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SHORT TECHNICAL NOTE Cryo-TEM liquid nitrogen splash guard B. E. GOWEN* & L. BURGER² *Structural Biology Programme and ²Mechanical Workshop, European Molecular Biology Laboratory, Postfach 10·22·09, Meyerhofstrasse 1, D69012 Heidelberg, Germany Key words. Cryo-TEM, liquid nitrogen, splash guard. Summary A simple aluminium splash guard provides protection for both microscopes and operators during cryo-transfer procedures. Introduction The development of cryotransmission electron microscopy has resulted in an enhanced ability for structural biologists to determine the structure of macromolecular complexes (Dubochet et al., 1988; Fuller et al., 1995). While the technique is becoming more common, microscope manu- facturers have tended not to tailor their equipment to the specific requirements of the technique. We have found that during cryotransfer, the unavoidable, repeated spilling of liquid nitrogen on the microscopes can result in the cracking of the coating on their table tops. This is contrary to the manufacturers’ claims that their equipment is ‘safe’ from such treatment. These cracks eventually enlarge and chips can break off. This can become an annoyance for the operator when focusing or changing magnifications as hands often come into contact with the cracked table top. Spilled liquid nitrogen can flow between the table top and column-viewing screen or microscope-control panel. Such spillage could conceivably cause damage to the electronics or control systems of the microscope located underneath the table top. A dangerous aspect of cryo-transfers results from the fact that spilled liquid nitrogen can pour off the front of the table top onto the legs/feet of the operator during the transfer process. We have designed a simple splash guard which is placed on the microscope during cryo-transfer procedures and provides a measure of safety for the microscope and operator. The splash guard is simple to make, lightweight, and stores easily beside the microscope when not in use. Materials and methods The splash guard dimensions and folds, as designed for use with Philips CM series TEMs, are shown in Fig. 1. The guard is manufactured using sheet aluminium 1·5 mm thick. The dimensions include the width of the table top (from the ends of the control knobs to the front edge) and the length (slightly longer than the table top as measured from the viewing chamber of the column to the end). The folded corners at the left-hand side of the guard are sealed using small pieces of the aluminium bent at right angles and held in place with metal screws. The left-hand side of the guard is raised slightly from the table top by the use of small bolts screwed through the bottom of the guard. Results and discussion During cryo-holder transfers a large amount of liquid nitrogen can be spilled on electron microscopes. The amount of liquid nitrogen spilled is a direct consequence of the amount of tilt that is available on the microscope specimen stage. ‘Standard resolution’ microscopes usually allow high tilts and so a cryo-holder can safely be loaded without spillage by first tilting the stage, then inserting the holder in an upright position, waiting until the prepumping is finished, resetting the stage to zero tilt, and inserting the holder fully into the microscope. The opposite procedure is done to unload the cryo-holder without spilling liquid nitrogen. However, many ‘high resolution’ microscopes have limited tilt capabilities (usually set as a safety mechanism to prevent damage to the expensive pole- pieces). On such microscopes there is no way to avoid spilling liquid nitrogen while loading/unloading the cryo- holder into/from the microscope. Pouring the liquid nitrogen from the holder in a safe location prior to loading Journal of Microscopy, Vol. 191, Pt 3, September 1998, pp. 320–322. Received 28 July 1997; accepted 15 January 1998 320 q 1998 The Royal Microscopical Society Correspondence to: Brent E. Gowen, Cryo-TEM Facility, Department of Biochem- istry, Imperial College of Science, Technology and Medicine, London SW7 2AY, U.K. E-mail: [email protected]

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Page 1: Cryo-TEM liquid nitrogen splash guard

SHORT TECHNICAL NOTE

Cryo-TEM liquid nitrogen splash guard

B. E. GOWEN* & L. BURGER†*Structural Biology Programme and †Mechanical Workshop, European Molecular BiologyLaboratory, Postfach 10·22·09, Meyerhofstrasse 1, D69012 Heidelberg, Germany

Key words. Cryo-TEM, liquid nitrogen, splash guard.

Summary

A simple aluminium splash guard provides protection forboth microscopes and operators during cryo-transferprocedures.

Introduction

The development of cryotransmission electron microscopyhas resulted in an enhanced ability for structural biologiststo determine the structure of macromolecular complexes(Dubochet et al., 1988; Fuller et al., 1995). While thetechnique is becoming more common, microscope manu-facturers have tended not to tailor their equipment to thespecific requirements of the technique. We have found thatduring cryotransfer, the unavoidable, repeated spilling ofliquid nitrogen on the microscopes can result in thecracking of the coating on their table tops. This is contraryto the manufacturers’ claims that their equipment is ‘safe’from such treatment. These cracks eventually enlarge andchips can break off. This can become an annoyance for theoperator when focusing or changing magnifications ashands often come into contact with the cracked table top.Spilled liquid nitrogen can flow between the table top andcolumn-viewing screen or microscope-control panel. Suchspillage could conceivably cause damage to the electronicsor control systems of the microscope located underneaththe table top. A dangerous aspect of cryo-transfers resultsfrom the fact that spilled liquid nitrogen can pour off thefront of the table top onto the legs/feet of the operatorduring the transfer process. We have designed a simplesplash guard which is placed on the microscope duringcryo-transfer procedures and provides a measure of safetyfor the microscope and operator. The splash guard is simple

to make, lightweight, and stores easily beside themicroscope when not in use.

