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Trace Evidence > Lecture 4&5 - Microscopic Techniques > Flashcards

Lecture 4&5 - Microscopic Techniques Flashcards

1
Q

what is optical microscopy

A

the use of visible light and a system of lenses to obtain magnified images of small samples

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2
Q

what range of dimensions does optical microscopy tend to deal with

A

1m (human dimensions) to 10-5m (diameter of a red blood cells) to 10-10m (radius of an atom)

last one is what electron microscopy deals with

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3
Q

what are the types of radiation in the electromagnetic spectrum

A

radio wave
microwave
Infrared
visible
ultraviolet
x-ray
gamma ray

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4
Q

what type of radiation in the magnetic spectrum does optical microscopy make use of

A

visible light
(with infrared, microwave and radio being used too but none to the right of visible light)

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5
Q

what is the definition of a wavelength

A

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

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6
Q

what is wavelength measured in

A

metres (m)

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7
Q

what is the definition of frequency

A

the number of vibrations of a given wavelength in a second

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8
Q

what are the units of frequency

A

Hertz (Hz)
1Hz = 1 wave completed per second

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9
Q

the longer the wavelength ……

A

the lower the frequency

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10
Q

WHY can the properties and wavelengths of light be useful in forensic science?

A

in identifying materials and their heterogeneity, comparisons between materials can be made and obscurities in a material can be identified

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11
Q

HOW can the wavelengths of light be useful in forensic science

A

by measuring the velocity of a wave as it travels through a material

light travels in a straight line and will travel at a constant speed in a homogeneous medium/material, as a wave enters a material it slows down

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12
Q

in a vacuum at what speed do all waves travel

A

3 x 10^8 m/s - due to a vacuum they all travel atthe same speed unless they encounter a sample or material where the speed is slowed down

different material slows the wave down different amounts

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13
Q

what is the equation realting velocity, frequency and wavelength of waves

A

velocity = frequency x wavelength

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14
Q

what three things could happen when light pass from one medium to another

A

absorption
reflection
refraction

(diffraction also can)

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15
Q

what happens in absorption when light pass from one medium to another

A

a photon of light enters a material but does not exit

involves an energy transfer in the form of thermal, chemical or electrical change

different colour surfaces absorb different amounts of certain wavelengths of light (the colour an object appears is reflected)

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16
Q

what happens in specular reflection when light pass from one medium to another

A

all light that hits a surface is reflected back off none is transferred or absorbed into the next medium

very few materials reflect ALL light

the incident angle = the reflection angle

impacted by surface texture, increase in roughness means more diffuse reflection rather than specular

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17
Q

what happens in refraction when light pass from one medium to another

A

the path of the light is bent as it passes into the next medium and here the velocity changes

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18
Q

what is birefringence

A

when an incident ray is split into two rays when a change in medium occurs

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19
Q

what is a black body

A

no reflection of light - everything is absorbed

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20
Q

what is diffuse reflection

A

light reflected in different directions

(specular - all light reflected in 1 direction)

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21
Q

what does total refraction mean

A

no light is reflected

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22
Q

relating to the refraction of light as the medium it passes through changes - what can be measured to help identify a material

A

the refractive index

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23
Q

what does the refractive index of two materials relate to in terms of how light behaves when it leaves a material and enters another

A

the difference between the two refractive index of the materials can help suggest the degree which the light bends and direction it bends

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24
Q

what three factors mostly affect refraction

A

the material
the angle of the incident ray
the wavelength of the incident ray

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25
Q

what impact does an increase in wavelength have on refraction of the light

A

increase wavelength = increase refraction angle

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26
Q

what impact does an increase in incident angle have on refraction of the light

A

increase in incident angle = increase in refraction angle

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27
Q

which law relates the angle of light and the refractive index

A

Snell’s law - allows us to gather more information about the properties of a material for us to make comparisons

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28
Q

what is another name for the magnification of a light microscope

A

numerical aperture

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29
Q

name three key specifications of light microscopes

A

resolution
depth of focus
field of view

(magnification)

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30
Q

what is the resolution of a light microscope

A

the ability to distinguish between two points on a specimen
(linked to magnification)
can reach value of 200nm

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31
Q

what is the key to improving the resolution and therefore magnification of a light microscope

A

understanding the focussing lenses of the microscope

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32
Q

what is the depth of focus of a light microscope

A

the ability to maintain focus over a range of depths within a specimen (how much of what we are looking at remains in focus at the same time)

