SAMPLE PREPARATION

NANOSCIENCE AND NANOTECHNOLOGY

• Nanoscience is the study of structures and

materials on an ultra-small scale.
• Nano – 10-9 m
• Nanotechnology is the branch of technology
that deals with dimensions and tolerances of
less than 100 nanometers, especially the
manipulation of individual atoms and
molecules.

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 OUTLINE
• Synthesis of Nanomaterials
• Characterization of Nanomaterials
• Analysis properties of Nanomaterials
• Application of the Nanomaterials

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 SAMPLE PREPARATION
Sample preparation :
• It is an extremely important part of the overall
chemical analysis.
• Purposes of sample preparation
1. Remove interferences
2. Increase sensitivity
• The prepared sample must be in a phase
compatible with the analytical instrument
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 SAMPLE PREPARATION
TECHNIQUES
 Chemical Fixation techniques
 Cryo Fixation techniques
 Dehydration
 Embedding biological samples
 Sectioning
 Staining
 Mechanical milling
 Chemical etching
 Ion etching
 Conductive coating
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 TYPES OF FIXATION
• The first step in any biological Electro
Microscopy (EM) sample preparation is Fixation.
• Fixation is the method that halts and preserves all
metabolic processes of a biological specimen as
close to the living state as possible through
subsequent processing and imaging stages.
• There are two main types of fixation:
1. Chemical fixation
2. Cryofixation

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 CHEMICAL FIXATIONS
• Chemical fixation is the most popular and
accessible fixation method for biological EM.
• There are several chemicals that, once applied to a
biological sample, will form cross-links between
amino acids, proteins, and lipids, usually in the
form of covalent bonds.
Example:
Proteins with aldehydes (formaldehydes
glutaraldehydes)
Lipids with osmium tetroxide

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 PRIMARY FIXATION
• Glutaraldehyde, Formaldehyde, Acrolein are widely
used chemicals for primary fixation.
• Glutaraldehyde and acrolein cause extensive, rapid
and permanent cross-linking of proteins
• It is possibly to apply glutaraldehyde as the only
fixative. however, it penetrates into a sample slowly
and during this period tissue degradation may occur.
For this reason, many researchers choose to combine
glutaraldehyde with formaldehyde.
• Formaldehyde penetrates a sample rapidly, providing a
temporary cross-linking of proteins that is subsequently
replaced by glutaraldehyde.

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 SECONDARY FIXATION
• A secondary fixation step is often applied, especially
for TEM sample preparation.
• Osmium tetroxide is the most common secondary
fixative and it has the advantage of preserving lipid
membranes, which are not preserved with aldehyde
fixation alone.
• Potassium dichromate, Chromic acid and potassium
permanganate used for histological preparation.
• Ethanol, methanol, acetone are precipitating fixatives.
• Alcohol causes shrinkage of tissues, acetic acid
causes tissue swelling, combination of these two gives
better preservation of tissue morphology.
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 Significant of Chemical Fixation
• Fixation process preserve a biological material
as close to its natural state upto examination.
• It is Preserve biological sample from decay.
• It terminates ongoing biochemical reaction in
sample
• It increase the mechanical strength and
stability of the biological samples

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 Demerits of Fixation
• After the fixation, samples is made unsoulble and
harden with chemical change
• Certain period of time, degree of fixation may varied
Over-fixation, Just fixation, under fixation.
• Based on this the sample results may vary.
• Light microscopy, the quality of fixation below its
resolving power does not provide the results.
• Even the most careful fixation may alter the sample
and introduce artifacts , that can interfere with
interpretation of the biological samples.
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 Intermediate Question
• What is Fixations ?

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 CRYOFIXATION
• The freezing of a specimen by immersion in liquid nitrogen.
• Definition: Cryofixation is a technique for fixation or stabilization of
biological materials as the first step in specimen preparation for electron
microscopy.
• Typical specimens for cryofixation include small samples of plant or
animal tissue, cell suspensions of microorganisms or cultured cells,
suspensions of viruses or virus capsids and samples of purified
macromolecules, especially proteins.
• The method involves ultra-rapid cooling of small samples to liquid
nitrogen temperature or below, thus stopping all motion and metabolic
activity, and preserving the internal structure by freezing all fluid phases
solid.
• The ultimate objective is to freeze the specimen so rapidly that ice crystals
are unable to form, or are prevented from growing sufficiently large to
cause damage to the specimen's ultrastructure.
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 DEHYDRATION (loss of water)
• Chemical Removal of Water and Fixative from the
Specimen
• Dehydration carried out gradually – sudden loss of
water does not distort the structure of sample.
• Dehydration carried out by maintaining the sample in
an atmosphere of very low pressure.
• Many dehydrants are alcohol , which are hydrophilic so
attract the water from the tissues, which progressively
decrease the concentration of water and increase the
concentration of dehydrant.
• Ethyl Alcohol, acetone, butyl alcohol, glycerin etc
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 Embedding biological samples
• After dehydration, Tissue observe in TEM is
embedded, then ready for viewing.
• Initially, tissue is passes through a transition solvent
(epoxy propane) then infiltrated with resin (Araldite
epoxy resin).
• Tissue embedded directly in water miscible acrylic
resin.
• Resin has been polymerized (hardened) the sample in
thin sectioned and stained – ready for viewing.

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 SECTIONING
• Produce thin slice of specimen, semitransparent to
electrons.
• Ultra microtome with a diamond knife to produce
ultrathin slices about 60-90 nm thick.
• Thickness of sections can be determined by the color
of the reflected light.
• Disposable glass knives – used in lab – Cheaper
• This method is used to obtain thin minimally
deformed samples that allow for the observation of
tissue samples.
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 STAINING
• Uses heavy metals like lead, uranium , tungsten to scatter
imaging electrons give contrast between different structure.
• In biology, specimens are stained before embedding and
after sectioning.
• In TEM samples of biological tissues can utilize high atomic
number stain to enhance contrast.
• The stain absorbs electrons or scatter part of the electron
beam which is projected onto the imaging system.
• Uranyl acetate, lead nitrate, lead hydroixe can use for
staining.
• Double staining (uranyl acetate with nitrate) give better
results

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Questions ???

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