Theory and practical preparation of buffers, fixatives, and other solutions required for chemical processing of biological samples for TEM.
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Individuals who are, or will be, responsible for chemical processing and sectioning of biological samples for TEM examination.
Lecture, demonstration and hands-on practice, as well as round table discussion. Participants are encouraged to bring their own samples, if possible.
- Theory and practical preparation of buffers, fixatives, and other solutions required for chemical processing of biological samples for TEM
- Fixation theory and application of primary and secondary fixatives
- Dehydration, choosing the correct one, ETOH, Acetone, or propylene oxide
- Infiltration and choice of embedment resin, epoxy, methacrylate, or acrylic
- Embedment and maintaining orientation if applicable
- Block evaluation
- Trimming for thick survey and thin sections
- Practical aspects of microtomy and instrument parameters affecting section quality
- Adjustment of clearance angle and block face/knife edge alignment
- Thick (0.5 µm) sectioning and chromatic staining for OLM assessment
- Thin (≤ 60 nm) sectioning and section retrieval
- Post staining Pb and UA
- Section assessment and troubleshooting potential artifacts such as chatter, knife marks, and tears
- Zeiss TEM
- Leica EM UC7 RT & Cryo
- Boeckeler RMC Power Tome PC RT & Cryo
- EMS 9000 Microwave
- Lynx II Auto Tissue Processor
Registration will be limited to a maximum of 15 participants.
EMS will provide samples to those who prefer not to bring their own.
Biological TEM: A Complete Picture
The proper chemical processing of biological samples for TEM observation is essential to maintain representative morphology and ultrastructural detail. This course will cover the buffers, fixatives, dehydrants, and embedment resins most often used for EM, with their individual advantages and disadvantages discussed. The preparation of these various solutions, when necessary, will be calculated and preformed. The microwave will be utilized for all steps except polymerization. The need for specialized protocols when using specific tissues, such as myleinated nerve which requires extended infiltration, will be discussed. Epoxy (Embed 812) and acrylic (LR White) resins will be available.
The TEM's ability to provide sub-nanometer resolution is dependent ultimately on sample thickness, typically 60 nm or less. To obtain sections of this dimension requires specialized Instruments Available, ultramicrotomes, high quality diamond knives, and a skilled technician. The process of trimming, thick sectioning for OLM observation, thin sectioning, section retrevial, and section assessment will be the major focus of this workshop. Basic operation of the TEM will include specimen insertion, condenser and objective astigmatism correction, and critical focusing.