Transcript ISvitrification2003.ppt

Electron Cryomicroscopy of Ice-Embedded Specimens
Only method that allows direct observation of frozen fully hydrated macromolecules
Other methods: Freeze-fracture
Freeze-drying
Freeze-substitution
A dry specimen is observed in EM
Problem: water evaporates in the electron microscope
How to preserve the hydrated state in the electron microscope?
- Hydrated chambers:
Parsons D. F. (1974). Structure of wet specimens in electron
microscopy. Science, 186, 407-414
- Glucose embedding:
Unwin P. N. T. & Henderson R. (1975). Molecular structure
determination by electron microscopy of unstained crytalline
specimens. J. Mol. Biol., 94, 425-440
- Vitrification:
Taylor K. A. & Glaeser R. M. (1974). Electron diffraction of frozen,
hydrated protein crystals. Science, 106, 1036-1037
Vitrification
Molecules of liquid become immobilized before they have time to crystallize
Luyet B.J. & Gehenio P. M. (1940)
I step
II step
obtaining a thin film of water on the grid prior to its freezing
freezing of the thin water film
- choice of grids
- choice of supporting film
- hydrophobisity
- blotting
- evaporation
- condensation
- choice of cryogen
Sample
- water
- salt, detergent, glycerol (or sucrose)……
- biological molecule
WATER
~ 70% of our body is water
covalent bond
0.0965 nm
400 kJ/mol
104.5
0.177 nm
10-20 kJ/mol
Structure of H2O
Properties
- V-shaped structure
- dipole
- tetrahedral structure
- negative volume of melting
- highest density (under normal pressure) at 4C
in the liquid range
- high surface tension
- high freezing, melting and critical temperature
- viscosity increases with pressure
- forms at least 10 solid polymorphs
‘flickering’ cluster
Crystal lattice of Ice
Temperature (C)
Vapour
Liquid
Phase diagram of water
Temperature (C)
Phase diagram around the triple point
Solid
Pressure (Pa)
Pressure (108 Pa)
Ice : - hexagonal ice (common ice)
- cubic ice (stable below -70C at normal pressure)
- vitreous (amorphous ice), -120 C)
- >10 solid polymorphs (at high pressure)
Liquid : - great mobility (exchange time ~10-12 s)
- long list of models
Water in the electron cryomicroscope
Time (t) required at T (C) for phase
(1) Hexagonal
- freezing of liquid H2O (~m)
- condensation of atmospheric H2O (20-30 nm)
- warming of cubic or vitreous ice
- density 933 kg/m3
vitreous -to- cubic
t = 1.58 x
1014
transition
cubic -to- hexagonal
e-0.126T
t = 1.22 x 1030 e-0.465T
(2) Cubic
Vitreous
Log time (s)
Cubic
Log time (s)
- warming of vitreous ice
- slow deposition of water vapour in vacuum
at -135 C and -100C
- sub-optimal conditions of freezing (not cold
enough cryogen; less efficient cryogen)
-once formed it is stable at T<Tv
- the same density as hexagonal ice
- appears as mosaic of small crystals (~0.1 m)
Hexagonal
Cubic
(3) Vitreous
- TV (vitrification) = -120 C
- rapid cooling
- slow deposition of water vapour at T< TV
- the same density as cubic ice or hexagonal ice
- smooth layer without internal structure
- polymorphous
Temperature (C)
Freezing
 Gs ~ r2
r – radius of ice crystal
 G =  Gv +  Gs
 Gv - expresses that water is more stable in
the crystal form than in the liquid
 Gs - expresses instability of liquid-solid interface
 Gv ~ r3
Temperature (C)
slow cooling
TO – homogeneous nucleation temperature, -35C
(nucleation,crystallization becomes unavoidable)
TE – heterogeneous nucleation temperature, TO < TE < TM
(nucleation by a seed contamination)
vitrification
more rapid
cooling
TG – glass temperature, TG = TV (TV - vitrification temp.),
-120C (no nucleation, high viscosity)
- Heat production & dissipation at the crystal surface
concentration solute
Freezing
Crystal size
Vitrification –
immobilization of H2O is achieved before its crystallization
- rapid cooling rate
- small mass of specimen
cooling speed
cooling speed = 106 C/sec (for 1 m thick layer)
tV = 10-4 sec
Growth of a hexagonal ice crystal in an aqueous solution
Ramified ice crystals
Choice of cryogen
Name
bp C
(1 atm)
mp C
cooling
efficiency
sublimation
rate
Ethane
-88.6
-172
good
rapid
Propane
-42.1
-188
good
slower
Nitrogen
-196
-210
poor
rapid
3-5 l
Liquid
ethane
-172°C
Liquid N2
-196°C
Evaporation
Log P (Pa)
- decreasing t of the sample
- changing in solute concentration
- changing pH
Mass flux (mg/m2 x s)
Temperature (C)
F = 18.5 P/ T
To prevent :
- humid chamber
- blotting paper from the both sides
- cooling the specimen to low t
Temperature (C)
Sublimation of vitreous water is very small at < Tv
Surface tension
L - surface tension of liquid
liquid tends to minimize its surface-to-volume ratio
(minimum surface tension)
s - surface tension of solid
SL – interfacial tension between liquid and solid
 - wetting angle
At equilibrium (Young condition):
s = Lcos  + SL
Perfect wetting conditions
=0
Changes in wetting angle
- Agents reducing interfacial tension
- Viscosity and velocity of solution
- Hydrophilic supporting film
1.2 m
Vitreous ice
Hexagonal ice
Cubic & hexagonal ice
hexagonal