Materials and methods

The splash guard dimensions and folds, as designed for usewith Philips CM series TEMs, are shown in Fig. 1. The guardis manufactured using sheet aluminium 1·5 mm thick. Thedimensions include the width of the table top (from the endsof the control knobs to the front edge) and the length(slightly longer than the table top as measured from theviewing chamber of the column to the end). The foldedcorners at the left-hand side of the guard are sealed usingsmall pieces of the aluminium bent at right angles and heldin place with metal screws. The left-hand side of the guardis raised slightly from the table top by the use of small boltsscrewed through the bottom of the guard.

Results and discussion

During cryo-holder transfers a large amount of liquidnitrogen can be spilled on electron microscopes. Theamount of liquid nitrogen spilled is a direct consequenceof the amount of tilt that is available on the microscopespecimen stage. ‘Standard resolution’ microscopes usuallyallow high tilts and so a cryo-holder can safely be loadedwithout spillage by first tilting the stage, then inserting theholder in an upright position, waiting until the prepumpingis finished, resetting the stage to zero tilt, and inserting theholder fully into the microscope. The opposite procedure isdone to unload the cryo-holder without spilling liquidnitrogen. However, many ‘high resolution’ microscopeshave limited tilt capabilities (usually set as a safetymechanism to prevent damage to the expensive pole-pieces). On such microscopes there is no way to avoidspilling liquid nitrogen while loading/unloading the cryo-holder into/from the microscope. Pouring the liquidnitrogen from the holder in a safe location prior to loading

Journal of Microscopy, Vol. 191, Pt 3, September 1998, pp. 320–322.Received 28 July 1997; accepted 15 January 1998

320 q 1998 The Royal Microscopical Society

Correspondence to: Brent E. Gowen, Cryo-TEM Facility, Department of Biochem-

istry, Imperial College of Science, Technology and Medicine, London SW7 2AY,

U.K. E-mail: [email protected]

Page 2: Cryo-TEM liquid nitrogen splash guard

into the microscope can result in a rapid rise in temperatureof the specimen. This can result in the devitrification of thespecimen before it is loaded into the microscope and liquidnitrogen replaced in the dewar.

As Fig. 2 demonstrates, the splash guard we havedesigned effectively prevents spilled liquid nitrogen from

settling on, and therefore damaging, the table top ofthe microscope. The guard also ensures that no liquidnitrogen flows into the areas beneath the table top. Inaddition, the guard prevents spilled liquid nitrogen frompouring onto the area where the operator is working.Having the splash guard raised slightly on the left-hand

q 1998 The Royal Microscopical Society, Journal of Microscopy, 191, 320–322

Fig. 1. Dimensions (mm) and folds for theliquid nitrogen splash guard. Top (left) andend (right) views are shown.

Fig. 2. The splash guard with liquid nitrogen being poured onto it. Notice that all the liquid nitrogen (arrows) is draining out of the end of theguard, off the table top, and away from the front of the microscope where the operator is located.

CRYO-TEM SPLASH G UAR D 321

Page 3: Cryo-TEM liquid nitrogen splash guard

side ensures that the liquid nitrogen quickly pours off theguard, away from the table top and operator to a safelocation. After the cryo-holder is loaded/unloaded into/fromthe microscope the guard is removed from the table top andstored beside it, allowing normal unobstructed use of themicroscope.

We have made the splash guard out of various types ofplastic, but when tested they all warped when exposed tothe liquid nitrogen. Only the aluminium splash guard didnot warp.

Acknowledgments

We thank Hans Wittman of the Drawing Office and Mah

Britt Hansen of the Photography Department of EMBL fortheir assistance.

References

Dubochet, J., Adrian, M., Chang, J.-J., Homo, J.C., Lepault, J.,McDowall, A.W. & Schultz, P. (1988) Cryoelectron microscopy ofvitrified specimens. Quart. Rev. Biophys. 21, 129–228.

Fuller, S.D., Berriman, J., Butcher, S.J. & Gowen, B.E. (1995) LowpH induces swivelling of the glycoprotein heterodimers in theSemliki Forest virus spike complex. Cell, 81, 715–725.

322 B. E. GOWEN AND L. BURGER

q 1998 The Royal Microscopical Society, Journal of Microscopy, 191, 320–322