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Trace evidence > Lecture 5: Microscopic Techniques > Flashcards

Lecture 5: Microscopic Techniques Flashcards

1
Q

What are the general features of a stereoscopic microscope?

A
  • Most frequently used in Forensic Science
  • 10-125x Range
  • Larger working distance
  • Good for bulky artefacts
  • Great first step when looking at physical features of trace evidence
  • Wide field of view
  • Great depth of focus
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2
Q

What types of illumantion does a compound microscope use?

A
  • Transmission illumination
  • Reflected illumnation
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3
Q

What is transmitted illumination?

A

When light passes from below the sample

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4
Q

What is reflected illumination?

A

Illumination of the sample from the objective side

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5
Q

What do diaphragms do?

A

They help to focus the incoming light

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6
Q

What does a comparison microscope allow?

A
  • Allows point-by-point and side-by-side comparison to determine if two samples are from the same source
  • This is because two identical microscopes are connected to a single comparison eyepiece or screen
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7
Q

What are the main features of a compound micrscope?

A
  • 40-450x range (up to 1000x)
  • Precise focus and light intensity control
  • X-Y stage to move around sample
  • Capable of reflected and transmitted illumination
  • Diaphragms
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8
Q

How is the fluorescence microscope different to a stereoscopic one?

A
  • It illuminates a sample in the ultraviolet wavelength range
  • Illumination causes some materials to fluoresce so they can be observed, counted, sized and mapped
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9
Q

What can a fluorescent micrscope be used for?

A

It is used to identify single cells and heavily used in biological sciences

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10
Q

How does normal light react?

A
  • Waves vibrating in every direction perpendicular to the direction of travel
  • It won’t have any polarisation, it’s not aligned in any direction.
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11
Q

What is linearly polarised light?

A

When waves are vibrating in one direction

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12
Q

How does polarised light microscopy work?

A
  • The polariser only allows a specific polarisation of light
  • As the light passes through the sample it gains some additional polarisation
  • The change in polarisation is measured by another polariser
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13
Q

In polarised light microscopy, what causes the change in polarisation?

A

The change in polarisation is as a result of the specimen which gives us information of the birefringence of the specimen and thus allowing us to link to a particular material.

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14
Q

Why do we get additional polarisation when the light passes through the sample?

A

The plane of light travels at a different speed when travelling through the material.

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15
Q

How is the change in polarisation measured in polarised light microscopy?

A

In order to measure it, you need another polariser and that polariser is rotated and we measure whatever is coming through the polariser on the other side as a function of the polarisation going in.

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16
Q

How can normal light become polarised?

A

Normal light can become polarised if it passes through a material that only allows transmission of rays in a particular direction, such as a crystal, or a film.

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17
Q

What is an anisotropic material?

A

An anisotropic material is something that has different properties in different directions of light.

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18
Q

What is an isotropic substance?

A

A substance that has the same properties in every direction that we pass light throught it.

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19
Q

How does brightfield microscopy illuminate a sample?

A
  • Brightfield microscopy uses light from the lamp source under the microscope stage to illuminate the specimen.
  • It is gathered in the condenser, then shaped into a cone where the apex is focused on the specimen.
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20
Q

What happens to the light in brightfield microcopy after it passes through the condenser?

A
  • After its passed through the condenser, everything is focused onto the sample as we want to illuminate it.
  • It passes through the sample and then everything is collected in the lens on the other side.
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21
Q

What is needed for a specimen to be seen in brightfield microscopy?

A
  • There needs to be contrast between the stage medium and the specimen
  • To view a specimen, the light rays that pass through it must be changed enough in order to contrast.
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22
Q

What can you do to a specimen if it can’t be seen in brightfield microscopy and what is the drawback of this?

A

A specimen can be stained in oder to visualise it but this can be destructive.

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23
Q

Where is ther light focussed in brightfield microscopy and where is it collected?

A

We want to illuminate the specimen so all teh light is focused onto it and then everything is collected by the lens on the other side.

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24
Q

What is contrast?

A

Contrast is the difference between the refractive index between two materials.

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25
Q

What determines the speed at which light passes through something?

A

The speed at which something passes through changes as a function of the refractive index of the material

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26
Q

If you get a change in refractive index what do you also get?

