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Goals of Specimen Preparation Observe specimen near natural state as possible. Preservation of as many features as possi PowerPoint Presentation
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Goals of Specimen Preparation Observe specimen near natural state as possible. Preservation of as many features as possi - PowerPoint PPT Presentation


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Goals of Specimen Preparation Observe specimen near natural state as possible. Preservation of as many features as possible. Avoid artifacts (changes, loss or additional information) Render specimen stable for examination in environment of SEM. Standard Preparation. Tissue. TEM. SEM.

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Presentation Transcript
slide1

Goals of Specimen Preparation

Observe specimen near natural state as possible.

Preservation of as many features as possible.

Avoid artifacts (changes, loss or additional information)

Render specimen stable for examination in environment of SEM.

slide2

Standard Preparation

Tissue

TEM

SEM

Chem.

Fixation

Cryo

Fixation

Chem.

Fixation

Cryo

Fixation

Rinse/store

Substitution

Rinse/store

En bloc

staining

Cryo-

sectioning

Dehydration

Dehydration

Dehydration

Drying

Resin

infiltration

Mounting

Sectioning

Coating

Post staining

slide3

SEM preparation

After dehydration with ethanol, samples are Critical Point Dried (CPD)

slide4

Critical point drying (CPD)

Purpose: To completely dry specimen for mounting while maintaining morphological details.

slide5

Method

1) Water exchanged for ethanol.

2) Ethanol exchanged for liquid CO2 (transitional fluid).

3) CO2 brought to critical point (31.1 C and 1,073 psi), becomes dense vapor phase.

4) Gaseous CO2 vented slowly to avoid condensation.

5) Dry sample ready for mounting.

slide7

Sample holders

-Keep samples separated

-Hold delicate or small

samples

-Ease of sample retrieval

slide8

Freeze Drying

-Sample is quick frozen in liquid nitrogen (LN2).

-Placed in vacuum evaporator on cold block (approx. -190 C).

-Left under vacuum for several days to sublimate water.

-Mounted and coated.

slide9

Hexamethyldisilizane

HMDS is a chemical method of “drying” the sample

Primarily used with insects, larger fleshy tissues, soft invertebrates, etc...

HMDS is a strong irritant and volitile (flammable).

Brief protocol:

-After fixation - ethanol dehydration to 100%

-Transition from ethanol to HMDS

-Two changes of pure HMDS

-Left overnight in dessicator with silica gel

slide10

Mounting the specimen onto stubs

“Stubs” are specimen holders specific for the instrument being used (e.g. Zeiss or Hitachi SEM)

Specimen is held to stub by conductive tape, paste or glue.

slide11

Conductivity of Samples

Charging results in:

deflection of the beam

deflection of some secondary electrons

periodic bursts of secondary electrons

increased emission of secondary electrons from crevices

slide12

Coating the Sample

a) Increased conductivity

b) Reduction of thermal damage

c) Increased secondary and backscattered electron emission

d) Increased mechanical stability

Accomplished by:

-Using OsO4 as fixative (biological)

-Painting a “grounding line” with silver or carbon paste

-Coating with nonreactive metal or carbon

slide13

Sputter coating

Gold, gold palladium target

-vacuum of approx. 2 millibar

-thickness 7.5 nm to 30nm

slide14

Thermal evaporation

-Typically used for shadowing

- 2 x10-7 torr

-From coarse to fine:

Carbon, gold, chromium, platinum, tungsten, tantalum

slide15

Evaporation

Trough for powders/cleaning

slide16

Used for high melting point metals (e.g. tantalum)

Similar to create emission of electrons from filament in microscope

Provides highest resolution

E-beam

slide17

Carbon Coating

For samples in SEM where x-ray information is needed.

TEM grids needing extra support

Support for replicas

Good vacuum required

Carbon rod may need outgassing

Do not look directly at heated electrodes

slide18

Carbon ribbon

Rotary device to

ensure uniform coating

Carbon Rods