An Automated System For Maintaining Liquid Nitrogen Levels In The Gatan Cryostage

For several years we have been developing a system for the automated control and acquisition of images from a transmission electron microscope ^1,2,3. The system has been developed around a Philips CM200 equipped with a Gatan cryostage and a Gatan MSC CCD camera. One of the primary motivations in developing this software is to provide for a system that can acquire many hundreds of images over several days of completely unattended operation.

The current implementation of our automated system allows data to be automatically collected from the TEM for more than 36 hours. There is no intervention required from the operator except for one critical exception, refilling the Gatan cryostage. The cryostage, which maintains the temperature of the vitreous ice specimens, must be refilled with liquid nitrogen (LN2) approximately every two hours.

We had previously extended the life of the LN2 dewar which cools the anti-contamination device on the TEM. This was achieved very simply by changing the size of the dewar and designing a new attachment to the microscope to support the larger container. The new container lasts up to 12 hours before it needs to be refilled. Extending the life of the cryostage poses far greater difficulties.

The cryoholder holds less than 0.25 liter of LN2, and the volume of liquid in the holder is critical to acquiring high resolution images. Ideally the level of the LN2 is set so that the rod that maintains thermal contact between the specimen and the cryogen is not fully immersed in the LN2. These appear to be ideal conditions to ensure that thermal drift is minimized. During replenishment of the cryogen it is also quite common that small particles of ice get introduced into the dewar. These will cause the LN2 to boil and result in unacceptable vibrations in the specimen unless they are removed. This is achieved using a simple device consisting of a glass tube passing through a rubber stopper. The glass tube is inserted into the LN2 while covering the aperture of the dewar with the stopper. This causes a sudden spurt of LN2 to be released and usually removes all vibrations from the system.

To our knowledge, there is currently no method to automatically replenish the cryogens in the Gatan cryostage. We must achieve the conditions described above while using a system that does not transfer vibration to the system or add excessive weight to the cryostage. Our initial approach has been to develop a simple refilling system that will allow the specimen to be kept cold but not require that good imaging conditions be maintained. This allows the operator to pause the experiment overnight and then resume again in the morning using the same grid.

The system we have developed is illustrated in figure 1. It is based on a combination of commercially available and fabricated components. A large (10-liter) double-walled and insulated plastic LN2 dewar (a) was modified for gravity-feed delivery by drilling through the base and inserting a fabricated fitting to which a vacuum-insulated braided stainless steel LN2 transfer hose (b) is attached. After the hose was connected, the joint between it and the dewar was covered with expanding insulating foam. A solenoid-operated valve (c) was then fitted to the free end of the hose, and a curved copper tube with a lesser internal diameter (d) was connected to the open end of the valve. The entire apparatus (ca. 12 kg), filled with LN2, is suspended from the ceiling adjacent to the electron microscope so that the copper tube is poised directly above the cryoholder (e). A single-sensor Cryo-Miser unit (f) (model T-11-A; Torr Vacuum Products, Inc., Van Nuys, CA) is used to monitor the level of LN2 in the cryoholder. The Cryo-Miser utilizes a LN2-immersible temperature sensor. When the level of LN2 in a container falls below that of the sensor, it opens the valve to the large dewar for a period of time which can be determined by the operator. The valve may be shut off as soon as the sensor is re-immersed, or it may remain on for as long as several minutes.

The current system allows the operator to leave the specimen unattended overnight and resume the automated data acquisition in the morning. We are now exploring whether a system can be designed that will allow the LN2 levels to be accurately set and the ice in the cryogen removed without adding excessive weight, vibration or expense to the system.

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