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Liquid nitrogen cryospray for biological applications
Physica B 284}288 (2000) 2047}2048
Liquid nitrogen cryospray for biological applications Franck Felisaz , Ricardo Steinmann *, Andrew Thompson , Vivian Stojano! European Molecular Biology Laboratory, Av. des Martyrs, 38043 Grenoble, France European Synchrotron Radiation Facility, Av. des Martyrs, 38043 Grenoble, France
Abstract One of the methods to study three-dimensional protein structures is via X-ray di!raction techniques. Radiation damage produced by the interaction of X-rays with the sample can be reduced by soaking the protein crystals in cryoprotectants and freezing them at liquid nitrogen temperature. The perfection of the frozen crystals depends critically on the choice of cryoprotectant, and the rate of freezing. We have developed a device to produce a "ne spray of liquid-nitrogen droplets smaller than 10 lm in order to increase the rate of freezing. 2000 Elsevier Science B.V. All rights reserved. Keywords: Cryogenic instrumentation; Cryoprotection; Nitrogen cryo spray; Protein structure
The mapping of protein structures by X-ray crystallography is an area of science that has seen a huge expansion in recent years. However, protein crystals are generally extremely radiation sensitive due to their large solvent content. The amount of radiation damage can be reduced by soaking them in cryoprotectants and freezing the crystals down to temperatures between 80}100 K. Di!erent methods have been tried to freeze samples [1], like using a cold gas stream or liquid propane, but in all cases the freezing rate plays a critical role. In order to maximise the thermal exchange between the freezing agent and the protein crystal we have developed the cryospray, an instrument capable of producing "ne atomisation of liquid nitrogen. Protein crystals can contain up to 75% solvent (e!ectively water) and, for cryoprotection to be successful, freezing has to be rapid enough for the solvent to vitrify, hence limiting mechanical damage of the crystal to the expansion of ice on crystallisation. Cooling protocols to do this include treatment with cryoprotectants such as glycerol, methyl-pentadiol (MPD) or polyethylene glycol (PEG) of various molecular weights, and rapid cooling in liquid nitrogen, propane or nitrogen gas streams. For a complete
* Corresponding author. E-mail address: [email protected] (R. Steinmann)
review of cooling rates and cryoprotectants, see Ref. [1]. Classical methods to produce a spray are well known and reliable, but none of them is suitable to get a dropsize well adapted for our application (the average size of samples varies from 50 to 150 lm). The device uses the technology of a coaxial injector (the same as used in the ARIANE V rocket engine) to produce "ne droplets [2]. The cryospray is generated using liquid nitrogen and helium gas. Fig. 1 shows a scheme of the instrument. Helium coming from the bottle (He) goes into the vacuum vessel (V) where it is thermalised on the nitrogen container (N) and driven into the gas chamber of the injector at a pressure of 300 mbar. The liquid nitrogen, pressurized at 150 mbar, is propelled through the central nozzle (0.4 mm ID, 16 mm length). The di!erential speed between the gas and the liquid generate a surface instability leading to the atomisation process. The whole droplet detachment is about 1.5 cm from the nozzle and, in contact with the sample, the crystal freezes at rates higher than 100 K/s. The liquid-nitrogen container has a volume of 1 l allowing 2.5 h of continuous operation. Because of the low temperature produced by the spray, a water-free environment is necessary to prevent ice from growing and, indeed, crystal damage. Fig. 2 shows the freezing chamber (F) made from an acrylic cylinder, one side tightly joined to the cryospray and the other one to a dewar (D) "lled with liquid nitrogen where frozen
0921-4526/00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 5 2 6 ( 9 9 ) 0 2 8 5 0 - 1
2048
F. Felisaz et al. / Physica B 284}288 (2000) 2047}2048
Fig. 1. General scheme of the instrument. See text for details.
samples are stored. The piston (P) is used to drive the sample holder (Sh) inside the chamber to the freezing position. A vertical translation platform (T), actuated by the knob (K), moves the sample from the spray to the storage container (D). A vent valve (S) regulates the internal pressure preventing any over-pressure. A typical freezing process starts by `"shinga a crystal from the mother liquid and transferring it to its cryoprotectant solution. Mounted in the loop of its experimental support, the crystal is placed into the spray using the piston (P). This operation takes a few seconds and is fast enough to avoid any damage to the sample. As soon as the crystal moves into the chamber, it comes in contact with the spray freezing in fraction of a second. The sample remains under the cryogenic spray all the way until being deposited in the liquid nitrogen. Accurate measurements of cooling rates of protein crystals on freezing have been hard to perform, but cooling from room temperature to 140 K in 0.16 s has been recorded and it is adequate to ensure that in most cases the vitri"ed state is attained. The device can be operated to freeze up
Fig. 2. Detail of the freezing chamber.
to six samples sequentially and to store them for later use.
Acknowledgements The authors would like to thank Franck Lapeyre for his invaluable help on the construction of the freezing chamber.
References [1] E.F. Garman, T.R. Schneider, J. Appl. Crystallogr. 30 (1997) 211. [2] W.O.H. Mayer, Exp. Fluids 13 (1994) 401.
