removal of free unbound Ab is not very effective and the
presence of extra unbound primary Ab in buffer may lead to
binding of secondary Au-IgG Ab and thus limit the amount of
secondary Ab available to bind to antigen entrapped in
archaeosomes.
5. Au-IgG Ab easily form aggregates at 37
C, so the incubation
should be carried out at RT or at 4
C. To avoid the collapse of
archaeosomes, the aqueous sample on TEM grids should be
kept in a humidity chamber during incubation.
6. In recent years, the design of plunge freezers has become more
sophisticated and considerably improved to make the freezing
process more efficient and reproducible. Nevertheless, whether
using an automated or manual control, the basic design of the
instrument is still the same and the production of high quality
cryo-EM specimens still depends on the skill of the individual
since the factors affecting cryo-EM, such as grid selection,
temperature, humidity, blotting pressure, and blotting duration, need to be optimized for each specimen.
7. The atmosphere moisture can condense on the surface of cryoTEM specimens as crystalline ice when transferring the frozen
samples from storage Dewar to workstation and loading them
to TEM holder or to the microscope. During transferring and
loading, the temperature of the frozen specimen can increase
and the ice phase can be changed from vitreous to cubic crystalline phase if the temperature is higher than À135
C
[1]. Shortening the exposure time of the frozen sample at
atmosphere, immersing the frozen sample in liquid nitrogen
and applying antifrosting shield on the holder can mitigate the
ice contamination and recrystallization.
8. The three-specimen cartridge on Gatan cryo-transfer holder
910 is removable. So the cryo-EM specimen can be loaded
onto the cartridge first before transferring the 910 holder
from TEM to cryo-workstation and thus the time exposed to
air can be shortened. This holder allows us to screen specimens
prepared at different freezing conditions to obtain optimal
conditions. Gatan cryo-transfer holder 914, with a narrow
holder tip allowing for high angle tilting up to Æ90
, is used
for tomography experiments. The cryo-EM specimen loading
direction is side entry so more caution should be paid to
specimen damage or crystalline ice contamination from cryoworkstation.
9. To avoid the spill of liquid N 2 from the Dewar while inserting
the holder to TEM column, the stage can be tilted to plus X
direction at about 25
. Thus, it can prevent the liquid N 2 from
splashing around and also the risk of the temperature rising.
However, the proper procedures and precautions should be
Cryo-TEM for Vaccine Delivery Carrier Evaluation
509
presence of extra unbound primary Ab in buffer may lead to
binding of secondary Au-IgG Ab and thus limit the amount of
secondary Ab available to bind to antigen entrapped in
archaeosomes.
5. Au-IgG Ab easily form aggregates at 37
C, so the incubation
should be carried out at RT or at 4
C. To avoid the collapse of
archaeosomes, the aqueous sample on TEM grids should be
kept in a humidity chamber during incubation.
6. In recent years, the design of plunge freezers has become more
sophisticated and considerably improved to make the freezing
process more efficient and reproducible. Nevertheless, whether
using an automated or manual control, the basic design of the
instrument is still the same and the production of high quality
cryo-EM specimens still depends on the skill of the individual
since the factors affecting cryo-EM, such as grid selection,
temperature, humidity, blotting pressure, and blotting duration, need to be optimized for each specimen.
7. The atmosphere moisture can condense on the surface of cryoTEM specimens as crystalline ice when transferring the frozen
samples from storage Dewar to workstation and loading them
to TEM holder or to the microscope. During transferring and
loading, the temperature of the frozen specimen can increase
and the ice phase can be changed from vitreous to cubic crystalline phase if the temperature is higher than À135
C
[1]. Shortening the exposure time of the frozen sample at
atmosphere, immersing the frozen sample in liquid nitrogen
and applying antifrosting shield on the holder can mitigate the
ice contamination and recrystallization.
8. The three-specimen cartridge on Gatan cryo-transfer holder
910 is removable. So the cryo-EM specimen can be loaded
onto the cartridge first before transferring the 910 holder
from TEM to cryo-workstation and thus the time exposed to
air can be shortened. This holder allows us to screen specimens
prepared at different freezing conditions to obtain optimal
conditions. Gatan cryo-transfer holder 914, with a narrow
holder tip allowing for high angle tilting up to Æ90
, is used
for tomography experiments. The cryo-EM specimen loading
direction is side entry so more caution should be paid to
specimen damage or crystalline ice contamination from cryoworkstation.
9. To avoid the spill of liquid N 2 from the Dewar while inserting
the holder to TEM column, the stage can be tilted to plus X
direction at about 25
. Thus, it can prevent the liquid N 2 from
splashing around and also the risk of the temperature rising.
However, the proper procedures and precautions should be
Cryo-TEM for Vaccine Delivery Carrier Evaluation
509