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FSCI6030 - Trace Evidence > Microscopic Techniques > Flashcards

Microscopic Techniques Flashcards

1
Q

define optical microscopy

A

use of visible light (400 - 700 nm) and a system of lenses to obtain magnified images of small samples

can be used to analyse anything to left on EM spectrum but nothing smaller to the right

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2
Q

define wavelength

A

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

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3
Q

define frequency

A

the number of vibrations of a given wavelength in one second
- measured in hertz
- 1 Hz = 1 wave completed per second

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4
Q

What is the correlation between wavelength and frequency?

A

higher wavelength
= vibrate fewer times
= lower frequency

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5
Q

For visible light what are the frequency range and therefore the equivalent wavelength range?

A

red: 4.3 x 1015 Hz
yellow: 5.4 x 1015 Hz
violet: 7.5 x 1015 Hz

  • equivalent 400 - 700 nm
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6
Q

What can be said about the movement of light in any given homogeneous medium or material?

What about in a vacuum?

what about from a vacuum into a material?

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 a vacuum where there is nothing interfering, all waves in EM spectrum travel at 3 x 10^8 ms-1
  • it slows down to a speed characteristic of that material
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7
Q

give the formula for the properties of waves

A

velocity = frequency x wavelength

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8
Q

What four things can happen when light passes from one medium to another?

A

1 - diffraction

2 - absorption

3 - reflection

4 - refraction

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9
Q

explain absorption (what happens, what materials absorb more light)

A
  • when a photon of light enters a material, but does not exit again
  • results in thermal, electrical or chemical changes - can measure this change in energy
  • dark materials absorb more light than light materials
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10
Q

Explain reflection (what happens, the two types, what can reflection tell us)

A
  • the light ray is turned back into the incident material instead of travelling on into the new material
  • two types:
  • specular reflection - perfect case where nothing is absorbed. angle of incidence = angle of reflection
  • diffuse reflection - all coming off at different angles
  • this gives us info on the-
  • colour - what is reflected back is the colour we see so if red surface all colours absorbed but red is reflected. perfect black body = every colour absorbed and non reflected (highly unlikely)
  • texture (rough or smooth) - as increase surface roughness, specular reflection peak broadens out and as increase it even more, diffuse reflection occurs. at 70/80 % surface roughness, all diffuse, no specular
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11
Q

Explain refraction (what is it, what happens in a perfect case, what three factors affect refraction)

A
  • the light ray’s path is bent when it passes from one transparent material to another transparent material where its velocity changes
  • in a perfect case, there is no specular reflection, all of the light is refracted
  • materials involved: the materials involved affect the angle of refraction e.g. in a vacuum there is very little refraction as there is no energy change
  • angle of incident ray of light affects the angle of refraction
  • wavelength of incident ray: as the wavelength increases, the refraction angle increases
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12
Q

In refraction, what does the refractive index values influence?

What does a higher refractive index mean for refraction?

A

the degree to which the light ray bends and the direction in which it bends

is is unique for any material we have

higher the RI = higher the refraction

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13
Q

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

Why is this important in forensic science?

A

By Snell’s Law:
sin01/sin02 = n21 = n2/n1 = v1/v2

it allows us to get more info about a material

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14
Q

What are the four key specifications of a light microscope?

What is comparatively low and what is comparatively good?

A

1 - magnification (numerical aperture)

2 - resolution - ability to distinguish between 2 points on the specimen

3 - depth of focus - ability to maintain focus over a range of depths within the specimen

4 - field of view - size of specimen that can be imaged at the same time

depth of focus comparatively low
field of view comparatively good

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15
Q

How does the field of view and depth of focus change when the resolution and magnification increases?

A

when resolution and magnification increases, field of view and depth of focus decreases

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16
Q

What are lens used for in optical microscopy?

What is the ability of lens to resolve details of a sample influenced by?

A

used to focus (refract) incoming light (from a sample) to a point

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

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17
Q

Define spot size

A

the spot size (d) is given by the diameter of the airy disk (when light is focussed to a point after passing through a circular aperture)

d = 1.22 x wavelength x (focal length/lens diameter)

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18
Q

How is resolution described when observing two features on a sample in terms of their airy disks?

What affects the lens resolution?

What can be said about this?

A
  • just resolved - centre of one’s airy disk coincides with the edge of the others
  • limit of resolution in a light microscope is given by the Rayleigh’s criterion, half the Airy disk diameter (d). The Airy disc diameter (i.e. spot size) is given by:
    d = 1.22 x wavelength x (focal length/lens diameter)
  • spot side and wavelength define the lens resolution
  • however this is idealised as in reality lenses have chromic and spherical aberrations
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19
Q

What is the optimum resolution for a light microscope?

A

on the order of 1 um

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20
Q

What is the purpose of the lamp at the base of the stand and the eyepiece lens in the light microscope?

