FIXATION

TEM SPECIMEN PREPARATION

===>SAMPLE ISOLATION
Collection of cells or isolation of tissue

===>FIXATION(S)

preservation of cellular components and organization ===>RINSES
To remove residual fixatives ===>DEHYDRATION To remove water and replace with suitable solvent for subsequent steps ===>INFILTRATION Replacement of dehydrating fluid with plastic ===>POLYMERIZATION Conversion of liquid plastic to solid ===>SECTIONING Slicing of samples thin enough for viewing(10,000 slices/mm) ===>STAINING Stain with aqueous heavy metals ===>VIEWING ===>RECORDING IMAGES ===>REPRODUCING IMAGES Negative Stain-- see objects light against dark background
for small particulates, don't need to fix, embed and slice

thin layer of sample is adsorbed on surface of coated grid
(grids need carbon or plastic (formvar) film to prevent particles from falling through)
add stain, then quickly blot off

soluble stain

uranium pH 4

phosphotungstic acid pH 6-8

beam stable

small size for penetration

electron dense

liquid phase keeps structure from collapsing

limit of resolution is 2 nm due to size of stain molecules

FIXATION
Goal of fixation==>approximate the living state as much as possible

1. preserves cell structure as close to in vivo as possible

A. no changes in volume, morphology, or spatial relationships

B. no losses in cellular constituents, no breaks in membranes

2. protects samples during processing steps such as dehydration, embedding etc. 3. stains samples for viewing 4. provides stability of sample in beam

Fixatives are not universal and omnipotent. Therefore one must balance preservation of organelles of interest vs. overall fixation. If a researcher is only interested in membrane structures, it may not be necessary to preserve all the rest of the cellular constituents. For example, the preservation of all the cellular ribosomes may make it difficult to observe the distribution of membranes in the cell.

Three main categories of fixatives:

1. coagulent fix ==> denatures or precipitates proteins (e.g., acid or alcohol to Îfixâ cells) 2. additive fix ==> adds into compound, links molecules together
It works by providing cross links between molecules, usually proteins, to hold everything together 3. cryogenic fix ==> rapidly freezes sample, locking everything in place
This techniques keeps everything locked in place only so long as the sample remains frozen.
Thawing the sample can lead to significant changes in structures Remember that cross linking of molecules, for example protein-protein interactions, can cause denaturation, bond shifting, release of H+, changes in ionization. All these can lead to changes in the material Coagulent fixatives:
alcohol, acetone at room temperature or on ice this fixation is not suitable for EM, can be useful for light alcohol, acetone at -80 C if the tissue is plunge frozen, then warmed to -80 in these solutions, can get excellent preservation acids, such as picric acid, or acetic acid not suitable for EM Additive fixatives 1. Osmium tetroxide
first used by Ruska in 1939

Palade (1952) buffered it

Osmium is reduced to a metallic form after fixation==> there are several different valent states for osmium, some of which are more reactive than others.

Osmium reacts with

1. double and triple bonds ==>lipids, fats, waxes

2. SH groups ==>disulfide bridges some amino acids

3. terminal OH groups or aldehyde groups ==>sugars

4. aromatics with 2+ OH groups ==> sugars, sterols

Doesn't react with peptide bonds, starch, glycogen, pectins, lignins, some sugars, nucleic acids
After fixation in osmium, cells generally are no longer selectively permeable and may no longer need buffers
2. Aldehydes
Because osmium doesn't react with many compounds,

Barnett and Sabbatini introduced glutaraldehyde in 1962

1. formaldehyde

2. glutaraldehyde

2 proteins-NH2 + glutaraldehyde==> protein-N=C-glut-C=N-protein + 2 water

aldehydes react best with proteins

1. amino groups ==> proteins

2. OH, SH, COOH ==> carbohydrates

3. nucleoproteins ==> DNA

4. Osmium fixation solution

5. glycogen

glutaraldehyde is a neutral fixative ==> it becomes ionized upon cross- reacting. It enters cells as an uncharged molecule, then is trapped inside as a charged molecule upon cross-reacting.

cells retain selective permeability in glutaraldehyde, so care must be taken to maintain ionic balance inside and outside of the cell during fixation

Chemical supplies for best results, formaldehyde is made fresh from paraformaldehyde
forms degradation products within 2 weeks
it can be purchased in sealed ampules with a long shelf life

glutaraldehyde--buy EM grade
OTHER FIXATIVE CONSIDERATIONS Fixative concentration low concentrations take longer to fix sample, which allows for extraction, rearrangement, volume change

high concentrations can 1) destroy enzyme activity,

2) damage fine structure

3) can cleave proteins

usually run a concentrations between 1-3%.

