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Chemistry for the Analysis of Trace Evidence > Microscopic Techniques > Flashcards

Microscopic Techniques Flashcards

1
Q

What is 1 wavelength?

A

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

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2
Q

What form of radiation is light and what type of light does optical microscopy make use of?

A
  • electromagnetic
  • visible
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3
Q

What is frequency?

A

the number of vibrations of a given wavelength in one second

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4
Q

What are the units of frequency?

A

Hz
* 1 Hz = 1 wave completed per second

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5
Q

What is the link between wavelengths and frequency?

A

longer wavelengths vibrate fewer times so the longer the wavelength the lower the frequency

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6
Q

What are the properties of waves?

A
  • light travels in a straight line from a source and reaches a definite and constant speed in any given homogenous medium or material
  • in vacuum, all waves in the electromagnetic spectrum travel at the same speed
  • not all waves slow down to the same speed outside a vacuum
  • as any wave enters a material from a vacuum it slows down
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7
Q

What formula relates the three properties of waves?

A

velocity (c) = frequency (f) x wavelength (λ)

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8
Q

What is absorption?

A

when a photon of light enters a material, but doesn not exit again
* results in thermal, electrical or chemical changes
* depends on colour (dark or light)

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9
Q

What is reflection?

A

the light ray is turned back into the incident material instead of travelling on into the new material
* dependant on colour, texture (rough or smooth)

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10
Q

What is refraction?

A

the light ray’s path is bent when it passes from one transparent material to another transparent material where its velocity changes
* if there is total refraction then nothing is reflected
* the bend at which the ray moves is representative of the material and a change in velocity

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11
Q

What factors affect refraction?

A
  • the material involved
  • the angle of the incident ray of light
  • the wavelength of the incident ray
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12
Q

How is the refractive index of the two substances related to refraction?

A
  • degree to which the light ray bends
  • direction it bends
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13
Q

How does wavelength relate to refraction?

A

increasing the wavelength (changing the colour) changes the amount of refraction

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14
Q

How are the light ray angles and refractive indices related to each other?

A

by Snell’s Law

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15
Q

What is Snell’s Law?

equation

A

sinθ1/sinθ2 = n21 = n2/n1
* n = refractive indices
* θ1 = angle of incidence
* θ2 = angle of refraction

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16
Q

What is resolution?

A
  • ability to distinguish between 2 points on the specimen
  • understanding focussing lenses are key to improve resolution
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17
Q

What are the key specifications of a light microscope?

A
  • resolution
  • depth of focus
  • field of view
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18
Q

What is depth of focus?

A
  • ability to maintain focus over a range of depths within the specimen
  • comparatively low - gets worse with higher magnifications
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19
Q

What is field of view?

A
  • size of specimen that can be imaged at the same time
  • good
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20
Q

What is focussing lenses?

A
  • the ability of the lens to resolve details of a sample is influenced by the quality of the lens but is limited by diffraction
  • as light passes through a circular aperture, it is focussed to a point. The spot size is given by the diameter of the airy disk which is dependent on the wavelength of the light
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21
Q

What is the equation for spot size in focussing lenses?

A

d = 1.22 x λ x focal length/lens diameter
* d = spot size
* λ = wavelength/resolution

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22
Q

What defines the lens resolution?

A
  • spot size
  • λ
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23
Q

How can the optimum resolution be improved to 200 nm?

A

use blue light and special objectives

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24
Q

What is the optimum resolution for a light microscope?

A

~ 1 um

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25
Q

What does higher magnification require?

A

more complicated objective systems in order to combat aberrations
* more lenses so you can increase the magnification = higher cost

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26
Q

Stereoscopic microscope

A
  • most frequently used in forensics
  • large working distance
  • good for bulky artefacts
  • wide field of view
  • great depth of focus
  • good first step when looking at physical features of trace evidence
  • 10-125x range
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27
Q

Compound microscope

A
  • precise focus and light intensity control
  • 40-450x range (up to 1000x)
  • transmitted and reflected illumination
28
Q

Comparsion microscope

A
  • allows point-by-point and side-by-side comparison
  • two identical microscopes are connected on a single comparison eyepiece or screen
29
Q

Fluorescence microscope?

A
  • similar design to a stereoscopic microscope or compound
  • illuminating light is in the UV wavelength range
  • causes some materials fluoresce
  • fluorescent tagging can also be used but less common for trace evidence
30
Q

Polarised light microscopy

A
  • linearly polarised light - waves vibrating in one direction
  • normal light can be polarised if it passes through a material tha only allow transmission of rays in a particular direction (crystal or a film)
  • useful in anisotropic substances
31
Q

What is an anisotropic substance?

A

having a physical property which has a different value when measured in a different direction

32
Q

Normal light vs linearly polarised light

A
  • normal: waves vibrating in every direction perpendicular to the direction of travel
  • polarised: waves vibrating in one direction
33
Q

Brightfield microscope

A
  • 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
  • light rays pass through it must be changed enough in order to contrast
  • if the RI of the specimen is similar to the medium, no image will be seen
  • to visualise, they must have a contrast with the medium or be stained - but staining can be destructive
34
Q

Darkfield microscopy

A
  • use special condenser which forms a hollow cone to collect only highly refracted light
  • objective lens sit in hollow of the cone and light directly transmitted through the sample misses the lens and isnt collected
  • field of view appears dark when there is no sample
  • sample appears bright against a dark background as only the scattered light is collected
35
Q

What does Snell’s Law dictates for isotropic substances?