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33
Q

what can be created from optical images from a light microscope

A

3D maps of a specimen

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34
Q

what is the field of view of a light microscope

A

the size of the specimen that can be imaged at the same time

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35
Q

what happens to the depth of focus with an increase in magnification

A

a decrease in depth of focus is observed

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36
Q

in optical microscopy what are the lenses used for

A

lenses are used to focus (by refracting) the incoming light from a sample to a point

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37
Q

where is the resolution of a light microscope defined

A

at the focal point

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38
Q

what is the ability of a lens to resolve details of a sample influenced by (4)

A

lens quality
diffraction
the diameter of the Airy disk
the wavelength of light

increase in diameter, increase in angle of aperture, increase magnification

39
Q

what is the angle of aperture

A

the max angle light can enter or exit a lens - determines the field of view and amount of light that can enter a system

40
Q

when are two features on a sample regarded as ‘just resolved’

A

when the centre of ones Airy disk coincides with the edge of another

41
Q

what do the use of focussing lenses allow in light microscopy

A

improved resolution

42
Q

in reality, what do lenses have that can affect the resolution of a specimen

A

chromic and spherical aberrations = inconsistencies in the lens and the light causing less resolution

43
Q

if we want to achieve a higher magnification with light microscopes what cost does this come with

A

using more complex lenses which use more than one lens in order to combat aberrations,

more lenses = higher cost

44
Q

name 9 types of microscopy we have considered

A

stereoscopic
comparison
polarised
reflected light
fluorescence
thermal
darkfield
brightfield
multispectral

45
Q

name 4 benefits of using stereoscopic microscopes (regular light microscopes)

A

large working distance (can fit bulkier items)
wide field of view
great depth of focus
good magnification range =10-125x

46
Q

what type of microscope is most used in forensic science

A

stereoscopic microscope

47
Q

when is a stereoscopic microscope generally used

A

as a initial step when looking at physical features of trace evidence

48
Q

what is the next microscope used after stereoscopic in forensic analysis and why

A

a compound microscope because it has an increased magnification range and resolution

49
Q

what is the magnification range of a typical compound microscope

A

40-450x

50
Q

what can be changed when using a compound microscope to help visualise a sample

A

the stage can be moved
the light intensity can be controlled
the focus can be adjusted

51
Q

what are the two modes (illumination) of compound microscopes

A

reflected illumination
transmitted illumination

52
Q

when are comparison microscopes used

A

to make point-point and side-by-side comparisons to suggest if two samples are from the same source

(setting and microscope should be the same for both sides to make a good comparison)

53
Q

what makes a fluorescence microscope different to a compound or stereoscopic microscope

A

the designs are the similar but fluorescence microscopes use illuminating light in the UV wavelength range

54
Q

when are fluorescence microscopes used in forensic analysis

A

in hope of something fluorescing to be observed, sized and mapped which then can undergo further testing

55
Q

why is fluorescent tagging often used in biological sample analysis, presumptive tests and fingermark identification but not trace evidence

A

as tagging the sample may interact with the sample and be destructive so better to not take the risk as there isn’t much of the evidence to begin with (as it is trace)

56
Q

what do polarised light microscopes use to analyse samples

A

polarised light - normal light is changed to polarised light using a polariser built into the microscope

57
Q

how is polarised light different from normal light

A

normal light waves are vibrating in every direction perpendicular to the direction of travel
linearly polarised light waves are vibrating in one direction

58
Q

when can normal light become polarised light

A

if it passes through a material that only allows the transmission of rays in a particular direction e.g crystal or film (called polarisers)

59
Q

how can the birefringence of a species be obtained using polarised microscopy

A

the change in polarisation of light observed when the light has interacted with the specimen gives the birefringence of a specimen

60
Q

when is polarised microscopy useful in forensic analysis

A

when analysing anisotropic substances = exhibit different properties in different directions when illuminated

61
Q

what is an isotropic species

A

exhibit the same properties regardless of the direction of observation/illumination

62
Q

what is brightfield microscopy

A

a type fo microscopy that uses light from the lamp source under stage to illuminate a specimen
the light is gathered into a condenser then shaped into a cone where the apex is focused onto the specimen

63
Q

what is needed in order for a specimen to be seen when using a brightfield microscope

A

contrast between the species and the medium the sample is mounted on

contrast = a difference in the refractive index between the two species

64
Q

why might an image of the specimen not be seen in a brightfield microscope

A

if there is no contrast (not a big enough difference in the refractive index) between the sample and the medium surrounding it

