Electron Microscopy
• The electron microscope (EM)
depends on the fact that
electron beams can be bent
and focused by electric or
magnetic fields, allowing them
to form magnified images in
much the same way as light in
light microscopy. The electron
beam originates from a
filament and, after passing
through
an
evacuated
microscope column, is focused
on a fluorescent screen or a
photographic plate.
F
Transmission Electron Microscopy
• the electron beam passes through the specimen. The denser areas in the
specimen scatter more electrons and the corresponding regions in the image
appear darker.
• Electron stains of high density are usually used to enhance the contrast.
• specimen preparation generally used for TEM resembles in principle the
fixation, embedding, sectioning, and staining used for the light microscope.
• fixatives such as OsO4, also serve as stains. Others, such as glutaraldehyde,
do not add to the contrast and additional treatment with electron stains is
needed.
• Used for isolated cell components, macromolecules, and macromolecular
assemblies such as ribosomes and viruses
• advance in TEM is the imaging of cells and macromolecules that are
embedded in amorphous ice at very low (liquid nitrogen) temperatures.
These specimens are unfixed and unstained, so the contrast arises directly
from differences in density of the cell components
Scanning Electron Microscopy
• a beam of electrons is focused to form a small diameter probe that is
scanned across the specimen in a process similar to that used to produce
television images
• This probe interacts with atoms in the specimen causing them to emit a
variety of signals.
• The microscope collects and displays electrons scattered from the surface
of the object (secondary electrons).
• Generally, the specimen has to be dried and coated with metal. The metal
increases the electrons scattering and also acts as a conductor to avoid
accumulation of charges.
• useful in observing the surfaces of three-dimensional objects.
• it is restricted in resolution to above 5 to 10 nm
• high-resolution SEM (HRSEM) has been used as an effective tool to study
subcellular detail the specimen is placed within the magnetic field of the
final lens (rather than below as in conventional SEM).
• The material is either fixed or rapidly frozen, the water is generally
removed or substituted.
• uncoated specimens can be used, generally they are coated with a metal
coat, optimally chromium or tantalum at a thickness of 1 to 12 nm.
Cryo-electron Tomography
• Electron cryo-electron tomography holds a promise of reconstructing the
actual structure of specimen in their native state.
• cells are preserved in their native states by vitrification
• The specimen is quickly exposed to a cryogen and then has to be kept in
the cold to avoid a transition to crystalline ice.
• In tomography, a three dimensional density distribution is arrived at by
combining two-dimensional projections viewed from different directions.
The specimen is tilted typically in the angular range of -70o to +70o. The
resolution (r) of a tomogram depends on the thickness (t) of the of the
specimen and the number of projections (n): r~pt/n.
Chromatography
• Chromatography is a powerful technique for separating
mixtures.
• There are different types of chromatography, such as paper,
thin layer, or column chromatography (amongst others), each
with its own strengths and weaknesses.
• Chromatography systems have a stationary phase (which can
be solid or liquid) and a mobile phase (usually liquid or gas).
• In column chromatography both phases are placed in a column
container.
Liquid Chromatography
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Useful for separating ions or
molecules that are dissolved
in a solvent.
If the sample solution is in
contact with a second solid or
liquid phase, the different
solutes will interact with the
other phase to differing
degrees due to differences in
adsorption, ion-exchange,
partitioning, or size.
These differences allow the
mixture components to be
separated from each other.
High-performance liquid chromatography
• form of liquid chromatography
• to separate compounds that are dissolved in solution.
• HPLC instruments consist of a reservoir of mobile phase,
a pump, an injector, a separation column, and a
detector.
• Compounds are separated by injecting a plug of the
sample mixture onto the column.
• The different components in the mixture pass through
the column at different rates due to differences in their
partitioning behavior between the mobile liquid phase
and the stationary phase.
Thin layer chromatography
• Thin layer chromatography (TLC) is a method for
identifying substances and testing the purity of
compounds.
• TLC is a useful technique because it is relatively quick
and requires small quantities of material.
• Separations in TLC involve distributing a mixture of two or
more substances between a stationary phase and a
mobile phase.
• The stationary phase is a thin layer of adsorbent (usually
silica gel or alumina) coated on a plate.
• The mobile phase is a developing liquid which travels up
the stationary phase, carrying the samples with it.
• Components of the samples will separate according to
how strongly they adsorb on the stationary phase
versus how readily they dissolve in the mobile
phase.
• Additional spots become visible when inspecting the TLC
plates with UV.
Paper chromatography
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In paper chromatography, like thin layer
chromatography
(TLC), substances are distributed between
a stationary phase and mobile phase.
The stationary phase is usually a piece of
high quality filter paper.
The mobile phase is a developing solution
that travels up the stationary phase,
carrying the samples with it.
Components of the sample will separate
readily according to how strongly they
adsorb on the stationary phase versus how
readily they dissolve in the mobile phase.
Prepare a chamber by pouring a solvent at
the bottom (up to 2 cm high) and let the
solvent saturate.
Electrophoresis
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Electrophoresis is a separation
technique that is based on the
mobility of ions in an electric field.
