Lecture 4&5 - Microscopic Techniques Flashcards
what is optical microscopy
the use of visible light and a system of lenses to obtain magnified images of small samples
what range of dimensions does optical microscopy tend to deal with
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
what are the types of radiation in the electromagnetic spectrum
radio wave
microwave
Infrared
visible
ultraviolet
x-ray
gamma ray
what type of radiation in the magnetic spectrum does optical microscopy make use of
visible light
(with infrared, microwave and radio being used too but none to the right of visible light)
what is the definition of a wavelength
the distance from a point in a cycle to the corresponding point in the next cycle
what is wavelength measured in
metres (m)
what is the definition of frequency
the number of vibrations of a given wavelength in a second
what are the units of frequency
Hertz (Hz)
1Hz = 1 wave completed per second
the longer the wavelength ……
the lower the frequency
WHY can the properties and wavelengths of light be useful in forensic science?
in identifying materials and their heterogeneity, comparisons between materials can be made and obscurities in a material can be identified
HOW can the wavelengths of light be useful in forensic science
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
in a vacuum at what speed do all waves travel
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
what is the equation realting velocity, frequency and wavelength of waves
velocity = frequency x wavelength
what three things could happen when light pass from one medium to another
absorption
reflection
refraction
(diffraction also can)
what happens in absorption when light pass from one medium to another
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)
what happens in specular reflection when light pass from one medium to another
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
what happens in refraction when light pass from one medium to another
the path of the light is bent as it passes into the next medium and here the velocity changes
what is birefringence
when an incident ray is split into two rays when a change in medium occurs
what is a black body
no reflection of light - everything is absorbed
what is diffuse reflection
light reflected in different directions
(specular - all light reflected in 1 direction)
what does total refraction mean
no light is reflected
relating to the refraction of light as the medium it passes through changes - what can be measured to help identify a material
the refractive index
what does the refractive index of two materials relate to in terms of how light behaves when it leaves a material and enters another
the difference between the two refractive index of the materials can help suggest the degree which the light bends and direction it bends
what three factors mostly affect refraction
the material
the angle of the incident ray
the wavelength of the incident ray
what impact does an increase in wavelength have on refraction of the light
increase wavelength = increase refraction angle
what impact does an increase in incident angle have on refraction of the light
increase in incident angle = increase in refraction angle
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
what is another name for the magnification of a light microscope
numerical aperture
name three key specifications of light microscopes
resolution
depth of focus
field of view
(magnification)
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
what is the key to improving the resolution and therefore magnification of a light microscope
understanding the focussing lenses of the microscope
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)
what can be created from optical images from a light microscope
3D maps of a specimen
what is the field of view of a light microscope
the size of the specimen that can be imaged at the same time
what happens to the depth of focus with an increase in magnification
a decrease in depth of focus is observed
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
where is the resolution of a light microscope defined
at the focal point
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
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
when are two features on a sample regarded as ‘just resolved’
when the centre of ones Airy disk coincides with the edge of another
what do the use of focussing lenses allow in light microscopy
improved resolution
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
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
name 9 types of microscopy we have considered
stereoscopic
comparison
polarised
reflected light
fluorescence
thermal
darkfield
brightfield
multispectral
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
what type of microscope is most used in forensic science
stereoscopic microscope
when is a stereoscopic microscope generally used
as a initial step when looking at physical features of trace evidence
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
what is the magnification range of a typical compound microscope
40-450x
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
what are the two modes (illumination) of compound microscopes
reflected illumination
transmitted illumination
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)
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
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
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)
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
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
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)
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
when is polarised microscopy useful in forensic analysis
when analysing anisotropic substances = exhibit different properties in different directions when illuminated
what is an isotropic species
exhibit the same properties regardless of the direction of observation/illumination
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
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
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
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
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
how the the field of view appear when there is no sample present in darkfield microscopy
dark (black)
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
what is a benefit of darkfield microscopy
no staining of the sample is generally required
what is cross polarised light microscopy able to do
pick out a samples density changes
which law does the contrast between materials relate to
Snell’s law
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
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
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
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
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
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
how are Becke Line immersion experiments achieved
by mounting the sample in media of varying refractive index’s until a little change is seen
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
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
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
what are the benefits of optical microscopy
good field of view
easy rapid sample prep
relatively low cost
what are the limitations of optical microscopy
resolution (200nm)
1000x magnification max
low depth of focus
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)
what limits the resolution achieved by light (optical) microscopy
the wavelength of the illuminating light
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
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
name 3 types of surface microscopy
SEM - scanning electron
TEM - transmission electron
Atomic Force
name one type of diffraction microscopy
X ray diffraction (XRD)
what is XRD used for
the find out the arrangement of atoms within a crystal structure and how they are stacked
which law is a simplistic model used to understand the conditions needed for XRD
Bragg’s Law
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)
what equation allows the average size of nanoparticles to be calculated
Scherrer equation