A

When light passes from one RFI to the next it will change and you you’ll get some change in speed and therefore change in angle of the material as it passes through.

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27
Q

Change in speed =
?

A

Change in speed = change in angle

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28
Q

Other than staining a specimen, how else can you create contrast between the specimen and stage media?

A

You can change the material of the media.

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29
Q

What does the special condenser do in darkfield microscopy?

A
  • Darkfield microscopy use special condenser which forms a hollow cone to collect only highly refracted light.
  • The objective lens sits in the dark hollow of this cone and light directly transmitted through the sample misses the lens and is not collected.
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30
Q

What does the field of view look like in darkfield micrscopy when there is no specimen present?

A
  • It will be entirely dark.
  • If there’s no sample, all of the light we’ve got illuminating is going to miss the lens and therefore when it passes through our stage into our eyepiece so we aren’t going to see anything.
31
Q

How does a sample appear on a stage in darkfield microscopy and why?

A
  • A sample placed on the stage appears bright against a dark background as only the scattered light is collected.
  • Thus providing contrast without staining.
32
Q

What does the filter do in darkfield microscopy?

A
  • In darkfield we put the filter in the middle so a cone of light is forced to pass around the sides.
  • This means as the light comes through the cone and is focused on the sample, you’ll be able to see the sample.
  • Due to the light being pushed to the edges we’ve created a case where non of the light is collected by the lens.
33
Q

How does darkfield microscopy work?

A
  • Once we put a sample in, its going to start scattering the light, the light is going to change its direction because its going to have a material and its going to have energy.
  • The small amount of light that is scattered comes up and passes through to where its collected in the lens.
  • We only see the light that has changed directions, it’s scattered and has interacted with the material some way so we have created our own contrast.
34
Q

What is the difference between brightfield microscopy and darkfield microscopy?

A
  • In darkfield microscopy we create our own contrast and we don’t see any light that is purely transmitted
35
Q

What is the problem with darkfield microscopy?

A
  • As we have to create our own contrast it takes along time to get a good resolution.
  • This is because depending on what our sample is, how much it scatters, how much is there, etc, it might take a long time for it to pass through.
  • This means it’s not a good technique for rapid analysis of samples or if you want something in high detail.
36
Q

How can you improve darkfield microscoy and what is the drawback of this?

A
  • You can ramp up the power of the lamp in order to get more photons of light and more light passing through that is scattered and collected allowing us to see the sample.
  • This can often lead to burning or overheating the sample.
37
Q

What is refraction dependant on?

A

Both the degree to which the light ray bends and the direction it bends are related to the refractive index values of the two substances.

38
Q

What does Snell’s law define?

A

Snells law defines how much change there is from a light hitting and interface and passing down.

39
Q

What does Snell’s law say about isotropic substances?

A

Snell’s Law dictates for isotropic substances, the change in the propagation direction of the incident light is related to the change in the velocity of the light transmitted into the crystal.

40
Q

What determines the change in propagation direction of the incident light?

A

This is determined by the RI difference between the particle and mounting medium.

41
Q

What is an isotropic substance?

A

A substance that only has 1 RI and no change in orientations.

42
Q

What is the only optical property that can be determined for isotropic substances?

A

Refractive Index Values

43
Q

What does changing the mounting media of an unknown sample allow?

A

Changing the mounting medium (change the RI of mounting media) of our unknown sample allows us to pick out particular particles and possibly identify what the RI of the unknown sample is.

44
Q

What does maximising the difference in RI lead to?

A

Maximising the change in RI leads to biggest deviation in our angle, if it deviates the most it means you’ll see the edges much more clearly.

45
Q

More deviation in angle = ?

A

More deviation in angle = Clearer edges

46
Q

Why does a larger difference in RI lead to defined edges?

A

With a large difference in refractive index, light incident on the particle deviates greatly from its original path and fails to enter the objective lens

47
Q

What happens to light if the RI changes?

A

The light will change energy as it’s passing through.

48
Q

What is reflected illumination useful for?

A

It is useful for paper materials or surfaces.

49
Q

What are the features of normal light?

A
  • Waves vibrating in every direction perpendicular to the direction of travel
  • It won’t have any polarisation, it’s not aligned in any direction
50
Q

How can normal light become polarised?

