Oils are widely used in crystallography – as cryoprotectants when samples are flash cooled, and as barriers to the dehydration, solvent evaporation, and oxidation that may occur in air near and above room temperature.

The ice rings seen in the diffraction patterns of flash cooled protein crystals arise primarily from crystallization of the aqueous solution surrounding the crystal, not of the internal solvent. Oils can displace and replace this surrounding solution with little risk of damage due to osmotic shock.
Advances in X-ray sources, optics, detectors and sample mounting technology have allowed protein crystals as small as 5 microns to be used in structure determinations. Crystals smaller than roughly 50 microns dehydrate rapidly in air, with sub-10 micron crystals dehydrating in seconds.

Substantial dehydration-induced damage can easily occur between removal from a drop and flash cooling. Dehydration can be minimized by first transferring to an oil drop (using the MicroSieves™ in Microtools™ Kit 1 and Microtools™ Kit 2 ) before mounting the oil-coated crystal on a MicroMount or MicroMesh.

However, surrounding oil scatters X-rays, reducing diffraction signal-to-noise, and contributes thermal mass that reduces crystal cooling rates and increases the need for penetrating cryoprotectants. These problems are especially acute for the smallest crystals. When mineral oil, Paratone oil and other high viscosity oils are used, the volume of surrounding oil can exceed the crystal volume by a factor of 10 or more, and can contribute excess drag that increases sample motion in a cryostream. For larger rod or plate shaped crystals, the surrounding oil exerts stresses when it freezes that can cause crystal cracking and mosaic broadening.

1. Mitegen’s LV CryoOil™ has the lowest viscosity of any available perfluoropolyether oil (1/10 that of Paratone oil and comparable to that of vegetable oil), and a very low surface tension (less than 1/3 that of water.) Consequently, a dip in this oil followed by gentle tapping to shake off excess can yield protective oil films on your crystal of as little as 10 microns thick.
2. The image at right, of an LV CryoOil™ coated crystal on a 75 micron MicroMount™, shows how cleanly crystals can be mounted using this oil.
3. Originally developed for use in vacuum diffusion pumps, LV CryoOil™ has extremely low vapor pressure, excellent chemical inertness and excellent thermal stability.
4. A note on using oils: After crystals have been transfered to oil they may seem to disappear, suggesting that they have dissolved. But don’t worry: your crystal is still intact. The refractive index of oil (n=1.5) is very close to that of protein crystals, and much closer than that of water (n=1.3). Once the oil has displaced all the water from the crystal’s surface, the interface between the oil and crystal surface will reflect very little light, making the crystal hard to see. Just adjust your illumination and look carefully.