The Microscope
The ability of the microscope to both magnify and resolve (or
allow small structures to be seen) firstly depends on the
refraction or bending of light. Refraction occurs when light
goes from a medium of one density or refractive index to one
of another. Parallel light rays from an infinitely distant source
enter a glass lens from air and are bent and made to converge
at the point F1. The distance from the centre of the lens to this
focal point is the Focal Length (f) of the lens.
The Microscope
The amount of refraction that
occurs depends on the
difference in Refractive Index
of the two media or materials
and is described by Snell's
law:η1 sin θ1 = η2 sin θ2
The refractive index (η) equals
the speed of light in vacuum
divided by its speed in the
material in question.
The Microscope
The exact refractive
index for a given
material varies with
the color or
wavelength of light.
This explains
dispersion, or the
ability of a prism to
separate out the
deferent colors of
light:
The Microscope
Unfortunately, an image made by a single lens suffers from a number of
optical defects. These can include:
chromatic aberration, resulting in different wavelengths or colors of
light being focused at different distances;
coma, resulting in the images of structures out from the center being
smeared outwards;
spherical aberration, resulting in light passing through the lens center
being focused at a different distance to light passing through the outer
portion of the lens;
The Microscope
In order to combat these defects and produce sharp images, microscope
objectives and eyepieces are far more complex and are comprised of
multiple lenses made of glass with differing refractive indices. Microscope
objectives come in several grades of correction. The left lens is an
achromat and is corrected for two colors of light, red and blue, but it still
suffers from chromatic aberration in the green region. The apochromat in
the middle is corrected for three colors, red, green and blue. On the right
the plan achromat is not fully corrected for color but is corrected for
spherical aberration and it has a flat field which is particularly important
for photography.
The Microscope
While the power of a lens indicates the magnification it gives,
the numerical aperture gives a relative indication of its
resolving power, which is more important than magnification.
Bigger is not always better, especially when it comes to
magnification, unless it is accompanied by increased resolution
of fine detail. The final magnification will be the product of the
objective magnification, the eyepiece magnification and perhaps
other factors such as the tube factor, the nose piece factor and
the camera factor. An old rule of thumb says that the final image
magnification should not be more than 1000 times the numerical
aperture of the lens used .
The Microscope
The numerical aperture, or N.A.,
of an objective results from the
sine of half of the entrance angle
of the light cone (shown as u' in
the figure below) multiplied by n,
the refractive index of the medium
between the cover slip and the
objective. When a lens is designed
to be used dry n = 0 but when a
lens is intended to be joined to
the prep with immersion oil (oel)
the refractive index is 1.515.
Numerical aperture generally
increases with magnification
and/or degree of optical
correction.
N.A. = n*sin u'
The Microscope
bulb
The basic light path of the
microscope can be clearly
seen at the left. Light from
the bulb in the base is
focused by the collector
lenses in the base and sent
upward, via a mirror or
prism, as an illuminating
cone of light which fills the
substage condenser with
light. The condenser then
focuses the light and the
image of the fields
diaphragm on the
specimen. If the aperture
diaphragm is set properly
the emerging light will fill
the objective and give
maximum resolution.
The Microscope
The preliminary image
produced by the objective
is deflected by the prism
into the eyetubes and
then it is further magnified
by the eyepieces which
project the image into the
eye or, if fitted, into the
camera.
The Microscope
This is a schematic of the optics and
light paths of the microscope. The
condenser focuses the image of the
field diaphragm into the specimen
plane and the plane of the eyepiece
field stop. It also focuses the
filament of the lamp into the plane of
the aperture diaphragm and the
objective exit pupil. The objective
produces a primary or intermediate
image of the specimen which is
further magnified by the eyepiece
and projected into the eye or
camera.
The Microscope
The condenser plays a critical role in image formation. Highly
corrected condensers are complex and are made of a number of lenses
as seen below. Like a microscope objective, a condenser has a
numerical aperture and it should equal or better that of the highest
magnification objective being used. The wavelength of light used
(which can be selected by a filter), the objective numerical aperture
and the condenser numerical aperture all affect the resolution of the
instrument.
The Microscope
There are six basic types of microscopes used in the
modern crime laboratory:
1) The
2) The
3) The
4) The
5) The
6) The
Compound Microscope
Comparison Microscope
Stereoscopic Microscope
Polarizing Light Microscope
Microscpectrophotometer
Scanning Electron Microscope (SEM)
The first five involve the use of focused light to achieve
image magnification. The last (SEM) involves the use
of focused electrons to achieve a magnified image.
