FSCI6030 - Trace Evidence > Microscopic Techniques > Flashcards

Microscopic Techniques Flashcards

(55 cards)

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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?

Study These Flashcards
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)

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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
Define compound microscope (type, magnification range, two modes, two features, some drawbacks)
- 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
Define comparison microscope (what does it allow, how does it work, how does the viewer observed it)
- 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
Define fluorescence microscope (describe it, how does it work, what else can be used)
- 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
How does polarised light microscopy work (difference between normal and linearly polarised light, how polarised light microscopy works, when it is useful)
- 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
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)
- 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
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)
- 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
What does Snell's law dictate for isotropic substances?
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
What are the only two optical properties to be determined for isotropic substances?
refractive index dispersion values
33
How does RI values tell us how defined edges will be?
- 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
In practice, a particle's RI matches what on the mounting medium? What does this mean?
- 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
How can you tell whether the particle has an RI higher or lower than that of the mounting medium?
- 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
How can make Becke line immersion measurements? What are the limitations of this?
- 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
What is the more precise version of Becke line immersion measurements? Describe this six step method What method is even more precise?
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
What are three limitations and three positives of optical microscopy?
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
How do we characterise nanostructure?
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
Describe the difference between light microscopy and electron microscopy
- 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
What is the role played by the beam of electrons in an electron microscope?
- 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
Why are electrons useful for imaging nanomaterials?
electrons are strongly interacting and have a wavelength that makes them useful for imaging nanomaterials
43
In TEM imaging, how does it work, what is transmission and scattering?
- 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
In SEM imaging, how does it work, what is scattering and what is secondary product?
- 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
What is the principle difference between SEM and TEM?
- 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
In spectroscopy, what is scattering and what is secondary product?
scattering: inelastic - energy loss secondary product: core electron lost and replaced
47
What is most useful for nanostructure characterisation (TEM/SEM/spectroscopy) ?
TEM as it is much higher resolution than SEM (1 nm compared to 10 nm)
48
Describe the difference between light, SEM and TEM in terms of resolution, depth of focus, field of view, specimen preparation and relative cost?
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
Which has best value in forensics (light, SEM, TEM)?
SEM has real value in analysis of forensic trace evidence
50
What does XRD establish?
- it is used to establish the arrangement of atoms within a crystal structure and how they stack together
51
Define Bragg's law
- 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
What four things can XRD determine?
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
What causes broadening in diffraction peaks?
crystallites smaller than around 120 nm create broadening in diffraction peaks
54
what enables the average size of nanocrystals to be calculated if there is no microstrain?
Scherrer equation
55
What five physical properties can be examined using microscopy?
1. morphology 2. cross section and diameter (dye penetration within it) 3. pleochroism 4. isotropy/anisotropy 5. refractive index/birefringence
FSCI6030 - Trace Evidence
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