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Brain Research Bulletin, Vol. IS, pp. 351-352, 1985. Q Ankho International Inc. Printed in the U.S.A. 0361~9230/85 $3.00 + .OO A Simple Jig Allows for a Rapid Mounting of Micropipette Tips for EM Viewing BRIEF COMMUNICATION JOHN R. LAVIOLE’ITE AND J. P. RAYNAULD’ Centre de recherche en sciences neurologiques, UniversitC de Montrkal, FacultC de mkdecine Case postale 6128, succursale A, Montrkal (Qutbec) Canada, H3C 3J7 Received 28 March 1985 LAVIOLETTE, J. R. AND J. P. RAYNAULD. A simple jig allows for a rapid mounting of micropipette tips for EM viewing. BRAIN RES BULL G(3) 351-352, 1985.-A simple jig to be used with a microscope allows rapid mounting of micropipettetips on electron microscope grids. Electron microscopy Micropipette tip Mounting jig THE advent of microprocessor controlled micropipette pul- lers has brought a new standard in micropipette reproduc- ibility. However, in order to verify this reproducibility, the micropipette tip has to be examined under (either scanning or transmission) the electron microscope. Although a number of methods to do this have appeared in the literature [l-5], electrophysiologists, who are not electron- microscopists, will likely appreciate this simple and rapid technique of mounting micropipette tips. The technique uses standard electron microscope grids to mount the electrode tips which can then be manipulated and stored as normal EM grids. The set-up illustrated in Fig. 1 is self explanatory. A small spring-loaded tungsten wire is used to clamp the EM grid which can be of the large hole (800 microns) or the long slot type; a small mount of modeling clay located in line with the optical axis of the microscope holds the micropipette during the delicate manipulations. Operationally, fmt the grid is centered and focused under a 4x objective then the slide is moved back using a single stage control in order to provide clearance for manipulation of the pipette. The pipette is then placed on the modeling clay and positioned manually so that its tip is at the center of the visual field, but slightly above the plane of focus. The slide is brought back and the EM grid is centered on the electrode tip. The pipette is Cmlly gently pressed down on the rim of the EM grid and a small drop of “5 minute Epoxy” glue is used to cement it there. After the required hardening, the pipette is broken off with a slight pressure on the micro- electrode. MODELING CLAY MtCROPlPETTE EM GRID NGSTEN WIRE 0.2 t-n-n DIA. FIG. 1. Illustration showing how the jig looks like on a Nikon L-Ke microscope stage. We normally prepare half a dozen such grids before coat- ing them with gold or gold-paladium at the sputtering facilities found in all scanning EM laboratories. A small layer of coating is usually sufficient since the purpose is just to drain electric charges during EM viewing. Viewing under either the scanning or transmission micro- scope is standard, although care must be taken not to over- heat the tip with the electron beam when using a transmis- sion electron microscope at high magnifications. ‘Requests for reprints should be addressed to Jean-Pierre Raynauld, Centre de rechereche en sciences neurologiques, Universite de Montreal, Faculte de medecine, Case postale 6128, succursale A, Montreal (Quebec) Canada, H3C 357. 351

A simple jig allows for a rapid mounting of micropipette tips for EM viewing

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Brain Research Bulletin, Vol. IS, pp. 351-352, 1985. Q Ankho International Inc. Printed in the U.S.A. 0361~9230/85 $3.00 + .OO

A Simple Jig Allows for a Rapid Mounting of Micropipette Tips for EM Viewing

BRIEF COMMUNICATION

JOHN R. LAVIOLE’ITE AND J. P. RAYNAULD’

Centre de recherche en sciences neurologiques, UniversitC de Montrkal, FacultC de mkdecine Case postale 6128, succursale A, Montrkal (Qutbec) Canada, H3C 3J7

Received 28 March 1985

LAVIOLETTE, J. R. AND J. P. RAYNAULD. A simple jig allows for a rapid mounting of micropipette tips for EM viewing. BRAIN RES BULL G(3) 351-352, 1985.-A simple jig to be used with a microscope allows rapid mounting of micropipette tips on electron microscope grids.

