EM Resource

	EM Resource: New User Information
CONTENTS Introduction Planning Experiments Submitting Samples Fixation Microscope Use Final Return to EM Home Page	INTRODUCTION The Research Electron Microscropy Resource is a full-service EM facility that supplies access to equipment, technical support, and expertise in electron microscopy. Efficient use of electron microscopy requires expertise in experiment planning, specimen processing, thin sectioning, microscope operation, and ultrastructural analysis. The staff of the facility is available to provide this expertise and, if desired, to help the researcher acquire these skills. Work is provided on a fee-for-service basis. Below is general information about using EM in your research. Click to see a schedule of fees. Click to download a powerpoint presentation on EM (46 MB) [Return to Top of Page] PLANNING EXPERIMENTS Specimens can be submitted directly to the EM facility as described below. However, for new experiments or for inexperienced researchers it is best to consult with the facility staff to insure that specimen preparation is optimized. A brief meeting between the Resource staff and the researchers laboratory to iron out details can be arranged by contacting the director. This meeting can help determine feasibility, provide suggestions for optimal use of the resource, and alert the resource staff to unique details of specimen preparation for your project. [Return to Top of Page] SUBMITTING SAMPLES Submitted specimens must be accompanied by a Research Electron Microscopy Resource work requisition. A copy of the requisition can be obtained from the Processing Lab (B-2103 MCN). A printable PDF version of the requisition is also available for downloading by clicking above or clicking on the "Sample Submission Form" highlight on the EM Resource Home Page index. (PDF files require Adobe Acrobat (tm) reader. You can download a copy from the Adobe web site.) Specimens should be fixed prior to submission and kept refrigerated until submitted. EM fixative and wash buffer can be obtained from the refrigerator in the Processing Lab (T-3208 MCN). [Return to Top of Page] GENERAL FIXATION PROCEDURE To maintain ultrastructure, specimens must be fixed rapidly after the oxygen supply has been cut off. The specifics of fixation (fixative, concentration, time, temperature) and the buffering medium will vary for specific experiments. However, in general, fixation will involve buffered aldehydes which cross link proteins, carbohydrates, and to a certain extent lipids. The fixatives will cross link similar molecules that surround the sample (body fluids, cell culture medium, etc. ) so the sample should be washed briefly in a buffered solution before fixation. Aldehyde fixatives penetrate tissue slowly, so samples must be small (less than 0.5 mm on two sides). For large specimens, mincing the sample directly in fixative after washing works best. This can be done by placing a drop of fixative on dental wax or parafilm and placing the excised and washed sample in the drop of fixative and then mincing with fresh razor blades. The EM staff can show you how this is done. Cold depolymerizes cytoskeletal elements, so whenever possible, buffer washes, fixatives, and sample should be at room temperature or warmer to initiate fixation. For temperature sensitive samples, such as enzyme cytochemistry, the samples can be briefly warmed placed in fixative and then, after a minute, re-cooled. To store fixed samples for more than a few hours, the specimen should be placed on ice or in the refrigerator after fixation. For storage of samples more than a few days before processing, the fixative should be replaced with buffer after 24 hours. [Return to Top of Page] MICROSCOPE USE TEM and SEM microscopes are available ONLY to those who have been trained in their use by a member of the RESOURCE STAFF. After training, you will be able to sign up and use the microscopes during normal business hours ( Monday through Friday 9:00 am until 6:00 pm). The scheduling link on our Home Page will direct you to the calendar to reserve the microscopes for your use. Use on weekends or after hours is limited to those who have extensive experience in their operation and have undergone additional training in how to shut down the microscope in case of a problem. Since no staff member is around after hours to help in an emergency, this policy is designed to avoid costly damage to the microscopes. [Return to Top of Page] FINAL NOTE The resource staff are happy to answer questions about our services and will help you plan your experiments. If you have questions please feel free to contact any member of the resource staff or the director: Jay Jerome U-2206 MCN E-mail: jay.jerome@vanderbilt.edu Phone: (615) 322-5530 [Return to Top of Page] [Return to EM Home Page]
ADDITIONAL USER INFORMATION
Created 11/12/01 by Jay Jerome. Copyright © 2001 Vanderbilt University. All rights reserved. Revised: March 31, 2008 .