Microscopy-Richard Flashcards
what is a converging lens?
focus parallel rays of light into a single point of convergence
the distance between the lens and that point is known as the focal length
the point of convergence is known as the focal point
What is a diverging lens?
cause parallel rays of light to diverge
focal point is in front of the lens which means that objects look smaller and nearer
Refraction
when lenses bend light
occurs whenever a mean of light moves from one medium to another at an angle other than 90 degrees and can be seen in many everyday objects
occurs because light slows down as it enters glass from air
the amount of bending or refraction relates to the refractive index of the medium which is a measure of how much our medium slows rays of light
the greater the refractive index, the slower the light travels through the medium compared to through air
What is the refractive index?
ratio of velocity of light in a vacuum to velocity of light in the medium and is always a number greater than 1
What law is used to calculate refraction?
snells law
Snell’s law
n1sin01=n2sin02
n1=refractive index of original medium
01=angle at which the rays enter the second medium from the first
n2=refractive index of second medium
02=angle at which the rays pass through the second medium after being refracted
Transverse magnification
occurs when an object is further away from the lens that its focal point
image produced is inverted
Angular magnification
when an object is closer to the lens than its focal point, a different form of magnification occurs
Real image formation
when an object is further away from the lens than its focal point it forms a real image
the rays of light converge to a single point
A virtual image
when an object is nearer to the lens than its focal point it forms a so-called virtual image
virtual images are not visible if we place a screen behind the lens as the rays do not come to focus
Compound microscope
imaging of transparent or semi-transparent objects
mag up to 1000x
relies on light transmitted through the subject by an inbuilt light source
short working distance and low depth of field
angular and transverse magnification techniques
objective lens produces a primary real image which is then enlarged by transverse mag
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
What are 8 parts of a microscope?
ocular lens objective lens turret stage condender diaphragm light source focussing knobs
What is the ocular lens?
eye pieces
What is the objective lens?
primary lens
improves resolution and removes distortions caused by chromatic and other optical aberrations
on lens it tells you mag and numerical aperture
What is the turret?
where the objective lens is mounted, holds 3 or 4 lenses
what is a stage?
sample is placed
what is a condenser?
focusses the light passing into your sample
what is a diaphragm?
control the amount of light and also the depth of field
what is a light source?
may or may not be colour corrected
what are the focusing knobs?
allow you to bring your samples into sharp focus
What is the numerical aperture?
measure of the angular range over which the lens can gather light but also affect magnification
as numerical aperture increases, mag and resolution increases
How do you increase numerical aperture?
place a drop of oil of R1 1.52 between our lens and our sample
we alter the way that light from the sample enters the lens, meaning that light takes a more direct path thereby increasing NA and hence the resolution
known as oil immersion
what is resolution?
the ability of a lens to clearly distinguish fine detail or resolve minute entities lying close together in the specimen as separate
How is resolution calculates?
r=1.22 x wavelngth / 2 x NA
resolution is therefore affected by numerical aperture and wavelength of light
Limits of resolution
to resolve an object, the object must be greater than half the wavelength of our light source in size
Bright field compound microscopy
produces an image with a bright background and specimen in relief
image is produced by the sample interacting with the rays of light
gives rise to differences in contrast and colour
when dealing with colourless or transparent samples, we can increase contrast further using stains
dark field compounds microscopy
produces an image with a dark background
used for specimens which do not generate enough contrast in bright-field (colourless or thin objects)
dark-field image is produced by the addition of an opaque dick in the light path
as light enters the sample, some is diffracted which passes into the eyepiece, but majority is transmitted unchanged
light is stopped by a direct illumination block
phase contrast compound microscopy
samples which lack contrast under bright field or even dark field microscopy
fragment of glass or unstained cells
enhances contrast and hence allows visualisation of specimens which would otherwise be virtually invisible
an image is formed by light that is absorbed, scattered, reflected, refracted, diffracted
thicker the 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 if all regions in the visible range are not absorbed evenly and attenuation is sufficient
how does the phase contrast technique work?
the light which passes through the sample is not just attenuated, it is slowed down or diffracted with respect to light which does not pass through the sample
when the light that passes through the sample and that which does not recombine, slight differences in phase between the two beams cause differences in brightness hence allowing visualisation
areas of our sample which absorb light or cause significant phase differences appear dark, whereas other areas will vary in brightness according to their thickness and the way which they interact with the light
recombined waves
when two light beams recombine, the respective brightness of the resulting beam depends upon the phase of the two beams
if the two beams recombine out of phase, they effectively cancel each other out resulting in a darker area on specimen
if the two beams recombine in phase, the two beams effectively amplify each other resulting in a brighter area on specimen
the difference between the two beams will be minimal with very slight increases and decreases in brightness
the greater the difference between bright and dark, the greater the contrast
increasing contrast
add annular ring between light course and condenser producing a hollowed-out ring of light brighter on the outside than the inside
any light which interacts with the sample is diffracted as per normal and becomes slowed by around 1/4 wavelength and then passes through the centre of a phase ring
light which does not interact with the sample is un-slowed and passes through a thinner part of the phase ring
the phase ring is rather clever and differentially slows down light passing through its centre more than light passing through its outside typically by 1/4 wavelength
so when the two beams recombine later on, they differ by 1/2 wavelength which massively increases contrast.
Polarising light compound microscopy
includes two polarising plates, the polariser and the analyser
along with retardation plate allow us to determine fundamental features of evidence such as the sign of elongation and the birefringence
stereo microscope
low-medium power mag
long working distance and high DOF
relied on light reflected from the subject so requires external or built-in reflective light source
has 2 separate light paths with different viewing angles which gives 3D visualisation
comparison microscope
2 microscopes connected via optical bridge
comparison macroscope
used for comparison of tool marks and firearms paraphernalia
electron microscopy
illuminate our sample with electrons rather than visible light allows us to produce images which become resolution limited at around 10 million times mag
all images are monochrome
transmission electron microscopy
ideal for high resolution imaging of thin slices of bio specimens
scanning electron microscope
ideal for determining the surface structure of samples
scans a beam of electrons across the sample some of which are reflected
reflected electrons are picked up using a complex detector producing a high-resolution image with 3D properties
both TEM and SEM
use electrons to illuminate the sample which takes away the resolution barrier which comes from using visible light allowing massive magnifications
electrons cannot easily travel through air which means the EM is rather complex
electrons will also not pass through a glass lens, which means we have to find an alternative means of mag and focusing
solving the air issue with EM
carried out under vacuum which means that air molecules cannot interact with electron beam
use of high vacuum means we can’t view anything living or anything that contains moisture
specimens undergo complex dehydration process
glass lens issue
electrons are charged particles which means we can influence them using magnetism
the lenses are electromagnets which allows us to bend the beam
sample preparation -EM
staining to increase contrast
stains nasty and stop electrons
samples must be coated with conductive layer
electrons tend to cause the sample to become negatively charged affecting image and coating allows charge to dissipate to earth
What is EDX?§
determine the elements present within materials
