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• 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 phaseversus how readily they dissolve in the mobilephase.

• Additional spots become visible when inspecting the TLC plates with UV.

Paper chromatography • 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 • 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 • 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 granules-

purple; 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 non-nuclear 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 carrying something that can be detected visually, chemically or fluorescently.

• Specific neuropeptides, such as somatostatin, can also be detected in this fashion

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.