A

Normal light can become polarised if it passes through a material that only allows transmission of rays in a particular direction, such as a crystal, or a film.

51
Q

Why does light gain additional polarisation in polarised light microscopy?

A

The light travels at different speed through the material.

52
Q

When is polarisation useful for forensics?

A

Polarisation is useful in forensic microscopy when applied to anisotropic substances

53
Q

What could measuring the RI of an unknown material help with?

A

For anything that changes it means the light will change its energy as its passing through. If we can measure the RI of a particular unknown evidence type of material it could help us identify the material or at least compare it to a known sample of something.

54
Q

What does snells law allow us to measure?

A

Snells law allows us to measure the refractive index of a variety of different things.

55
Q

How is a materials refractive index be determined?

A

This can be rapidly obtained by how close (or far away) the refractive index value of a particle is to that of its mounting medium

56
Q

What is the contrast of a particle in a particular mounting media a qualitative assessment of?

A

The contrast of a particle in a particular mounting medium is essentially a qualitative assessment of how clearly defined its edges are

57
Q

What edges do you get in brightfield microscopy?

A

In brightfield you get good contrast of the edges as there was refraction around the edges.

58
Q

What happens when two refractive indices are equal in Brightfield microscopy?

A

In the event that the two refractive indices are equal, the light passing through the particle does not deviate at all, and the particle remains invisible.

59
Q

What happens when the two refractive indices are far apart?

A
  • When the refractive indices are far apart, the light passing through will change direction substantially.
  • If refracted sufficiently, they miss the objective lens and these areas of the particle become dark, resulting in high contrast
60
Q

How many wavelengths will match the RI of a particle?

A
  • In practice, a particle will only have a refractive index that matches that of the mounting medium for one wavelength.
  • All other wavelengths will be refracted.
61
Q

What is the result of a particle RI only matching one wavelength?

A

As a result, coloured fringes become visible in areas of the particle having other than normal incidence (typically most pronounced near the edges of the particle).

62
Q

What does bioinfringence mean?

A

Bioinfringence means that change allows us to bring out even more information because the amount that different wavelengths are scattered ends up splitting and a creation of a rainbow going through the sample.

63
Q

How can you find out whether a particle has a a higher or lower RI compared to its mounting media?

A

Becke Line Test

64
Q

What happens when particles have a higher RI than it’s mounting media.

A
  • A particle with a higher RI mounted in a medium of lower RI, will focus axial illuminating rays toward a point above the particle
  • When the media has a lower RI, the light that passes through will be focused.
65
Q

What happens when particles have a lower RI than it’s mounting media.

A
  • Lower RI particle in higher RI medium will direct light in opposite direction.
  • Moving the line outside the particle.
  • When the sample has a RI lower than the media, its refracted into the opposite direction, its dispersed and moves the other way.
66
Q

How can Becke Line immersion measurements be made?

A

Becke Line immersion measurements can be made by mounting the substance in media of varying RI’s until little change is observed

67
Q

What is the limitation of the Becke Line Test?

A

It will only be true for one wavelength of light at a time.

68
Q

When the particle RI is higher than the medium…

A
  • When the particle RI is higher than the medium we get the refraction going in, its blurrier because we’re no longer in focus
  • Around the edges there is a little white light and that’s because its being refracted inwards, its moved inwards.
69
Q

If the particle has a lower RI than the medium…

A
  • If the particle has a lower RI than the medium, it’s refracted outwards and gets pushed.
  • You get this halo effect of these things moving around.
70
Q

What is a better method to use in comparison to becke line test?

A

Variation method

71
Q

What is the variation method?

A
  • Mount in a special High RI medium above that of sample
  • Fix light at a single wavelength (typically 589nm - Sodium Line)
  • Slowly heat the sample on a hot stage
  • The medium RI changes from the heating much faster than the sample
  • At some put the sample is going to disappear as the RI is going to match and then it will pass out the other side.
  • The temperature of the lowest contrast is noted
  • RI value is compared to a table of values
72
Q

When is 200nm resolution possible for optical microscopy?

A

200nm resolution only possible with blue/violet light & oil immersion

73
Q

What is the max magnifcation for optical microscopy?

A

1000x magnification

74
Q

What is the typical white light microscope resolution?

A

1 micron

Trace evidence (22 decks)

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