Part 1: Sample Chamber - G. Jensen

Summary

This video explains the intricate process of sample insertion and manipulation within an electron microscope (EM) column, specifically focusing on cryo-electron microscopy (cryoEM). It covers how samples are inserted, sealed in a vacuum, and positioned precisely using mechanical adjustments to ensure optimal imaging. The role of the pole pieces in the objective lens, the importance of the pole piece gap for resolution, and the impact of water contamination in cryoEM are explored. The video also discusses the concept of eucentric height, ensuring the object remains stable during rotations for accurate imaging.

Takeaways

• 😀 The electron microscope column operates under high vacuum to enable precise imaging.
• 😀 Sample insertion is typically done via side entry, although top entry is another possibility.
• 😀 Some electron microscopes use automatic sample loaders, while older models may require manual insertion.
• 😀 The sample holder has a fine tip and an EM grid, with a pin and O-ring to ensure a vacuum seal.
• 😀 A vacuum is established in the anti-chamber before connecting the sample to the microscope column.
• 😀 The ball valve rotates to connect the sample holder with the microscope column once the vacuum is stable.
• 😀 Objective lens coils are carefully wrapped and enclosed with metals to focus the electron beam accurately.
• 😀 The pole piece gap affects resolution; a smaller gap offers higher resolution, but cryo-EM microscopes typically have a larger gap for better sample handling.
• 😀 In cryo-EM, water molecules in the vacuum can freeze on the sample, so the microscope is equipped with cryo boxes to reduce contamination.
• 😀 The eucentric height is a key concept: the sample must be positioned along the optical axis to prevent vertical movement during rotation.
• 😀 The sample holder is designed to move the grid in multiple directions, including left/right and up/down, to achieve optimal positioning for imaging.

Q & A

What is the significance of the high vacuum inside the microscope column?

The high vacuum inside the microscope column is crucial because it minimizes the interference from air molecules, which would scatter the electron beam and degrade the image quality. A vacuum allows electrons to travel freely from the source to the sample without significant deflection.

What are the differences between side-entry and top-entry electron microscopes?

In side-entry electron microscopes, the sample is inserted from the side of the column, while in top-entry microscopes, the sample is inserted from the top. Side-entry microscopes can either have manual or automatic sample loaders, with the manual ones typically being more common in cryoEM.

What role do the O-rings play during sample insertion?

The O-rings are critical for creating a vacuum seal between the sample holder and the insertion tube. Once a vacuum is established, the O-rings ensure that no air leaks into the microscope column, maintaining the integrity of the high vacuum environment needed for proper imaging.

How does the ball valve work in the sample insertion process?

The ball valve is a mechanical component that seals the microscope column from the outside environment. After the vacuum is established in the anti-chamber, the ball valve rotates, allowing the sample holder to pass through it and enter the microscope column.

Why is the pole piece gap important in electron microscopes?

The pole piece gap determines the precision of the magnetic field lines used to focus the electron beam. A smaller gap allows for better control of the magnetic field and higher resolution, but it also limits the space available to rotate the sample. This is why cryoEM microscopes typically have a larger pole piece gap.

How does water contamination occur in cryoEM, and how is it mitigated?

In cryoEM, water molecules in the microscope column can freeze onto the cold sample (held at around 80 K). To mitigate this, cryoEM systems typically use cryo boxes above and below the sample, cooled by liquid nitrogen, which trap most of the water molecules before they can reach the sample.

What is the function of the cryo box in electron microscopy?

The cryo box serves to protect the sample from water contamination by trapping water molecules before they can freeze onto the sample. It contains apertures that allow some molecules to pass through, but the cold surfaces help reduce the likelihood of contamination.

Why is it important to position the sample at the eucentric height in electron microscopy?

Placing the sample at the eucentric height ensures that when the sample is tilted, the object of interest remains in the same position within the microscope column. This prevents the sample from moving up or down during tilting, which can distort the image.

What is the relationship between the specimen holder and the objective lens in terms of alignment?

The specimen holder is designed to align the sample's object of interest along the optical axis of the objective lens. This ensures that the electron beam remains as focused and centered as possible, optimizing imaging conditions.

How do electron microscopes achieve rotation and movement of the sample holder?

Electron microscopes allow the sample holder to rotate and move by using mechanical elements within the holder that adjust the position of the sample in various directions—left, right, up, and down—allowing precise alignment and rotation within the column.