Positively charged ions migrate
towards a negative electrode and
negatively-charged ions migrate
toward a positive electrode.
Examples: gel electrophoresis
capillary electrophoresis
In this technique the
support gel maintains a pH gradient.
As a protein migrates down the gel, it
reaches a pH that is equal to its
isoelectric point. At this pH the
protein is netural and no longer
migrates, i.e, it is focused into a sharp
band on the gel.
• Capillaries are typically of 50 µm inner diameter and 0.5
to 1 m in length. The applied potential is 20 to 30 kV.
• A small volume of sample (10 nL) is injected at the
positive end of the capillary and the separated
components are detected near the negative end of the
capillary.
• Due to electroosmotic flow, all sample components
(positively-charged ion) migrate towards the negative
electrode and and carry solvent molecules in the same
direction.
Cell culture and Single cell Isolation
Histology Techniques
• Histology is the study of tissue. To examine the tissue components, it is
necessary to process the tissue
• steps of processing involve: fixation, sectioning, and visualization
Fixation
• involves processing the tissue to prevent its decomposition; cell
metabolism is stopped.
• most common fixative is Formalin, a 37% aqueous solution of
formaldehyde, which is either used on its own or in combination with
other chemicals.
• Formaldhyde reacts with the amino groups of proteins, and thus
preserves the general structure of the cell and extracellular
components.
Sectioning
• To allow a specimen to be examined, it must be thinly sliced on the level of
micrometers (1/1000th of a millimeter).
• cryosectioning- tissue can be frozen and sectioned at a low temperature,
using a cryostat with a razor blade knife. The fresh or fixed tissue is frozen
to a "chuck" which is placed in a holder in a refrigerated chamber (-20
degrees). When a wheel is turned, the holder advances towards the blade by
the required width of the tissue; if 7 um thick sections are wanted, the
holder moves 7 um. During this process, the holder also moves up and
down, which brings the edge of the tissue down on the top of the blade.
• microtome. A microtome works similarly to a cryostat but does not require
cold. Instead, the tissue is fixed, dehydrated and completely permeated with
paraffin. It is then embedded in a paraffin block. This block is stuck to a
chuck, and then placed in a holder, which is progressed by a rotating wheel.
Visualization
• various dyes, staining and imaging techniques are available
that allow different cell types, or structural or molecular
components, to be preferentially visualized.
• Stains can also be used in combination on a single tissue
sample. When a stain is dissolved in ethanol, all of the water in
the tissue must be replaced by ethanol before the staining can
take place.
Chemical Stains
• Hematoxylin and Eosin.
• quite useful for the display of
general structural features of the
tissue.
• Hematoxylin stains nuclear
substances, chromosomes,
mitochondria and muscle
striations blue to black.
• Eosin stains the cytoplasm and
other structures various shades
of red.
• Cresyl Violet is a Nissl stain, used
for staining neurons which
contain Nissl bodies (rough
endoplasmic reticulum). The cell
bodies are stained a violet color.
Silver staining
• Neuroscience
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•
Ramon y Cajal first demonstrated the
shapes and the interconnections in neural
tissue
impregnation with a silver dye, silver
staining can be used to stain neurons and
glial cells.
• neurons appear a
yellow/orange color,
neurofibrils are brown to black,
and neuroglia will be black. In
the silver stained tissue, only
the glial elements remain
stained a black color.
Other Chemicals
• Carmine: Dyes nuclei red.
• Gold Chloride/Formic Acid. Muscle fibers red, myelin black; nerve fibers
brown/red
• HPS: Hematoxylin, Phloxine, Safron. Nuclei- blue; cytoplasm, muscle,
myelin- red; connective tissue- yellow
• Mason Stain: chromatin- blue to black; nuclei- red; zymogen granulespurple; cytoplasmic elements- red to mauve; collagen, mucus or connective
tissue- green.
• MB&P: Methylene Blue and Phloxine: Methylene blue is also a Nissl stain
which stains the cell body blue. Phloxine stains collagen and other nonnuclear tissue elements bright rose
• Nuclear Fast Red: Stains nuclei red
• Osmic acid: Myelin is stained black.
• Wolke's Myelin Sheath: Myelin sheath, blue; background, clear; glial cells
and nucleoli of neurons, black
Other Staining Techniques
Immunohistochemistry &
Immunocytochemistry
• Use of antibodies against a specific
target (antigen) to detect the target's
presence.
• The target is usually a protein. The
antibody will have another
substance conjugated to it, which is
targeted by another antibody that is •Specific neuropeptides, such as
somatostatin, can also be detected in
carrying something that can be
this fashion
detected visually, chemically or
fluorescently.
Direct Injection
• can also be directly injected with
substances which then fluoresces
and allows them to be visualized
• Neurons will also readily pick up
horseradish peroxides (HRP) and
pass it on to the next cell up their
pathway; early tract-tracing
experiments were often carried
out using HRP.
Transmission Electron Microscopy
• the tissues are stained with a heavy metal, such as lead citrate, to make the
structures more electron dense. Electrons are then streamed through the
tissue, creating an image. Can be used to visualize sub-cellular elements.