lower the frequency.

Question 4

What are the frequencies for visible light?

Answer 4

Red: 4.3 x 10 ¹⁵ Hz
Yellow: 5.4 x 10¹⁵ Hz
Violet: 7.5 x 10¹⁵ Hz

Question 5

How does light travel in a homogenous material?

Answer 5

Light travels in a straigth line from a source and reaches a definite and constant speed in any given homogenous medium or material

Question 6

What happens to waves in a vacuum?

Answer 6

In a vacuum, all waves in the electromagnetic spectrum travel at the same speed as nothing is interacting with it.
3 x 10⁸ m/s-1

Question 7

What happens when a wave enters a material after a vacuum?

Answer 7

As any wave enters a material from a vacuum it slows down. Whilst it’s slowing down, as long as it’s in the same material, it will stay at the same speed.

Question 8

What is the equation for Velocity?

Answer 8

Velocity (c) = Frequency (f) x Wavelength (λ)

Question 9

What is absorption?

Answer 9

When a photon of light comes from one material and hits the interface of a material but does not exit again as it’s all absorbed.

Question 10

What can you learn from absorption?

Answer 10

The colour of the matieral as different colours absorb different amounts of light.

Darker materials absorb more light.

Question 11

What is reflection?

Answer 11

The light ray is turned back into the incident material instead of travelling on into the new material.

Question 12

What is specular reflection?

Answer 12

Specular reflection is when a light path hits a material and all of it is turned backed (reflected away).

Nothing travels through the material and nothing is absorbed.

Question 13

What happens when you change the wavelength in specular reflection?

Answer 13

• If you change wavelength (changing the colour) changing the angle means the incidence reflected angle matches exactly the reflected angle on the same side, they remain the same.
• This is the case for perfect specular reflection however few materials actually have this.

Question 14

What happens when you increase surface roughness is specular reflection?

Answer 14

• If you increase surface roughness, multiple colours begin to show and broaden out.
• It comes of at different angles and you begin to get diffuse reflection.

Question 15

What happens as you increase surface roughness in specular reflection?

70-80% surface roughness

Answer 15

As you increase surface roughtness, diffuse reflection increase and specular reflection decreases.

Once you get to a surface roughness of around 70-80% you’ll get virtually no specular reflection and all is diffusely reflected in multiple directions

Question 16

What is refraction?

Transparent material

Answer 16

• The light ray’s path is bent when it passes from one transparent material to another transparent material where its velocity changes.
• Light passes through at an angle, the angle is representative of the material and as a result of a change in velocity.

Question 17

The difference in refractive index…

Answer 17

The difference in refractive index defines how much refraction occurs.

Question 18

What does Snells law tell us?

Answer 18

The relationship between two materials and the ratio between them.

Question 19

Equation for n21

Answer 19

sinθ1 / sinθ2

Question 20

n2/n1 = ?

Answer 20

n2/n1 = v1 / v2

Question 21

sinθ1/sinθ2 = ? = ? = ?

Answer 21

sinθ1/sinθ2 = n21 = n2 / n1 = v1 / v2

Question 22

What is resolution?

Answer 22

Ability to distinguish between 2 points on the specimen.

Question 23

What is the resolution on a light microscopy?

Answer 23

Can reach values close to 200nm

Question 24

What defines the lens resolution?

Answer 24

Spot size (& hence λ) defines the lens resolution.

Question 25

Increased diameter of spot size =

Answer 25

increased diameter = increased magnification + increased angle aperture

Question 26

For a light microscope the optimum resolution is on the order of ?

Answer 26

For a light microscope the optimum resolution is on the order of ~1μm

Question 27

What do lenses have that affect the resolution?

Answer 27

Spherical and chromic abberations

Question 28

What is depth of focus?

Answer 28

• Ability to maintain focus over a range of depths within the specimen.
• Depth of focus is how much of what we’re looking at remains in focus at the same time.
• How many different focal planes you can pick out?

Question 29

How is depth of focused affected when magnication increases?

Answer 29

In a light micrscope depth of focus decreases when magnification increases

Question 30

What is the depth of focus on a light micrscope?

Answer 30

Comparatively low

Question 31

What is field of view?

Answer 31

The Size of the specimen that can be imaged at the same time.

Question 32

What are lenses used for in optical microscopy?

Answer 32

In optical microscopy, lenses are used to focus (refract) incoming light (from a sample) to a point.

Question 33

What influences the ability of the lens to resolve details of a sample?

Answer 33

The ability of the lens to resolve details of a sample is influenced by the quality of the lens but is ultimately limited by diffraction.

Question 34

What is the diameter of the airy disk depedant on?

Answer 34

The spot size (d) is given by the diameter of the Airy disk which is dependent on the wavelength of the light, focal length and lens diameter.

Question 35

Increasing the lens diameter…

Answer 35

Increasing the lens diameter increases the angle aperture therefore increasing magnification.

Question 36

How does wavelength affect resolution

Answer 36

Shorter wavelengths you can get an improved resolution.

Question 37

What affect does using a single wavelength have?

Answer 37

Often if you’re changing down to a single of wavelength you’ll have fewer photons and much less light going through. This makes it more difficult to see.

Question 38

Equation for spot size (d)

Answer 38

d = 1.22 x λ x focal length / lens diameter

Question 39

Higher magnification…

Answer 39

Higher magnification requires more complicated objective systems in order to combat aberrations.

Question 40

What do more lenses allow you to do and what is the drawback of this?

Answer 40

• More lenses allow you to focus and move closer and control the light in order to improve resolution.
• More lenses = Higher cost

Question 41

What is the stereoscopic microscope good for examining?

Answer 41

• Good for bulky artefacts
• Great first step when looking at physical features of trace evidence

Question 42

How do we obtain magnification images of small samples?

Answer 42

We use visible light and a system of lenses to obtain magnification images of small samples

Question 43

What is absorption?

Answer 43

When a photon of light comes from one material and hits the interface of a material but does not exit again as it’s all absorbed.

Question 44

What does absorption result in?

Answer 44

Results in thermal, electrical or chemical changes as the energy in the light has to go somewhere.

Question 45

What materials absorb more light?

Answer 45

Darker materials

Question 46

What happens when you get to 70-80% surface roughness?

Answer 46

• Once you get to a surface roughness of around 70-80% you’ll get virtually no specular reflection and all is diffusely reflected in multiple directions
• This varies, diffuse reflection will vary by wavelength going in.

Question 47

What is refraction afffected by?

Answer 47

It is affected by the materials involved, the angle of the incident ray of light and the wavelength of the incident ray.

Question 48

What is the angle of incidence?

Answer 48

This is the angle the light is coming in at.