(d) Apply 5 μl droplet of archaeosomes solution on the carbon
side of the grid.
(e) Mount the tweezer securing the TEM grids loaded with
aqueous solutions or the grids with the immunolabeled
archaeosomes prepared in step 3 onto the releasing anvil.
(f) Blot the TEM grid with filter paper from the back side of
carbon film for about 4 s; press the anvil releasing button
and plunge the grids into liquid ethane rapidly.
(g) Transfer the frozen grid from liquid ethane cup to the
cryo-EM grid storage box immersed in liquid nitrogen.
(h) Transfer and store the TEM grid storage box in a large
nitrogen cryo-storage Dewar (50 L). It can be stored for
several days or weeks until transferred for imaging.
3.3 Cryo-TEM
Imaging
For imaging frozen-hydrated specimens in a TEM, additional precautions and proper procedures should be followed to obtain high
quality cryo-TEM micrographs. Ice contaminations or ice phase
change during specimen transferring/loading and electron beam
damage to frozen specimen are unavoidable but can be mitigated.
1. Loading cryo-EM specimen to TEM column (see Note 7).
(a) Transfer the storage box containing frozen TEM grids
from cryo-storage Dewar (50 L) to a 2 L liquid N 2
Dewar quickly.
(b) Precool down the cryo-transfer holder in TEM column to
À180
C.
(c) Cool down the cryo-workstation with liquid N 2 for about
30 min. Immerse tweezers and tools for fixing specimen in
liquid N 2 .
(d) Transfer the precooled holder into the cryo-workstation
and monitor the temperature below À160
C.
(e) Transfer storage box containing frozen grids from the
liquid N 2 Dewar to the cryo-workstation.
(f) Load the frozen specimen onto holder in liquid N 2 or just
above liquid N 2 (see Note 8).
(g) Close the anti-frosting cover on the cryo-holder and
transfer it to the TEM column. Top up liquid N 2 to the
Dewar on holder as needed (see Note 9).
(h) Wait for about 20–30 min to let the temperature and stage
stabilized before opening the beam.
2. Image acquisition using low dose imaging (see Note 10).
(a) At low magnification, evaluate the quality of the cryo-EM
specimen (see Note 11).
Cryo-TEM for Vaccine Delivery Carrier Evaluation
505
side of the grid.
(e) Mount the tweezer securing the TEM grids loaded with
aqueous solutions or the grids with the immunolabeled
archaeosomes prepared in step 3 onto the releasing anvil.
(f) Blot the TEM grid with filter paper from the back side of
carbon film for about 4 s; press the anvil releasing button
and plunge the grids into liquid ethane rapidly.
(g) Transfer the frozen grid from liquid ethane cup to the
cryo-EM grid storage box immersed in liquid nitrogen.
(h) Transfer and store the TEM grid storage box in a large
nitrogen cryo-storage Dewar (50 L). It can be stored for
several days or weeks until transferred for imaging.
3.3 Cryo-TEM
Imaging
For imaging frozen-hydrated specimens in a TEM, additional precautions and proper procedures should be followed to obtain high
quality cryo-TEM micrographs. Ice contaminations or ice phase
change during specimen transferring/loading and electron beam
damage to frozen specimen are unavoidable but can be mitigated.
1. Loading cryo-EM specimen to TEM column (see Note 7).
(a) Transfer the storage box containing frozen TEM grids
from cryo-storage Dewar (50 L) to a 2 L liquid N 2
Dewar quickly.
(b) Precool down the cryo-transfer holder in TEM column to
À180
C.
(c) Cool down the cryo-workstation with liquid N 2 for about
30 min. Immerse tweezers and tools for fixing specimen in
liquid N 2 .
(d) Transfer the precooled holder into the cryo-workstation
and monitor the temperature below À160
C.
(e) Transfer storage box containing frozen grids from the
liquid N 2 Dewar to the cryo-workstation.
(f) Load the frozen specimen onto holder in liquid N 2 or just
above liquid N 2 (see Note 8).
(g) Close the anti-frosting cover on the cryo-holder and
transfer it to the TEM column. Top up liquid N 2 to the
Dewar on holder as needed (see Note 9).
(h) Wait for about 20–30 min to let the temperature and stage
stabilized before opening the beam.
2. Image acquisition using low dose imaging (see Note 10).
(a) At low magnification, evaluate the quality of the cryo-EM
specimen (see Note 11).
Cryo-TEM for Vaccine Delivery Carrier Evaluation
505