Cryo-EM University: Sample Preparation

Sample preparation

4:27:41

Sample Preparation

Detailed overview of the process to prepare samples for cryo electron microscopy analysis and the conditions that are needed to produce quality vitrified samples.

Preparing hydrophilic grids

4:20

Manufacturer's grid boxes

Review of different manufacturer grid boxes, the orientation of the grids, and how to remove the grids.
1:26

Recognizing the carbon and copper sides of a grid

Demonstration of how to identify the different sides of grids- the copper and carbon sides of the grid.
1:02

How to grab grids with tweezers

Demonstration of how to properly hold grids with tweezers.
5:41

Glow discharging_Plasma cleaning

Demonstration and step by step instruction of how to glow discharge and plasma clean grids using a Pelco easiGlow.
3:28

Hydrophobic and hydrophilic grids

Demonstration of the difference between a glow discharged and nonglow discharged grid.

Plunge-freezing with a Vitrobot Mark IV System

7:30

Plunge-freezing tools

Review of the tools needed for plunge-freezing samples with the Vitrobot System.
1:30

Vitrobot System connections

Walk through of the Vitrobot System connections and cables.
1:18

Mounting the Vitrobot humidifier

Demonstration of how to properly mount the Vitrobot System's humidifier.
1:34

Filling the Vitrobot humidifier

Step by step demonstration of how to properly fill the Vitrobot System humidifier.
3:29

Tour of the Vitrobot humidifying chamber

Walk through of the Vitrobot System humidifying chamber, including the different components and their purpose.
7:31

The Vitrobot user interface and options

Demonstration of the operating software of the Vitrobot System.
1:29

Replacing Vitrobot blot pads

Step by step instructions for replacing the Vitrobot System blot pads.
3:01

Mounting and unmounting Vitrobot tweezers

Demonstration on how to properly mount and unmount tweezers from the Vitrobot System.
3:38

Aligning the Vitrobot tweezers between the blot pads

Step by step demonstration of how to align the Vitrobot System tweezers between the blot pads before sample application.
3:29

Preparing the ethane gas source

Preparing the ethane gas source and safety considerations.
15:05

Filling the ethane cup

Demonstration of how to carefully fill the ethane cup with liquid ethane for plunge freezing with the Vitrobot System.
13:25

Plunge-freezing with the Vitrobot

Step by step walk through of plunge freezing a sample with the Vitrobot System.
4:47

Reviving the ethane

Demonstration of how to revive solid ethane to use it again for plunge freezing samples.
6:17

Sources of contamination

Walk through of the different sources of contamination in liquid nitrogen and recommended ethane quality.

Assembling autogrids

7:28

Autogrid assembly tools

Run through of the tools required to assemble standard plunge frozen EM grids into an autogrid cartridge.
1:32

Loading C-clips into insertion tools

Step by step instructions on how to properly insert C-clips into the insertion tool.
7:03

Assembling autogrids - room temp demo

Run through of how to assemble autogrids step by step and the tools required without liquid nitrogen.
13:54

Assembling autogrids - cryo conditions

Step by step instructions on how to assemble autogrids with liquid nitrogen.

Loading autogrids into cassettes

6:50

Cassette loading tools

Walk through of the components needed to put the cartridges in the cassette.
3:37

Assembling the cassette-loading station

Demonstration on how to properly load and mount a capsule onto the loading station and assembly of the cassette-loading station.
2:29

Structure of the cassette

Walk through of the autoloader cassette and the different features of the cassette.
4:54

Inserting autogrids into the cassette

Demonstration on how to insert the autogrids into the cassette and optimal conditions for the cassette.
3:15

Loading the cassette - room temp demo

Run through of loading the autoloader cassette with autogrids without liquid nitrogen using the loading station.
5:50

Loading the cassette - cryo conditions

Demonstration of loading autogrids into the autoloader cassette in the loading station with liquid nitrogen.

Docking and undocking capsules

6:29

Talos Arctica Cryo-TEM on-system display

Walk through of the on-system display of the Talos Arctica Cryo-TEM for docking and undocking cassettes.
3:46

The capsule pin and its purpose

Demonstration of the capsule pin and its purpose for proper function of the autoloader.
1:30

Docking a cassette

Demonstration of docking a cassette into the autoloader on a Talos Arctica Cryo-TEM.
1:29

Undocking a cassette

Demonstration of undocking a cassette out of the autoloader on a Talos Arctica Cryo-TEM.

Safety

8:53

Biological hazards

Discussion of the biological hazards and precautions that can be used when working with hazardous agents.
2:09

Compressed gas hazards

Review of the hazards from compressed gases used in cryo electron microscopy and mitigation.
8:49

Explosive gas hazards

Discussion of the combustion hazards of ethane.
3:07

Can the liquid ethane in the cup catch fire

Demonstration of how flammable liquid ethane is.
9:10

Large liquid nitrogen tank hazards

Discussion and walk through of the hazards around large liquid nitrogen tanks.
2:08

Small liquid nitrogen dewars

Walk through of the hazards of different small liquid nitrogen Dewars.
3:50

Liquid nitrogen on the skin hazards

Safety considerations when handling and working with liquid nitrogen.
11:51

Ethane splash and cold cup hazards

Review of the safety hazards of handling ethane and the cold cup.

For Research Use Only. Not for use in diagnostic procedures.

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