A
  • lamp at base of stand = supplies light to specimen, without it the overall illumination and contrast would not be sufficient for imaging
  • eyepiece lens = further magnifies the image from the objective and puts it in a form suitable for viewing
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21
Q

What is role of light in light microscope

A
  • a reflected light microscope a light source (usually visible) is directed through a tube, reflects off the surface of the sample, and is then sent through a series of lenses to magnify the sample
  • the image is relayed to the eyepiece which puts it into a form suitable for viewing for the operator looking through the eyepiece
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22
Q

Why are focussing lens important?

A

they help improve our resolution

can be improved to 200 nm if using blue light and special objectives

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23
Q

What does a higher magnification require?

A

requires more complicated objective systems in order to combat aberrations

more lenses = higher cost

24
Q

Define stereoscopic microscope (most common use, magnification range, three advantages, next step up)

A
  • most frequently used in forensic science
  • 10 - 125 x range
  • large working distance (useful for bulky artefacts
  • wide field of view and great depth of focus
  • once used this might want to increase resolution and use compound microscope
25
Q

Define compound microscope (type, magnification range, two modes, two features, some drawbacks)

A
  • binocular microscope
  • 4 - 450 x range (up to 1000 x)
  • transmitted illumination - light is coming through
  • reflected illumination - light reflected mode
  • precise focus and light intensity control (various lenses allow us to focus)
  • X-Y stage to move around sample
  • field of view and depth of focus starting to be reduced as we are focusing in
26
Q

Define comparison microscope (what does it allow, how does it work, how does the viewer observed it)

A
  • it allows point-by-point and side-by-side comparison to determine if two samples are from the same source
  • two identical microscopes are connected to a single comparison eyepiece or screen (basically two compound microscopes joined together)
  • the viewer sees the images from both microscopes next to one another as an inset image to compare
27
Q

Define fluorescence microscope (describe it, how does it work, what else can be used)

A
  • it is similar in design to a stereoscopic/ compound microscope, but the illuminating light is in the ultraviolet wavelength range
  • illumination causes some materials to fluoresce so they can be observed, counted, sized and mapped
  • fluorescent tagging can also be used but less common for trace evidence compared to it being used more commonly with biological sample or fingermark identification
28
Q

How does polarised light microscopy work (difference between normal and linearly polarised light, how polarised light microscopy works, when it is useful)

A
  • normal light = waves vibrating in evert direction perpendicular to direction of travel whereas linearly polarised light = waves vibrating in one direction
  • normal light can be polarised if it passes through a material that only allows transmission of rays in a particular direction (polariser) such as a crystal or a film
  • can then measure change in polarisation
  • this will give us info on infringements of a specimen (can then link to particular material)
  • polarisation is useful in forensic microscopy when applied to anisotropic substances (having a physical property that has a different value when measured in different directions
29
Q

Define Brightfield microscopy (describe how it works, how is it used to visualise a material, what materials can it not visualise, why is this bad in forensics)

A
  • it 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
  • to view a specimen, the light rays that pass through it must be changed enough in order to contrast
  • if a specimen has a refractive index similar to the surrounding medium then the image cannot be seen
  • in order to visualise these materials, they must have contrast with medium or be stained
  • staining can be destructive to specimens - very bad for forensic science where integrity of samples is important
30
Q

define darkfield microscopy (describe how it works, how is it used to visualise a material, give a benefit over brightfield microscopy, what is a disadvantage)

A
  • it uses a 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
  • the entire field of view appears dark when there is no sample in the microscope stage
  • when a sample is placed on the stage it appears bright against a dark background as only the scatted light is collected
  • this provides contrast without staining
  • it takes a long time to get any real resolution on our sample so we have to ramp up power of lamp (need to be careful not to burn/overheat sample)
31
Q

What does Snell’s law dictate for isotropic substances?

A

isotropic - things that only have one R.I and no change of orientation

  • says that the change in the propagation direction of the incident light is related to the change in velocity of the light transmitted into the crystal
  • this is determined by the RI difference between particle and mounting medium
32
Q

What are the only two optical properties to be determined for isotropic substances?

A

refractive index
dispersion values

33
Q

How does RI values tell us how defined edges will be?

A
  • by how close/far away the RI value of a particle is to the mounting medium
  • the difference in RI values are essentially qualitative assessment of how clearly defined edges are
  • large difference in RI = light incident on particle deviates greatly from original path and fails to enter objective lens = biggest deviation in angle = leads to defined edges = high contrast
  • medium difference in RI = light incident on particle deviates from original path and fails to enter objective lens = medium deviation in angle = leads to defined edges = medium contrast
  • equal RI values = light passing through particle does not deviate at all (goes straight through) and particle remains invisible
34
Q

In practice, a particle’s RI matches what on the mounting medium?

What does this mean?

A
  • a particle will only have one refractive index that matches that of the mounting medium for one wavelength (one colour)
  • all other wavelengths will be refracted
  • coloured fringes become visible in some areas of the particle having other than normal incidence (most pronounced near edges of particle)
  • this is useful as shows how light transmits through sample
35
Q

How can you tell whether the particle has an RI higher or lower than that of the mounting medium?

A
  • using becke line test
  • get in focus in F1 then move up to F2 but keep focus
  • particle RI higher than medium - focuses axial illuminating rays towards a point above the particle
  • particle RI lower than medium - direct light in opposite direction moving the line outside the particle - causing halo effect
36
Q

How can make Becke line immersion measurements?