Electron microscopy Micropipette tip Mounting jig

THE advent of microprocessor controlled micropipette pul- lers has brought a new standard in micropipette reproduc- ibility. However, in order to verify this reproducibility, the micropipette tip has to be examined under (either scanning or transmission) the electron microscope. Although a number of methods to do this have appeared in the literature [l-5], electrophysiologists, who are not electron- microscopists, will likely appreciate this simple and rapid technique of mounting micropipette tips. The technique uses standard electron microscope grids to mount the electrode tips which can then be manipulated and stored as normal EM grids. The set-up illustrated in Fig. 1 is self explanatory. A small spring-loaded tungsten wire is used to clamp the EM grid which can be of the large hole (800 microns) or the long slot type; a small mount of modeling clay located in line with the optical axis of the microscope holds the micropipette during the delicate manipulations.

Operationally, fmt the grid is centered and focused under a 4x objective then the slide is moved back using a single stage control in order to provide clearance for manipulation of the pipette. The pipette is then placed on the modeling clay and positioned manually so that its tip is at the center of the visual field, but slightly above the plane of focus. The slide is brought back and the EM grid is centered on the electrode tip. The pipette is Cmlly gently pressed down on the rim of the EM grid and a small drop of “5 minute Epoxy” glue is used to cement it there. After the required hardening, the pipette is broken off with a slight pressure on the micro- electrode.

MODELING CLAY

MtCROPlPETTE

EM GRID

NGSTEN WIRE 0.2 t-n-n DIA.

FIG. 1. Illustration showing how the jig looks like on a Nikon L-Ke microscope stage.

We normally prepare half a dozen such grids before coat- ing them with gold or gold-paladium at the sputtering facilities found in all scanning EM laboratories. A small layer of coating is usually sufficient since the purpose is just to drain electric charges during EM viewing.

Viewing under either the scanning or transmission micro- scope is standard, although care must be taken not to over- heat the tip with the electron beam when using a transmis- sion electron microscope at high magnifications.

‘Requests for reprints should be addressed to Jean-Pierre Raynauld, Centre de rechereche en sciences neurologiques, Universite de Montreal, Faculte de medecine, Case postale 6128, succursale A, Montreal (Quebec) Canada, H3C 357.

351

LAVIOLETTE AND RAYNAILI~

FIG. 2. Photograph of micropipette tips. Top, pipette used for patch clamp studies; bottom, pipette used for intracellular recording. Scale is for one micron in both cases.

The end results are illustrated in Fig. 2. On a routine basis, the electrode tip geometrical parameters may now be

obtained, published or used as reference whenever neces- sary.

ACKNOWLEDGEMENTS

The authors are grateful to K. C. Watkins for use of the electron EM pictures. The typing and word-processing expertise of Joanna microscope facilities, to G. Filosi for graphic art, to M. Lauzon for advice on sputtering, and to A. Diel-Calamba for taking the scanning

Frongillo is also acknowledged. This work was supported by grants from the Medical Research Council of Canada.

REFERENCES

Baldwin, D. J. Non-destructive electron microscopic examina- tions with rotation of beveled micropipette electrode tips. J Neurosci Methods 2: 163-167, 1980. Bils, R. F. and M. Lavallee. Measurement of glass microelec- trodes. Experimentia 20: 231- 232, 1964. Frank, K. and M. C. Becker. Microelectrodes for recording and stimulation. In: Physical Techniques in Biological Research, vol 5, part A, edited by W. L. Nastuk, New York: Academic Press, 1964, pp. 22-87.

Fry, D. M. A scanning electron microscope method for the examination of glass microelectrode tips either before or after use. Experimentia 31: 695-696, 1975. Tupper, J. T. and H. Tedeschi. Microelectrode studies on the membrane properties of isolated mitochondria. Proc Nat1 Acad Sci USA 63: 370-377, 1969.