Troubleshooting; Guidelines For Optimal Specimen Preparation - Thermo Scientific Vitrobot Mk IV User Manual

Chapter | Troubleshooting
4

Troubleshooting

4.1 Guidelines for Optimal Specimen Preparation

The instrument performance has been tested with phospholipid vesicles
(dipalmitoyl-phosphatidylcholine; DPPC 10mg/ml) prepared by extrusion through a 100 nm
filter.
The shape of these vesicles varies with temperature; spherical above 41 °C (lipid in the L
phase), facetted and rippled between 37 and 41 °C (lipid in the P ' phase), and facetted
below 37 °C (lipid in the L phase). To obtain a large area for cryo-electron microscopy
(thickness below 100 nm, good vitrification) two short blotting actions were employed. With
low viscosity samples, such as a DPPC suspension, we recommend to increase the number
of blotting actions (two blotting actions instead of one) when vitrified films are too thick. With
high viscosity samples we recommend to increase the blotting time (e.g. 2-3 sec) before
deciding for an increase in the number of blotting actions.
Also note the critical effects of the environmental humidity prior to a vitrification action. With
an unsaturated environment the actual temperature of the sample will be lower than the set
temperature of the chamber (dew-point effect, demonstrated with the shape of DPPC
vesicles!). Evaporation will also accelerate the thinning of aqueous thin films. Thinning may
result in concentration of particulate material by a sweeping action of two concave air -water
interfaces approaching each other and eventually flattening out in an equilibrium
configuration. The effects of thinning are recognized in the image after some experienc e with
cryo-observation. Temperature effects and osmotic effects are often not readily observed in
vitrified specimens. An evaporation of 50 % of the water volume during preparation of a thin
film (10 sec, 40 % rH) was experimentally determined from volume changes in liposomes
(pictured the collapse of spherical liposomes into two interconnected concentric spheres).
Confidential, limited rights
PN 103261 | Revision D | 23-Oct-2019
User Manual
Page 32