What are the limitations of this?

A
  • can be made by mounting the substance in media of varying RI’s until little change is observed
  • will only be true for one wavelength of light at a time (so averaged for white light) - need a more precise method
37
Q

What is the more precise version of Becke line immersion measurements?

Describe this six step method

What method is even more precise?

A

single variation method

1 - mount in a special high RI medium above that of sample
2 - fix light at a single wavelength (typically 589 nm (sodium line))
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 contrast is noted (usually computationally)
6 - compare to table of RI values corresponding to temperature

double variation method

38
Q

What are three limitations and three positives of optical microscopy?

A

1 - 1000 x magnification max

2 - 200 nm resolution only possible with blue/violet light and oil immersion
- typical white light microscope resolution is 1 micron

3 - low depth of focus

1 - good field of view

2 - easy and rapid specimen prep

3 - low relative cost

39
Q

How do we characterise nanostructure?

A

done with a combination of techniques to study their chemical and physical properties:
- surface (microscopy: scanning electron, transmission electron, atomic force)
- bulk (diffraction: x-ray powder, optical)

40
Q

Describe the difference between light microscopy and electron microscopy

A
  • resolution of light microscopy is limited by the illuminating wavelength
  • higher resolutions are achievable using electrons instead of light
  • non-destructive analysis of very small quantities of material is possible this way (although beam damage can occur for sensitive samples)
  • this allows for the rapid accumulation of results in high resolution
  • can even give elemental composition of a material
41
Q

What is the role played by the beam of electrons in an electron microscope?

A
  • electron microscopes use a beam of electrons rather than visible light to visualise the object
  • the beam interacts with the sample and produces several types of electron signals
  • some of the electrons that are scattered are collected by a detector to produce an image, with magnifications of the order of 100,000x
42
Q

Why are electrons useful for imaging nanomaterials?

A

electrons are strongly interacting and have a wavelength that makes them useful for imaging nanomaterials

43
Q

In TEM imaging, how does it work, what is transmission and scattering?

A
  • electrons pass through sample (must be thin)
  • whole images collected at the same time
  • lenses after sample to enable high resolution
  • transmission through sample
  • scattering: elastic - diffraction
44
Q

In SEM imaging, how does it work, what is scattering and what is secondary product?

A
  • scanning approach builds up image of one point at a time
  • samples can be large (as long as there is space in the sample chamber
  • scattering: inelastic - back scattering
  • secondary product: secondary electron knocked out of atom (these are fluorescence information)
45
Q

What is the principle difference between SEM and TEM?

A
  • in SEM the focussed electron beam is scanned across the specimen and is reflected from the surface
  • in TEM the beam passes through the specimen
46
Q

In spectroscopy, what is scattering and what is secondary product?

A

scattering: inelastic - energy loss
secondary product: core electron lost and replaced

47
Q

What is most useful for nanostructure characterisation (TEM/SEM/spectroscopy) ?

A

TEM as it is much higher resolution than SEM (1 nm compared to 10 nm)

48
Q

Describe the difference between light, SEM and TEM in terms of resolution, depth of focus, field of view, specimen preparation and relative cost?

A

resolution: TEM has highest resolution

depth of focus: SEM best, TEM medium, light low

field of view: good for light and SEM, limited for TEM

specimen preparation: easy and rapid for light and SEM (SEM slightly longer than light) whereas TEM skilled and slow

price: TEM most, SEM still a lot, light the least

49
Q

Which has best value in forensics (light, SEM, TEM)?

A

SEM has real value in analysis of forensic trace evidence

50
Q

What does XRD establish?

A
  • it is used to establish the arrangement of atoms within a crystal structure and how they stack together
51
Q

Define Bragg’s law

A
  • it is a simplistic model to understand what conditions are required for diffraction
  • for parallel planes of atoms, with a space of (d) between the planes, constructive interference only occurs when Bragg’s law is satisfied
  • ny = 2dsin0

where
- n = integer (often 1)
- y = x-ray wavelength
- d = interplanar spacing
- 0 = angle between plane and beam

52
Q

What four things can XRD determine?

A

1 - lattice parameters (by indexing the position of the peaks)
- gives info on alloying/doping/strain in material

2 - phase composition of the sample (given by the relative amounts of overlaid diffraction patterns)
- giving compositional information

3 - crystal structure (by refining the whole diffraction pattern)
- giving texture and orientation of crystals in the bulk

4 - crystallite size (by looking at peak broadening)
- giving even more bulk structural information of the material

53
Q

What causes broadening in diffraction peaks?

A

crystallites smaller than around 120 nm create broadening in diffraction peaks

54
Q

what enables the average size of nanocrystals to be calculated if there is no microstrain?

A

Scherrer equation

55
Q

What five physical properties can be examined using microscopy?

A
  1. morphology
  2. cross section and diameter (dye penetration within it)
  3. pleochroism
  4. isotropy/anisotropy
  5. refractive index/birefringence

FSCI6030 - Trace Evidence (13 decks)

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