forensic microscopy Flashcards
the compound microscope
most common form of microscope used in science
- versatile allowing a number of specialist techniques
- imaging of transparent or semi transparent objects
- allows up to 1000x magnification
- relies on light transmitted through the subject by an inbuilt light source
parts of the microscope
the ocular lenses- or eye pieces
objective lenses- primary lenses which ,magnify your specimen
the turret- where the objective lenses are mounted it typically hold 3 or 4 lenses to allow various magnifications
a stage- onto which your sample is placed
a condenser- which focusses the light passing into your sample
a diaphragm- which control the amount of light and also the depth of field
a light source- which may or may not be colour corrected
focussing knobs- which allow you to bring your samples into sharp focus
the compound microscope consists of several magnifying systems
- able to magnify by use of angular and transverse magnification techniques
- objective lens produces a primary real image which is then enlarged by transverse magnification
- the image is projected into the ocular lens which acts as a ,magnifying glass and enlarges by angular magnification
- resulting image is enlarged but is inverted
compound microscope overall magnification
affected by the chosen combination of objective and ocular lens
compound microscope objective lens
housed in 4 position turret enabling the selection of objectives of increasing magnification
- colour coded lenses egg 4x in yellow, 10x in red
compound microscope ocular lens
simply be removed and replaced by on of higher magnification
objective lens
- consists of a number of glass lenses designed to improve performance
- multiple lens system improves resolution and removes distortions caused by chromatic and other optical aberrations
the lens
- contains the magnification
- its numerical aperture
numerical aperture
- a measure of angular range over which the lens can gather light but it also affects magnification
- higher the aperture better resolution, although means less bright so more illumination
- increase numerical aperture u placing drops of oil between our lens and sample
resolution
ability of a lens to clearly distinguish fine detail or resolve minute entities
- affected by numerical aperture and wavelength of light
- calculated by r= 1.22x wavelength of light/ 2 x NA (numerical aperture)
limits of resolution
- light microscopes are limited to a maximum magnification of around 1000-1500x
- to resolve an object it must be greater than half the wavelength of our light source size
- effective resolution reaches a limit around 200nm
- resolution becomes limited by the wavelength visible light
bright field compound microscopy
- produces an image with a bright background and your specimen in relief
- the image is produced by the sample interacting with the rays of light
- this gives rise to differences in contrast and colour
- when dealing with colourless or transparent samples we can increase contrast further using stains
dark field compound microscopy
- produces an image with a dark background
- used for specimens that do not generate enough contrast in bright field
- produced by the addition of an opaque disk in the light path
- as the light enters the sample some is diffracted which passes into the ey piece but the majority is transmitted unchanged
- transmitted light stopped by a ‘direct illumination block’
phase contrast microscopy
- used for samples which lack contrast under bright field or even dark field microscopy
- e.g fragments of glass or unstained cells
- technique enhances significantly and hence allows visualisation of specimens
- allows visualisation of certain types of cells without the need for staining
- this is an advantage as staining kills living cells
phase contrast microscopy- understanding the technique requires
- an image is formed by light that is absorbed, scattered, reflected, refracted, diffracted or in some way altered by our specimen
- absorption, scattering and reflection causes attenuation or loss of the light that passes through
- the thicker an object the more attenuation that occurs and thus the darker the object appears in the microscope
- absorption may even change the colour of our sample
phase contrast microscopy- how does the phase contrast technique work
areas of our sample which absorb light or cause significant phase differences appear dark, whereas other areas will cary in brightness according to their thickness and the way in which they interact with the light
phase contrast compound microscopy - recombined waves
- when two light beams recombine the respective brightness of the resulting beam depends upon the phase of the two beams
- recombine out of phase, cancel each other out resulting in a dark specimen
- recombine in phase, amplify each other resulting in brighter specimen
- the different between the beams will be minimal
phase contrast microscopy- increasing contrast
to do this must increase phase difference between light which passes thought hr sample and light which doesn’t
- add annular ring between light source and condenser
phase ring slows down light passing through the centre more than light passing through its outside typically by 1/4
so when the two beans recombine they differ by 1/2 wavelength which increases contrast
polarising light compound microscopy
polarising light to bring out more detail and use the microscope as a true analytical instrument
- includes two polarising plates, the polariser and the analyser
- These along with a retardation plate allow us to determine fundamental features of evidence such as the sign of elongation and the birefringence
- valuable in fibre analysis and gemmology
the stereo microscope
-Unique in design and utility
-Used for low-medium power magnification (0.7 to 100x) of objects
-Long working distance and high depth of field
-Relies on light reflected from the subject and hence does require an external or built in reflective light source
-Has 2 separate light paths with slightly different viewing angles
which gives a 3D (stereoscopic) visualisation of the subject
- Used frequently
For visual analysis of trace evidence, item search and evidence recovery
comparison microscope
- Essentially 2 microscopes connected via an optical bridge
- The optical bridge allows the user to view samples on the
left stage, the right stage, to overlay the images or to view the images side by side… - allows side by side analysis of features and colours
comparison macroscope
Used for the comparison of tool marks and firearms paraphernalia
- Essentially 2 low-medium power microscopes (macroscopes)
connected via an optical bridge
-low magnification
electron microscopy
- Illuminating our sample with electrons rather than visible light allows us to produce images which become resolution limited at around 10 million times magnification giving phenomenal performance
- start all images are monochrome (black and white)
transmission electron microscopy
- Ideal for high resolution imaging of thin slices of (normally) biological specimens
- Works in a similar manner as a light microscope except that electrons rather than light passes through a specimen and through a series of ‘lenses’
- The magnified image is then projected onto a screen
scanning electron microscopy
- Ideal for determining the surface structure of samples
- Scans a beam of electrons across the sample some of which are reflected
- These reflected electrons are picked up using a complex detector producing a high resolution image
electron microscopes (TEM and SEM)
- Use electrons to illuminate the sample which takes away the resolution barrier allowing massive magnifications
- Electrons will also not pass through a glass lens
- Electron microscopy is carried out under vacuum which means that air molecules cannot interact with our electron beam
- this means that we are unable to view anything living or in fact anything that contains any moisture
- Typically, specimens have to undergo a complex dehydration process and guarantees the death of any living organism
electron microscope- the glass lens issue
-A glass lens is simply a device which allows us to bend the path of a beam of light, so we need only find some other means of carrying out this process
- electrons are charged particles which means that we can influence them using magnetism
- The lenses themselves are simply electro-magnets which allows us to bend the beam as we wish
electron microscopes- other issues
- inability to produce colour images – electrons are after all monochrome
- Sample Preparation
In addition to dehydration many samples undergoing TEM require staining to increase contrast - Samples which undergo SEM are typically larger entities and must
normally be coated with a conductive layer
SEM
- GSR and pollen is simply too small to be able to visualise readily with conventional microscopy
- Scanning electron microscopy allows for further analysis using a technique called EDX
- EDX allows us to determine the elements present within our materials which is hugely valuable
