A large number of variables influence blotting-based Cryo-EM sample preparation, which can lead to poor repeatability in sample quality. The manual transfer of vitrified samples to the grid box is a critical time during which the sample is influenced by the ambient environment and by the expertise of the user. The grid is exposed to air during the transfer - users report that they observe variations in sample quality based on time of year, weather, location, etc., which suggests ambient properties are influencing the sample. Manually transferring the grid requires dexterity while simultaneously tending to multiple things (liquid nitrogen levels, ethane quality, etc.) – it’s stressful for experienced users, and for beginners trashed grids are the norm.
NanoSoft's Igloo aims to better control the manual transfer when preparing samples with the Thermo Fisher Vitrobot. A more enclosed environment provides protection from contaminating ambient humidity, while various system features transform the manual transfer process into a user friendly experience. The Igloo is beneficial for all users, but less experienced users in particular will be able to prepare viable grids sooner.
Igloo Cover: The styrofoam ring from the Vitrobot dewar aims to maintain an environment containing cold, dry nitrogen gas above the liquid nitrogen to protect vitrified grids during their transfer. However, the ring is open and susceptible to humidity in the external environment. Furthermore, the ring is cold and collects ice. As it is submerged into the liquid nitrogen with every plunge of the Vitrobot, ice particulate is deposited into the liquid nitrogen, which can contaminate grids already placed in the grid box.
The Igloo is a nearly enclosed cover that sits atop the Vitrobot dewar, with an opening large enough for comfortable grid button access and grid transfer. The volume enclosed by the Igloo fills with cold gaseous nitrogen, creating a dry space with improved protection versus the ambient environment. The Igloo is designed to fit comfortably over the floating styrofoam ring, minimizing the time that the styrofoam ring is exposed to ambient humidity and significantly reducing the potential for ice particulate contaminating the liquid nitrogen. The Igloo material is compatible with liquid nitrogen temperatures, allowing for easy refill with the Igloo on the dewar. Furthermore, the top surface of the Igloo remains comfortable to the touch, providing a convenient surface upon which the user can rest their hands for steady transfer.
The Cobra: Upon removing the tweezers from the Vitrobot dovetail, users must carefully hold the tweezers closed (to hold grid), keep the tips of the tweezers submerged in liquid ethane and simultaneously lift the dewar to transfer it away from the Vitrobot to transfer the sample to the grid box.
The Cobra is integrated into the top surface of the Igloo and flips up to provide a slot into which tweezers can be placed. When resting in the slot, the tips of the tweezers remain submerged in liquid ethane. The Cobra slot is compatible with both NanoSoft's Cryo-Tweezers and other Vitrobot tweezers. The Cobra engages with magnets in the Plunge-Clamp of NanoSoft's Cryo-Tweezers, providing a more secure attachment. The Cobra provides freedom to the user to focus both hands on holding the dewar when moving it from the Vitrobot, the ability to fill the dewar with liquid nitrogen, etc.