Cryo-electron Microscopy Requires a Special Environment for the Microscope
Working at cryo-temperatures is more demanding than working at room temperature so special consideration must be taken with regard to designing the space that will house the electron microscope. In a transmission electron microscope, the tiny specimen grid is suspended between the objective lens pole pieces of the TEM by means of a specialized low temperature specimen holder. Although one might think that the specimen is well protected from the environment outside of the robust electron optical column, it is actually quite susceptible to many factors that can degrade the overall resolution of the final projected image. It is important to minimize or totally eliminate any environmental issues that would adversely affect the operation of the electron microscope and ancillary instrumentation used in conjunction with the microscope. Three key factors are vibration, climate control and stray electromagnetic fields.
Vibration can be transmitted to the cryo-specimen holder either mechanically through the microscope or acoustically through the air. Mechanical vibrations can occur at low and high frequency. Specimen holders for cryo-TEM come in several forms: side entry, fixed top entry and holders that combine attributes of both. Although fixed top entry cryo-holders are known to be extremely stable due to their design characteristics; all cryo-holders can suffer the ill effects of vibration resulting in degradation of the image. Some side entry cryo-holders that incorporate an external liquid nitrogen dewar can be susceptible to high frequency vibration if the level of liquid nitrogen within the dewar is not maintained a constant level. In addition, if the cooling water that flows through the objective lens is not properly dampened, it will also cause vibration to be transmitted to the specimen. Low frequency vibrations can originate from traffic on a near-by streets, geoseismic activity, activity at a near-by construction site, or even foot traffic near the microscope. Unfortunately, vibrations in the low frequency range are the most difficult to eliminate. Vibration that is transmitted through the air is generally caused by acoustic noise that comes from, for example, noisy air handling equipment that services the microscope room. These services should be supplied to the microscope room in a very quiet and controlled manner. Even small air currents in the vicinity of the specimen holder can degrade resolution. No matter what the source, vibrations can limit resolution by causing blurring of the image in the horizontal plane, especially in the direction perpendicular to x axis for specimen translation (e.g., perpendicular to the long axis of the specimen rod of a side entry cryo-holder), or blurring of the image along the vertical axis. Many high resolution electron microscopy laboratories go to great lengths to eliminate all sources of vibrations that might affect the performance of the microscope.
Ideally, the electron microscope should be housed in a room that has stringent humidity control (preferably 15% R.H. or lower) and temperature control. This is especially true if one is going to use side entry cryo-holders. Depending upon the design of the holder, the tip where the specimen is located is very cold when in use (approximately -180 °C) and it acts as a cryo-sorption pump. If the humidity in the room is very high, a significant amount of water vapor in the air will condense (as frost) onto the surface of the shields that protect the cryo-grid during transfer to the microscope airlock. This condensed water vapor is then carried into the airlock mechanism, and subsequently into the optical column, eventually degrading the vacuum as well as acting as a source of contamination to the cryo-specimen. If the humidity in the microscope room is maintained to a very low level, this effect is minimized. Once the cryo-specimen is loaded into the microscope, it is necessary to use devices to prevent the buildup of contamination onto the surface of the specimen. Contamination can be minimized by using anticontamination devices provided by the microscope manufacturer or by third-party instrument designers who specialize in instrumentation for cryo-EM. such as Gatan. In addition, cryo-specimens are particularly susceptible to drift from temperature gradients, and variations in these gradients, that can originate from both inside and outside of the microscope. Drift occurs in a direction parallel to the long axis of the mechanism that holds the specimen holder. Room air temperature variation caused by the cycling of heating or cooling air entering the microscope room, or the variations in the temperature of the cooling water used to cool the lenses of the microscope, can all result in specimen drift which will limit the final resolution of the image.
External AC and changing DC fields can affect the high resolution performance of the microscope. The electron optical column is composed of a series of electromagnetic lenses that are used to focus and to control the position of the electron beam that is emitted from the electron gun. Although the microscope column is shielded, external fields can still penetrate the structure of the column. This results in movement and subsequent blurring of the projected image. Although AC and changing DC fields degrade images, changing DC fields are particularly troublesome and have a greater effect on the ultimate performance of the microscope than AC fields. This field can originate from the movement of a nearby elevator, commuter rail trains and even movement of metal chairs in the vicinity of the microscope. In addition, even a large constant DC field, such as from the superconducting magnets found in NMR instruments, can affect performance.
Microscope manufacturers have stringent guidelines regarding acceptable limits for the microscope site, especially when the microscope will be used for collecting high resolution cryo-data. Many high resolution cryo-TEM laboratories may also employ outside testing firms to ensure that their facility fully meets the required specifications. Meeting or exceeding the microscope installation requirements when designing a cryo-electron microscopy laboratory will help to insure success in implementing this exciting technique.