65
Q

what can be done in brightfield microscopy if the image cant be seen in order to see an image

A

change the medium so there is greater contrast
stain the specimen - but this can be destructive so not desirable in trace analysis

66
Q

what is darkfield microscopy

A

a type of microscopy that uses a condenser to form a hollow cone with no light, light is scattered from the sample and collected to form the image that is bright against a dark backgroun

light transmitted through the sample misses the lens and is not collected

67
Q

how the the field of view appear when there is no sample present in darkfield microscopy

A

dark (black)

68
Q

what is one of the main problems encountered when using darkfield microscopy

A

achieving high resolution (high detail) because this depends on how a sample scatters light and how long this takes

can increase the amount of light to try and overcome this but this can burn the sample

69
Q

what is a benefit of darkfield microscopy

A

no staining of the sample is generally required

70
Q

what is cross polarised light microscopy able to do

A

pick out a samples density changes

71
Q

which law does the contrast between materials relate to

A

Snell’s law

72
Q

what does Snell’s Law suggest for isotropic substances

A

the change in light direction is related to its change in velocity when it enters a new medium

this is determined by the difference in refractive index between two media

73
Q

when are defined edges of specimens observed

A

when there is a larger difference in the refractive index between the sample and the mounting medium = greater contrast

74
Q

what happens when the refractive index of the sample and the mounting medium are equal

A

light passing through the sample will not change direction and so it remains unseen in the microscope

75
Q

what happens when the refractive index of the sample and the mounting medium are very different

A

the light passing through the sample will change direction enough for an image of the specimen to be seen

76
Q

in practice when is the only time a particle will have a refractive index that matches the mounting media

A

for one wavelenghts/colour of light
all of wavelengths will be refracted

77
Q

what can be used to know whether the particle in a sample has a high or lower refractive index compared to the mounting medium

A

the Becke Line test

78
Q

how are Becke Line immersion experiments achieved

A

by mounting the sample in media of varying refractive index’s until a little change is seen

79
Q

what is a limitation of Becke Line tests

A

the observations will only be true for one wavelength of light at a time so is averaged for white light - there is the need for a more precise method

80
Q

what is a method that is more precise than the Becke Line test

A

the Single Variation Method or the Double Variation Method is even more precise

81
Q

explain the process of the single variation method

A

1 - mount sample in a medium with a higher RI
2 - set a certain wavelength of light (normally 589nm)
3 - slowly heat sample on a hot stage
4 - the RI of the medium will change on heating faster than the sample
5 - temp of the lowest contrast is noted

the sample will ‘disappear’ when the RI of the medium and sample are the same

82
Q

what are the benefits of optical microscopy

A

good field of view
easy rapid sample prep
relatively low cost

83
Q

what are the limitations of optical microscopy

A

resolution (200nm)
1000x magnification max
low depth of focus

84
Q

what methods are used to characterise nanostructures

A

a combination of surface microscopy (scanning electron, transmission electron and atomic force) and bulk diffraction (x-ray powder or optical)

85
Q

what limits the resolution achieved by light (optical) microscopy

A

the wavelength of the illuminating light

86
Q

what are the benefits of electron microscopy

A
  • higher resolution achieved compared to light/optical microscopy
  • non destructive (beam damage can occur for sensitive samples though)
    fast
  • can give elemental composition
  • can use small quantities of material
87
Q

is TEM or SEM higher resolution

A

TEM so therefore more used for nanostructure characterisation

in TEM electrons pass through the sample and the lens is after the sample

88
Q

name 3 types of surface microscopy

A

SEM - scanning electron
TEM - transmission electron
Atomic Force

89
Q

name one type of diffraction microscopy

A

X ray diffraction (XRD)

90
Q

what is XRD used for

A

the find out the arrangement of atoms within a crystal structure and how they are stacked

91
Q

which law is a simplistic model used to understand the conditions needed for XRD

A

Bragg’s Law

92
Q

name 4 things XRD can determine

A

lattice parameters (by indexing the position of peaks)

phase composition of the sample (looking at relative amounts of overlaid diffraction patterns)

crystal structure (looking at whole diffraction pattern)

crystallite size (looking at peak broadening)

93
Q

what equation allows the average size of nanoparticles to be calculated

A

Scherrer equation

Trace Evidence (16 decks)

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