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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
what impact does an increase in wavelength have on refraction of the light
increase wavelength = increase refraction angle
26
what impact does an increase in incident angle have on refraction of the light
increase in incident angle = increase in refraction angle
27
which law relates the angle of light and the refractive index
Snell's law - allows us to gather more information about the properties of a material for us to make comparisons
28
what is another name for the magnification of a light microscope
numerical aperture
29
name three key specifications of light microscopes
resolution depth of focus field of view (magnification)
30
what is the resolution of a light microscope
the ability to distinguish between two points on a specimen (linked to magnification) can reach value of 200nm
31
what is the key to improving the resolution and therefore magnification of a light microscope
understanding the focussing lenses of the microscope
32
what is the depth of focus of a light microscope
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)
33
what can be created from optical images from a light microscope
3D maps of a specimen
34
what is the field of view of a light microscope
the size of the specimen that can be imaged at the same time
35
what happens to the depth of focus with an increase in magnification
a decrease in depth of focus is observed
36
in optical microscopy what are the lenses used for
lenses are used to focus (by refracting) the incoming light from a sample to a point
37
where is the resolution of a light microscope defined
at the focal point
38
what is the ability of a lens to resolve details of a sample influenced by (4)
lens quality diffraction the diameter of the Airy disk the wavelength of light increase in diameter, increase in angle of aperture, increase magnification
39
what is the angle of aperture
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
when are two features on a sample regarded as 'just resolved'
when the centre of ones Airy disk coincides with the edge of another
41
what do the use of focussing lenses allow in light microscopy
improved resolution
42
in reality, what do lenses have that can affect the resolution of a specimen
chromic and spherical aberrations = inconsistencies in the lens and the light causing less resolution
43
if we want to achieve a higher magnification with light microscopes what cost does this come with
using more complex lenses which use more than one lens in order to combat aberrations, more lenses = higher cost
44
name 9 types of microscopy we have considered
stereoscopic comparison polarised reflected light fluorescence thermal darkfield brightfield multispectral
45
name 4 benefits of using stereoscopic microscopes (regular light microscopes)
large working distance (can fit bulkier items) wide field of view great depth of focus good magnification range =10-125x
46
what type of microscope is most used in forensic science
stereoscopic microscope
47
when is a stereoscopic microscope generally used
as a initial step when looking at physical features of trace evidence
48
what is the next microscope used after stereoscopic in forensic analysis and why
a compound microscope because it has an increased magnification range and resolution
49
what is the magnification range of a typical compound microscope
40-450x
50
what can be changed when using a compound microscope to help visualise a sample
the stage can be moved the light intensity can be controlled the focus can be adjusted
51
what are the two modes (illumination) of compound microscopes
reflected illumination transmitted illumination
52
when are comparison microscopes used
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
what makes a fluorescence microscope different to a compound or stereoscopic microscope
the designs are the similar but fluorescence microscopes use illuminating light in the UV wavelength range
54
when are fluorescence microscopes used in forensic analysis
in hope of something fluorescing to be observed, sized and mapped which then can undergo further testing
55
why is fluorescent tagging often used in biological sample analysis, presumptive tests and fingermark identification but not trace evidence
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
what do polarised light microscopes use to analyse samples
polarised light - normal light is changed to polarised light using a polariser built into the microscope
57
how is polarised light different from normal light
normal light waves are vibrating in every direction perpendicular to the direction of travel linearly polarised light waves are vibrating in one direction
58
when can normal light become polarised light
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
how can the birefringence of a species be obtained using polarised microscopy
the change in polarisation of light observed when the light has interacted with the specimen gives the birefringence of a specimen
60
when is polarised microscopy useful in forensic analysis
when analysing anisotropic substances = exhibit different properties in different directions when illuminated
61
what is an isotropic species
exhibit the same properties regardless of the direction of observation/illumination
62
what is brightfield microscopy
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
what is needed in order for a specimen to be seen when using a brightfield microscope
contrast between the species and the medium the sample is mounted on contrast = a difference in the refractive index between the two species
64
why might an image of the specimen not be seen in a brightfield microscope
if there is no contrast (not a big enough difference in the refractive index) between the sample and the medium surrounding it
65
what can be done in brightfield microscopy if the image cant be seen in order to see an image
change the medium so there is greater contrast stain the specimen - but this can be destructive so not desirable in trace analysis
66
what is darkfield microscopy
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
how the the field of view appear when there is no sample present in darkfield microscopy
dark (black)
68
what is one of the main problems encountered when using darkfield microscopy
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
what is a benefit of darkfield microscopy
no staining of the sample is generally required
70
what is cross polarised light microscopy able to do
pick out a samples density changes
71
which law does the contrast between materials relate to
Snell's law
72
what does Snell's Law suggest for isotropic substances
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
when are defined edges of specimens observed
when there is a larger difference in the refractive index between the sample and the mounting medium = greater contrast
74
what happens when the refractive index of the sample and the mounting medium are equal
light passing through the sample will not change direction and so it remains unseen in the microscope
75
what happens when the refractive index of the sample and the mounting medium are very different
the light passing through the sample will change direction enough for an image of the specimen to be seen
76
in practice when is the only time a particle will have a refractive index that matches the mounting media
for one wavelenghts/colour of light all of wavelengths will be refracted
77
what can be used to know whether the particle in a sample has a high or lower refractive index compared to the mounting medium
the Becke Line test
78
how are Becke Line immersion experiments achieved
by mounting the sample in media of varying refractive index's until a little change is seen
79
what is a limitation of Becke Line tests
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
what is a method that is more precise than the Becke Line test
the Single Variation Method or the Double Variation Method is even more precise
81
explain the process of the single variation method
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
what are the benefits of optical microscopy
good field of view easy rapid sample prep relatively low cost
83
what are the limitations of optical microscopy
resolution (200nm) 1000x magnification max low depth of focus
84
what methods are used to characterise nanostructures
a combination of surface microscopy (scanning electron, transmission electron and atomic force) and bulk diffraction (x-ray powder or optical)
85
what limits the resolution achieved by light (optical) microscopy
the wavelength of the illuminating light
86
what are the benefits of electron microscopy
- 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
is TEM or SEM higher resolution
TEM so therefore more used for nanostructure characterisation in TEM electrons pass through the sample and the lens is after the sample
88
name 3 types of surface microscopy
SEM - scanning electron TEM - transmission electron Atomic Force
89
name one type of diffraction microscopy
X ray diffraction (XRD)
90
what is XRD used for
the find out the arrangement of atoms within a crystal structure and how they are stacked
91
which law is a simplistic model used to understand the conditions needed for XRD
Bragg's Law
92
name 4 things XRD can determine
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
what equation allows the average size of nanoparticles to be calculated
Scherrer equation

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