however, there are reasons for low fixative concentrations==>preserves antigienicity (at a cost of poorer preservation)

Buffers
1. should not have effects on cell constituents

2. should not react with fix or added reagents

3. should not extract cell material

Aldehyde fixation releases significant H+ ions. Usually buffer animals at about pH7.2-7.4., plants around pH 6.8 However, keep in mind that in the same cell, the cytoplasm can be pH 7, the nucleoplasm 7.6-7.8

Use a concentration of 0.05-0.2M

phosphate is commonly used, but can be extractive and may precipitate solutions (e.g., sea water).
Avoid if doing phosphatase cytochemical reactions

cacodylate is excellent, but arsenic based

Zwitterionic buffers (HEPES, MOPS, PIPES) more recently have been used with good results

Osmolarity

Cells may expand or contract if the ion balance between fix and cell interior is not similar.
Fixation can shut down membrane pumps, so ion balance may change dramatically

If the buffer is adjusted to exactly mimic the ionic concentration in of cell, get very slow penetration of fixative into cell. It is therefore best to set the fixative slightly hypotonic for rapid uptake of fixative

use sucrose, salt, and buffer concentration to adjust.

for phosphate and cacodylate, osmotic potential is approximately twice the concentration (0.1M is about 200mOsm)

Animal cells run about 300 mOsm; marine cells can be 1000 mOsm (milli Osmoles).

Temperature
high temperature ==>faster penetration
==>higher enzyme autolysis

use lower temperature since diffusion trade off is less deleterious than degredation (enzyme activity doubles every 10 C)

Cryogenic Fix
ADVANTAGES
1. physical process
few biochemical reactions involved

2. little alteration of cellular constituents

3. no loss of soluble materials

4.very rapid preservation, far faster than chemicals

DISADVANTAGES

1. Ice, ice ice

2. poor heat conductivity limits size of good freeze area

To minimize ice damage, use croprotectants
reduce the amount of water present

increase the number of ice nucleating sites (==>.more, smaller rather than 1 bigger ice crystal)

There are two types of cryoprotectants
penetrating

10-20% glycerol

DMSO

ethylene glycol

non-penetrating==>pulls water out of tissue and may reduce the extracellular ice, which may be nucleating intracellular ice sucrosedextran
Freezing solvents
In the freezing process, want to vitrify ice==>conversion of water to ice
want to get amorphous ice==> very small crystals

to accomplish this, need a cooling rate of 10 4-105 C/sec cooling rate

Several different solutions have been employed to this end:

Freezing material	cryogen temp	freezing rate	boiling point
propane	-190 C	98K C/sec	-42 C
	high thermal conductivity and heat capacity
isopentane	-160 C	45K C/sec
nitrogen slush	-207 C	21K C/sec
liquid nitrogen	-196 C	16K C/sec	-184 C

Freezing devices 1. plunge freezing superficial layer, 10 um
use cryoprotectants, which can cause artifacts2. spray freezing individual cells can generate shear forces that disrupts cells
3. slam freeze against mirror==> 10-15 um can flatten tissue4. propane jet ==> 40 um (20 from each side) 5. high pressure freezing ==> 1.6 x 10 6 Torr
Subsequent Steps
The cryofix lasts only so long as the sample remains frozen. With a freezing stage in the microscope, it is possible to look at a frozen sample. With a cryostat, it is possible to cut slices of frozen tissue for the light microscope. With the proper machine, it is possible to slice a frozen sample thin enough to view in the transmission electron microscope. A more convenient way to work with the sample is to preserve the structure while it is frozen, then bring the sample up to room temperature in the fixative. This is called freeze substitution ==> this process requires small samples and several days. The frozen sample is immersed in ÷80 C solvent (alcohol or acetone+/- glutaraldehyde and/or osmium)) for several days. As the sample warms at ÷80, the ice in the sample is replaced by the fixative. There is little opportunity for the sample to rearrange, since everything remains frozen until fixed
Advantages:

1) less shrinkage

2) waxy coating better preserved

3.) less movement of soluble elements

Disadvantages

1) takes days->weeks

2) no 'significant' improvement in preservation

alternatively, the frozen sample can be put under vacuum and freeze dried. The water will sublime from solid to gas, leaving behind a dry sample. The dry sample can be viewed in the SEM, or embedded in plastic and sliced for TEM. This process has many the same advantages and disadvantages as freeze substitution.