Transmission electron microscopy (TEM) is

a microscopy technique in which a beam of electrons is transmitted

through an ultra-thin specimen, interacting with the specimen
as it passes through. An image is formed from the interaction
of the electrons transmitted through the specimen; the image
is magnified and focused onto an imaging device, such as
a fluorescent screen, on a layer ofphotographic film, or to be detected by
a sensor such as a CCD camera.
TEMs are capable of imaging at a significantly
higher resolution than light microscopes, owing to the small de Broglie
wavelength of electrons. This enables the instrument's user to
examine fine detaileven as small as a single column of
atoms, which is thousands of times smaller than the smallest
resolvable object in a light microscope. TEM forms a major
analysis method in a range of scientific fields, in both physical
and biological sciences. TEMs find application in cancer
research, virology, materials science as well aspollution, nanotechnology,
and semiconductor research.
At smaller magnifications TEM image contrast is due to
absorption of electrons in the material, due to the thickness
and composition of the material. At higher magnifications
complex wave interactions modulate the intensity of the
image, requiring expert analysis of observed images.
Alternate modes of use allow for the TEM to observe
modulations in chemical identity, crystal orientation,
electronic structure and sample induced electron phase shift
as well as the regular absorption based imaging.
The first TEM was built by Max Knoll and Ernst Ruska in 1931, with this
group developing the first TEM with resolution greater than
that of light in 1933 and the first commercial TEM in 1939.
Cell Fractionation
Cell fractionation is the process of producing pure fractions of
cell components. The process involves two basic steps:
disruption of the tissue and lysis of the cells, followed by
centrifugation.
Tissue Disruption and Lysis.
The first step in cell fractionation is tissue disruption and cell
lysis. The objective is to disaggregate the cells and break
them open with minimum damage to the cellular fraction of
interest (i.e., don't use a hammer). Tissues can be broken up
and cells lysed in a number of ways. The three basic methods
of breaking up the tissues and cells are 1) homogenization, 2)
sonication, and 3) osmotic lysis. The particular method one
chooses depends on the tissue, the cell type, and the
particular cell fraction of interest. Most animal and plant
tissues must be homogenized. Homogenization involves the
use of a mechanical homogenizer, like a blender or a mortor
and pestle, to break the tissue apart and lyse the cells.
Sonication involves the use of ultrasound to disrupt the cells.
Sonication is often used when prokarytic cells are to be lysed.
Osmotic lysis is often the method of choice when dealing with
cells that are vulnerable to osmotic stresses. Red blood cells
are a perfect example of a cell that can easily be lysed
through osmotic stress.
Staining is an auxiliary technique used in microscopy to enhance
contrast in the microscopic image. Stains and dyes are frequently
used in biology and medicine to highlight structures in biological tissues for
viewing, often with the aid of different microscopes. Stains may be
used to define and examine bulk tissues (highlighting, for

example, muscle fibers or connective tissue), cell populations (classifying

different blood cells, for instance), or organelles within individual cells.
In biochemistry it involves adding a class-specific
(DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or
quantify the presence of a specific compound. Staining
and fluorescent tagging can serve similar purposes. Biological
staining is also used to mark cells in flow cytometry, and to
flag proteins or nucleic acids in gel electrophoresis.
Simple staining is staining with only one stain/dye. There are
various kinds of multiple staining, many of which are
examples ofcounterstaining, differential staining, or both, including double
staining and triple staining.
Phase contrast microscopy is an optical microscopy
technique that converts phase shifts in light passing through a
transparent specimen to brightness changes in the image.
Phase shifts themselves are invisible, but become visible
when shown as brightness variations.
Figure 1: The same cells imaged with traditional bright field
microscopy (left) and with phase contrast microscopy (right).
When light waves travel through a medium other than
vacuum, interaction with the medium causes the wave
amplitude and phase to change in a manner dependent on
properties of the medium. Changes in amplitude (brightness)
arise from the scattering and absorption of light, which is
often wavelength dependent and may give rise to colors.
Photographic equipment and the human eye are only
sensitive to amplitude variations. Without special
arrangements, phase changes are therefore invisible. Yet,
phase changes often carry important information.
Phase contrast microscopy is particularly important in biology.
It reveals many cellular structures that are not visible with a
simpler bright field microscope, as exemplified in Figure 1.
These structures were made visible to earlier microscopists by
staining, but this required additional preparation and killed the
cells. The phase contrast microscope made it possible for
biologists to study living cells and how they proliferate
through cell division.[1] After its invention in the early 1930s,
[2] phase contrast microscopy proved to be such an
advancement in microscopy, that its inventor Frits Zernike
was awarded the Nobel prize (physics) in 1953.[3]
The basic principle to make phase changes visible in phase
contrast microscopy is to separate the illuminating
background light from the specimen scattered light, which
make up the foreground details, and to manipulate these
differently.
The ring shaped illuminating light (green) that passes the
condenser annulus is focused on the specimen by the
condenser. Some of the illuminating light is scattered by the
specimen (yellow). The remaining light is unaffected by the
specimen and forms the background light (red). When
observing an unstained biological specimen, the scattered
light is weak and typically phase shifted by -90 relative to
the background light. This leads to the foreground (blue
vector) and background (red vector) having nearly the same
intensity, resulting in a low image contrast (a).
A scanning electron microscope (SEM) is a type of electron
microscope that produces images of a sample by scanning it
with a focused beam of electrons. The electrons interact with

atoms in the sample, producing various signals that can be

detected and that contain information about the sample's
surface topography and composition. The electron beam is
generally scanned in a raster scan pattern, and the beam's
position is combined with the detected signal to produce an
image. SEM can achieve resolution better than 1 nanometer.
Specimens can be observed in high vacuum, in low vacuum,
in wet conditions (in environmental SEM), and at a wide range
of cryogenic or elevated temperatures.

The most common SEM mode is detection of secondary

electrons emitted by atoms excited by the electron beam. The
number of secondary electrons depends on the angle at which
beam meets surface of specimen,[citation needed] i.e. on
specimen topography. By scanning the sample and collecting
the secondary electrons with a special detector, an image
displaying the topography of the surface is created.
[clarification needed]