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The SEM and TEM microscopes are calibrated several times each week with the aid of a magnification calibration diffraction grating replica. In addition, representatives from JEOL's technical service department conduct a rigorous twice-yearly performance evaluation/testing of the electron microscopes to ensure proper function. Preventive maintenance on the equipment is also scheduled during the performance evaluation period. Test samples submitted from investigators are always compared with control specimens.

A detailed set of protocols is provided to the investigator regarding the preparation of samples for SEM and TEM (see below). In the majority of cases, the investigator fixes the sample with fixative prepared by SIMR staff, and SIMR staff performs subsequent processing. Screening of samples with the assistance of technical support is recommended at all times. Background references on the particular investigation are usually requested by SIMR to ensure that the correct fixatives and specific information regarding the specimen is known before the analysis.

For grant or paper submission:

Fixed samples containing 3% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) were washed with 0.1M cacodylate buffer (pH 7.3) and then post-fixed with 1% cacodylate buffered osmium tetroxide. Samples were washed with 0.1M cacodylate buffer and then with distilled water. Samples were then sequentially treated with Millipore-filtered 1% aqueous tannic acid, washed in distilled water, treated with Millipore-filtered 1% aqueous uranyl acetate, and then rinsed thoroughly with distilled water. The samples were dehydrated with a graded series of increasing concentrations of ethanol and then transferred to graded series of increasing concentrations of hexamethyldisilazane (HMDS) and then allowed to air dry overnight. Samples were mounted on double-stick carbon tabs (Ted Pella Inc., Redding, CA), that had been previously mounted to aluminum specimen mounts (Electron Microscopy Sciences, Ft. Washington, PA). The samples were then coated with platinum under vacuum using a Balzer MED 010 evaporator (Technotrade International, Manchester, NH) until they reached a thickness of 25 nm. Samples were then flash carbon coated under vacuum and transferred to a desiccator for examination at a later date. Samples were examined with a JSM-5910 scanning electron microscope (JEOL, USA, Inc., Peabody, MA) at an accelerating voltage of 5 kV.

For grant or paper submission:

Samples were fixed with a solution containing 3% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3). Samples were then washed in 0.1M sodium cacodylate buffer and treated with 0.1% Millipore-filtered cacodylate buffered tannic acid, post-fixed with 1% buffered osmium and stained en bloc with 0.1% Millipore-filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol and then infiltrated and embedded in LX-112 medium. The samples were then polymerized in a 60?C oven for approximately three days. Ultrathin sections were cut using a Leica Ultracut microtome (Leica, Deerfield, IL) and then stained with uranyl acetetate and lead citrate in a Leica EM Stainer. The stained samples were examined in a JEM 1010 transmission electron microscope (JEOL USA, Inc., Peabody, MA) using an accelerating voltage of 80 kV. Digital images were obtained using an AMT imaging system (Advanced Microscopy Techniques Corp., Danvers, MA).