make things look bigger and
clearer to the naked eye
microscope
objective lens
lens closest to the object or
specimen. the information
gathering lens of an optical
system
eye piece
projection lens system
projecting an image onto the
retina of the eye
substage condenser
focuses light
onto the
specimen
controls resolution
versus contrust
iris diaphragm
resolution
versus
contrast
controls the ratio
between resolution and
contras
light intensity control is the
sole means to adjust the
brightness.
Light Microscopy
uses electromagnetic radiation in
the ultraviolet or visible wavelength
range to obtain a magnified
imagine of an object
the resolution of the
imaging is limited by the
minimum focus of the
radiation due to
difraction
the difraction limit is
approximately 1um
magnification and
imagine formation
magnification is the
enlargement of an image
magnification restricts clear
image formation the
following are affected
resolution
the ability to see objects
as being separate
if you have two objects and and put them closer and
closer together at some point you will not be able to see
them as separate objects. and that is the limit of
resolution.
the ABBE equation
D relates the distance
between two objects
as alpha increases D
gets smaller and
resolution increases
as d decreases,
resolution increases
as wavelength becomes
smaller, D becomes smaller,
increasing resolution
you can improve the
resolution by changing the
wavelength. i.e by
decreasing the wavelength
relates refractive index
and wavelenght
defines resolution
as wavelength decreases,
d decreases and resolution
increases
depth of field
affects the
image you
are actually
seing.
contrast
relates to the colour light absorption of one material
in comparison to another e.g black on white would
have high black on black will have no
contrastcontrast whereas
staining helps to
eliminate the issue
with no contrast
used when
looking at
proteins, lipids or
antybody
stains can damage living cell samples,
limiting problem as it is inappropriate for
delicate or living material
optical aberrations
spherical abberation
for monochromatic light the
focus point varies with lens
position
may result in fuzzy images
that are not clear
can be solved by reducing the amount of light. or reducing the amount of lens
that is being exposed to light, can use apertures, this narrows the light source,
ligtht more likely to hit the centre of the lends and focus in one point
chromatic aberation
for polychromatic light the
focus point varies with
wavelegth
rainbow effect but the red diffracts less than
the blue because its a shorter wavelength.
the imaging will be the product of two
different points. the light focuses over a
range therefore you get a fuzy image
can be resolved by using a combination of lens with different refractive indices allowing
you to recombine the colour, put it back together to white light
using a second lens, however this method is expensive
wavelength of
light restricts
image resolution
visible light: phase contrast
microscopy
light paths varies therefore phase varies.
converts invisible phase
difference into intensity
differences
manipulates phase differences to improve
contrast using interference. manipulating
the waves to get them in the right place
and size
two unique components in
phase contrast system
annular diaphragm which
directs a hallow cone of light
through specimen
where the light comes
from, (shines through a
ring and forms a ring of
light)
a diffraction or phase plate in
the objective lens
cone of light converges on he sample, both
directed light and diffracted light passes up
into the objective lends and then to the phase
plate, phase plate separate the diffracted and
the direct light, and it alters their relative
intensity (so they are roughly the same) and
phase so they combine in the eye piece to
form a visible image resulting in interference
between the two beams. where you se no
light in the microscope you have destructive
interference and where you have constructive
interference you get bright light. (contrasting)
UV light: fluoresence microscopy
UV light is directed onto the sample
(BUT allows only visible light to return to
the eye)
Fluorescent materials re-emits visible light
Fluorescent microscopy gives specificity of
detection since only fluorescent material is
observed
can only see
things that are
fluorescent,
objects can be
marked with
flourescent
markers or stain
with fluorescent
Tissue specific, cell specific and
molecule specific fluorescent
probes are commercially available
useful with antibodies
excite the fluorescent material
with UV light, electrons go from
normal state to excited state
because they take in a photon of
energy from the UV light, and
they fall back to a lower state,
the energy diference is given out
as light in the visible spectrum. the
longer the wavelength the lower
the energy
electron microscopy
wavelength of an electron beam is much
smaller than light hence theoretically vastly
improved resolution
Light microscopy can achieve up to 97% of
theoretical resolution Electron microscopy
achieves only 5% of theoretical resolution due to
technical problems
the electrons travel in waves. the waves are
much smaller wavelengths. the only reason EM is
better is because the wavelengths are shorter
Electron beam is easily attenuated, so sample
sections need to be very then (<1m)
due to having very short
wavelength
EM Sample preparation
1 Fixation: chemical stabilisation 2. Dehydration:
baths of increaseing alcohol content 3. Infiltration:
infusion of plastic monomer into tissue 4.
Polymerisation: catalysis 5. Sectioning:
ultra-microtome (< 1) 6. Mounting: on small copper
or nickel grid
fixing means you locking it in
place.
if you have water in your sample it will boil
due to the conditions and the sample may
start to smell. the way to do this is to wash
the sample with alcohol and then you wash
the alcohol out with the infusion of plastic
monomer
by the time you finish the sample preparation the sample looks
nothing like what you started with. so when you view it you have to
try to come up with reasons why that is the live image of what you
started with earlier. ARTIFACTS. big problems especially with
biological materials. causes change or damage to the sample which
causes artifacts which can be very misleading.
method dependent on electron density of the sample.
most unstained samples look the same, need to bring
about some contrast in the electron density in the
sample. tend to use metal salts to increase electron
density.
EM goes on under high vacuum condition
scanning electron microscopy
Primary electrons hit sample surface and cause emission of
secondary (back scattered) electrons Electrostatic grid
collects secondary electrons Intensity mapped as a function of
scan position Reveals surface structures of 3-D objects
For low density surfaces it is necessary to coat
the surface with a very thin dense gold layer. This
is done by evaporation.
SEM typical resolving power ~10nm (T EM
~0.2nm) This is largely due to reliance on electron
scatter to produce an image TEM is limited only by
the ability of the sample to transmit electrons SE
micrographs therefore often relatively low
magnification but excellent resolution because of
the short wavelengths. low vacuum ddoesnt
harm live animals.
used generally to look at surfaces. electrons are fired
at the surface,depending on what they hit and what
angle they hit, eletrons are remmitted at the surface,
the electrons have higher energy, the electrons are
gathered by an electron grid which then maps out the
electrons in term of current. what you measure is the
attenuation of the flow of electrons.
atomic force microscopy
The tip (very fine) is dragged across the surface
Deflections of the cantilever are monitored by a laser The
cantilever magnifies the movement of the tip by <1000X An
image of the surface is built up Reads like a record player
AFM is often used on soft materials e.g. polymer films, biological surfaces The
dragging tip was found to dig into the surface Artifacts resulted, image not
representative ‘Tapping’ approach was developed the tip is set close to but not
touching the surface it is then vibrated the effect of the surface on the frequency of
the vibration is then used to build up an image damage to the surface is greatly
reduced
resolution is dependent on tip sharpness, cantilever length and the laser optics.
AFM or SEM surfaces: both are good high resolution techniques
but give you different information about the surfaces. AFM can
measure in all 3 dimensions in one scan. SEM has a larger depth
of field. SEM has low magnification