Liquid nitrogen cryospray for biological applications Franck Felisaz , Ricardo Steinmann *, Andrew Thompson , Vivian Stojano! European Molecular Biology Laboratory, Av. des Martyrs, 38043 Grenoble, France European Synchrotron Radiation Facility, Av. des Martyrs, 38043 Grenoble, France
Abstract One of the methods to study three-dimensional protein structures is via X-ray di!raction techniques. Radiation damage produced by the interaction of X-rays with the sample can be reduced by soaking the protein crystals in cryoprotectants and freezing them at liquid nitrogen temperature. The perfection of the frozen crystals depends critically on the choice of cryoprotectant, and the rate of freezing. We have developed a device to produce a "ne spray of liquid-nitrogen droplets smaller than 10 lm in order to increase the rate of freezing. 2000 Elsevier Science B.V. All rights reserved. Keywords: Cryogenic instrumentation; Cryoprotection; Nitrogen cryo spray; Protein structure
The mapping of protein structures by X-ray crystallography is an area of science that has seen a huge expansion in recent years. However, protein crystals are generally extremely radiation sensitive due to their large solvent content. The amount of radiation damage can be reduced by soaking them in cryoprotectants and freezing the crystals down to temperatures between 80}100 K. Di!erent methods have been tried to freeze samples [1], like using a cold gas stream or liquid propane, but in all cases the freezing rate plays a critical role. In order to maximise the thermal exchange between the freezing agent and the protein crystal we have developed the cryospray, an instrument capable of producing "ne atomisation of liquid nitrogen. Protein crystals can contain up to 75% solvent (e!ectively water) and, for cryoprotection to be successful, freezing has to be rapid enough for the solvent to vitrify, hence limiting mechanical damage of the crystal to the expansion of ice on crystallisation. Cooling protocols to do this include treatment with cryoprotectants such as glycerol, methyl-pentadiol (MPD) or polyethylene glycol (PEG) of various molecular weights, and rapid cooling in liquid nitrogen, propane or nitrogen gas streams. For a complete
* Corresponding author. E-mail address: [email protected] (R. Steinmann)
review of cooling rates and cryoprotectants, see Ref. [1]. Classical methods to produce a spray are well known and reliable, but none of them is suitable to get a dropsize well adapted for our application (the average size of samples varies from 50 to 150 lm). The device uses the technology of a coaxial injector (the same as used in the ARIANE V rocket engine) to produce "ne droplets [2]. The cryospray is generated using liquid nitrogen and helium gas. Fig. 1 shows a scheme of the instrument. Helium coming from the bottle (He) goes into the vacuum vessel (V) where it is thermalised on the nitrogen container (N) and driven into the gas chamber of the injector at a pressure of 300 mbar. The liquid nitrogen, pressurized at 150 mbar, is propelled through the central nozzle (0.4 mm ID, 16 mm length). The di!erential speed between the gas and the liquid generate a surface instability leading to the atomisation process. The whole droplet detachment is about 1.5 cm from the nozzle and, in contact with the sample, the crystal freezes at rates higher than 100 K/s. The liquid-nitrogen container has a volume of 1 l allowing 2.5 h of continuous operation. Because of the low temperature produced by the spray, a water-free environment is necessary to prevent ice from growing and, indeed, crystal damage. Fig. 2 shows the freezing chamber (F) made from an acrylic cylinder, one side tightly joined to the cryospray and the other one to a dewar (D) "lled with liquid nitrogen where frozen
0921-4526/00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 5 2 6 ( 9 9 ) 0 2 8 5 0 - 1
2048
F. Felisaz et al. / Physica B 284}288 (2000) 2047}2048
Fig. 1. General scheme of the instrument. See text for details.
samples are stored. The piston (P) is used to drive the sample holder (Sh) inside the chamber to the freezing position. A vertical translation platform (T), actuated by the knob (K), moves the sample from the spray to the storage container (D). A vent valve (S) regulates the internal pressure preventing any over-pressure. A typical freezing process starts by `"shinga a crystal from the mother liquid and transferring it to its cryoprotectant solution. Mounted in the loop of its experimental support, the crystal is placed into the spray using the piston (P). This operation takes a few seconds and is fast enough to avoid any damage to the sample. As soon as the crystal moves into the chamber, it comes in contact with the spray freezing in fraction of a second. The sample remains under the cryogenic spray all the way until being deposited in the liquid nitrogen. Accurate measurements of cooling rates of protein crystals on freezing have been hard to perform, but cooling from room temperature to 140 K in 0.16 s has been recorded and it is adequate to ensure that in most cases the vitri"ed state is attained. The device can be operated to freeze up
Fig. 2. Detail of the freezing chamber.
to six samples sequentially and to store them for later use.
Acknowledgements The authors would like to thank Franck Lapeyre for his invaluable help on the construction of the freezing chamber.
References [1] E.F. Garman, T.R. Schneider, J. Appl. Crystallogr. 30 (1997) 211. [2] W.O.H. Mayer, Exp. Fluids 13 (1994) 401.