This allows for magnification far beyond what is
available through visible light microscopes.
The Microscope
Most of the initial
discussion of
microscope optics was
made for the
compound
microscope. As
described the main
parts of a compound
microscope are:
1) The base
2) The arm
3) The stage
4) The tube
5) Coarse adjust
6) Fine adjust
7) Illuminator
8) Condenser
9) Objective lens
10)Eyepiece lens
The Microscope
A comparison
microscope is a
device used to
observe side-by-side
specimens. It consists
of two microscopes
connected to an
optical bridge, which
results in a split view
window.
The Microscope
The idea behind the
comparison microscope is
simple. Two microscopes are
placed next to each other and
the optical paths of each
microscope are connected
together by the optical bridge.
The Microscope
The stereomicroscope is the
most frequently used
microscope in the crime lab.
It allows a 3-dimensional
view of objects at high
magnification (something you
can’t get using a magnifying
glass). The stereomicroscope
is the primary tool for
characterizing physical
evidence as diverse as paint,
soil, gunpowder residues and
marijuana.
The Microscope
Stereoscopic Vision
The Microscope
http://www.microscopyu.com/tutorials/flash/smz1500/index.html
The Microscope
The polarizing light
microscope is used
to study materials
that polarize light.
Often this
characteristic is
unique to a particular
material such as
crystals in soil or
fibers. By shining
polarized light
through a specimen,
one can detect the
degree to which it
further polarizes light
generating a
birefringence.
The Microscope
Natural sunlight and almost every other form of artificial
illumination transmits light waves whose electric field vectors
vibrate in all perpendicular planes with respect to the direction of
propagation. When the electric field vectors are restricted to a
single plane by filtration, then the light is said to be polarized with
respect to the direction of propagation and all waves vibrate in the
same plane.
The Microscope
Polarized light coming up off the horizontal surface of a
highway is often termed glare and can be easily demonstrated
by viewing the distant part of a highway on a sunny day. Light
reflected by the flat surface of a highway is partially polarized
with the electric field vectors vibrating in a direction that is
parallel to the ground. This light can be blocked by polarizing
filters oriented in a vertical direction with a pair of polarized
sunglasses.
The Microscope
Polarizing microscopy provides a vast amount of information about the
composition and three-dimensional structure of a variety of samples.
Observations under plane-polarized light (Figure 9(a)) reveal refractive
index differences between the fiber and the mountant and the presence
of opacifying titanium dioxide particles. The image under crossed polars
(Figure 9(b)) shows third order polarization colors and their distribution
across the fibers indicates that this is a cylindrical and not a lobate fiber
useful in predicting mechanical strength. The use of the quartz wedge
(Figure 9(c)) enables the determination of optical path differences for
birefringence measurements.
The Microscope
The microspectrophotometer
combines the latest technologies
to allow the user to measure UVvisible-NIR range transmission,
absorbance, reflectance, emission
and fluorescence spectra of
samples as small as one micron.
One can look at a crystal through
the microscope and, at the same
time, do a spectrophotometric
analysis of the crystal to
determine what it is. It’s
especially useful for drugs and
explosives.
The Microscope
The Microscope
The Scanning Electron Microscope
1)The "Virtual Source" at the top represents the electron gun, producing a stream of
monochromatic electrons.
2)The stream is condensed by the first condenser lens (usually controlled by the
"coarse probe current knob"). This lens is used to both form the beam and limit the
amount of current in the beam. It works in conjunction with the condenser aperture
to eliminate the high-angle electrons from the beam
3)The beam is then constricted by the condenser aperture (usually not user
selectable), eliminating some high-angle electrons
4)The second condenser lens forms the electrons into a thin, tight, coherent beam
and is usually controlled by the "fine probe current knob"
5)A user selectable objective aperture further eliminates high-angle electrons from
the beam
6)A set of coils then "scan" or "sweep" the beam in a grid fashion (like a television),
dwelling on points for a period of time determined by the scan speed (usually in the
microsecond range)
7)The final lens, the Objective, focuses the scanning beam onto the part of the
specimen desired.
8)When the beam strikes the sample (and dwells for a few microseconds)
interactions occur inside the sample and are detected with various instruments
9)Before the beam moves to its next dwell point these instruments count the
number of interactions and display a pixel on a CRT whose intensity is determined
by this number (the more reactions the brighter the pixel).
10)This process is repeated until the grid scan is finished and then repeated, the
entire pattern can be scanned 30 times per second.
http://www.mos.org/sln/SEM/