A
  • the change in the propagation direction if the incident light is related to the change in the velocity of the light transmitted into crystal
  • determined by the RI difference between the particle and mounting medium
36
Q

What are the only optical properties that can be determined for isotropic substances?

A

refractive index and dispersion values

37
Q

How can you qualitatively say how defined a samples edges are?

A
  • the constast of the particle in a particular mounting material
  • with a large difference in RI, light incident on the particle deviates greatly from its original path and fails to enter the objective lens
38
Q

What happens when there is no contrast in the RI of the sample and the mounting medium?

A
  • light passing through the particle does not deviate at all and the particle remains invisible
39
Q

What happens when the RI of a sample and the mounting media are far apart?

A

the light passing through will change direction substantially - mainly at the edges

40
Q

What happens if the RI of the sample and mounting media are drastically different?

A
  • refractive light misses the objective lens completely and these areas become dark resulting in a high contrast
41
Q

Why do coloured fringes become visible in area of a particle?

A
  • a particle will only have a refractive index that matches that of the mounting medium for one wavelength
  • all other wavelengths will be refracted
  • coloured fringes become visible in areas of the particle having other than normal incidence (usually near the edge)
42
Q

How do you work out if the RI of the sample is higher or lower than the mounting medium?

A

Becke Line Test

43
Q

How does the Becke Line test work?

A
  • small particles behave much like lenses, refracting light that depends on the relative RI values of the particle and mounting media
  • a particle with a higher RI mounted in a medium of lower RI, will focus axial illuminating rays toward a point above the particle
  • lower RI particle in higher RI medium will direct light in iposite direction, moving the line outside the particle
44
Q

How are Becke line immersion measurments made?

A

by mounting the substance in media of varying RI’s until little change is observed

45
Q

What is the limitation of Becke Line?

A

will only be true for one wavelength of light at a time (so averaged for white light)

46
Q

What are variation methods?

A

more precise way to work out the RI of a substance

47
Q

How do you do a single variation method?

A
  1. mount in a special high RI medium above that of sample
  2. fix light at a single wavelength
  3. slowly heat the sample on a hot stage
  4. the medium RI changes on heating much faster than the sample
  5. the temperature of lowest contranst is noted (usually computationally)
  6. compare to table of RI values corresponding to temperature
48
Q

Double variation method compared to single?

A

more precise

49
Q

What are the limitations of optical microscopy?

A
  • 1000x magnification max
  • typically white light resolution ~1micron
  • ~200nm resolution only possible with blue/violet light
50
Q

How do we characterise nanostructures?

A
  • surface: microscopy - SEM, TEM, atomic force
  • bulk: diffraction - X-ray power, optical
51
Q

Electron microscopy

A
  • higher resolutions are achievable using electrons instead of light
  • non-destructive analysis of very small quantities - beam damage can occur for sensitive samples
  • rapid accumulation of results
  • give elemental composition
52
Q

What is used in SEM imaging?

A
  • inelastic - backscattering
  • secondary electron knocked out of atom
53
Q

What is used in TEM imaging?

A
  • transmission through sample
  • elastic scattering - diffraction
54
Q

What is used in spectroscopy?

A
  • inelastic scattering - energy loss
  • core electron lose and replaced
55
Q

SEM

A
  • samples can be large
  • scanning approach builds up image of one point at a time
  • high depth of focus
  • good field of view
  • easy and rapid
  • expensive
56
Q

TEM

A
  • best resolution - most used for nanostructure characterisation
  • medium depth of focus
  • limited field of view
  • skilled and slow
  • very expensive
  • electrons pass through sample so they must be thin
  • whole image is collected at the same time
  • lenses after sample to enable high resolution of image
57
Q

XRD

A
  • used to establish the arrangement of atoms within a crystal structure and how they stack together
  • Bragg’s law is the simplest model to understand what conditions are required for diffraction
  • for parallel planes of atoms, with a space (d) between the planes, constructive interference only occurs when Bragg’s law is satisfied
58
Q

What is Bragg’s law?

A

nλ = 2dsinθ
* n = integer (often 1)
* λ = x-ray wavelength
* d = interplanar spacing
* θ = angle between plane and beam

59
Q

What two parts of Bragg’s law can we control and what does this mean?

A

λ and θ, can give us d from the space between peak positions

60
Q

What can XRD determine?

A
  • lattice parameters
  • phase composition of the sample
  • crystal structure
  • crystallite sizw
61
Q

How does XRD determine lattice parameters?

A

by indexing the positions of the peaks

62
Q

How does XRD determine phase composition of a structure?

A

given by the relative amounts of overlaid diffraction patterns

63
Q

How does XRD determine crystal structure?

A

by refining the whole diffraction pattern

64
Q

How does XRD determine crystallite size?

A

by looking at peak broadening

65
Q

What size of crystal creates broadening in diffraction peaks?

A

smaller than ~120nm

66
Q

What does the Scherrer equation enable?

A

for the average size of nanocrystals to be calculated

Chemistry for the Analysis of Trace Evidence (16 decks)

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