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Electron Microscopy Sciences

EMS Academy Applications

arrow13The Biological TEM Workshop: A Complete Picture

Theory and practical preparation of buffers, fixatives, and other solutions required for chemical processing of biological samples for TEM.

Examples of the endless possibilities in the field of Microscopy

Bone Marrow   Bone Marrow
Bone Marrow: Transmission electron microscope image of a thin section cut through an area of bone marrow area near the cartilage/bone interface in a mouse kneecap. Image shows small opening in the thin endothelium of the vascular sinus wall, where a blood cell is crossing the thin vascular sinus wall and into the sinus lumen.
Louisa Howard, Dartmouth College.
Pancreas   Pancreas
Pancreas: Transmission electron microscope image of a thin section cut through the pancreas (mouse). Image shows a cross section through part of a nerve cell and part of a capillary within the pancreatic tissue. The capillary lining consists of long, thin endothelial cells, connected by tight junctions. The image shows a fenestration in this endothelial cell. Basal lamina is present at the edges of the acinar cells.
Louisa Howard, Dartmouth College.
region in the Drosophila germarium   synaptonemal complex in nuclei of Drosophila germarium cell
Transmission electron microscope image of a region in the Drosophila germarium. Specific cells in the germarium contain synaptonemal complex in their nuclei.   Transmission electron microscope image of synaptonemal complex in nuclei of Drosophila germarium cell.
Louisa Howard, Dartmouth College.
Zebra Fish muscle   Zika Virus
Transmission electron micrograph of Zebra Fish muscle.
Specimen courtesy of Dr J Leslie.
  Transmission Electron micrograph of Zika Virus. Virus particles are 40 nm in diameter, with an outr envelope and an inner dense core.
Courtesy of Cynthia Goldsmith, CDC.

Targeted Participants

Individuals who are, or will be, responsible for chemical processing and sectioning of biological samples for TEM examination.

Facility

The EMS Microscopy Academy
Located in Hatfield, Pennsylvania, the Academy provides electron microscopy classes, workshops and training sessions for all fields of microscopy, including materials science and biological science.

Scope of Class

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 equipment, 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.

Format

Lecture, demonstration and hands-on practice, as well as round table discussion. Participants are encouraged to bring their own samples, if possible.

Main Curriculum

  • 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
  • Trimmimg for thick survey and thin sections
  • Practical aspects of microtomy and instrument parameters affection 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 retrevial
  • Post staining Pb and UA
  • Section assessment and troubleshooting potential artifacts such as chatter, knife marks, and tears

Equipment

Leica UC7 Ultramicrotome Boeckeler Autotome
EMS 9000 Microwave Vacuum Oven

Faculty

Al Coritz
Al has been doing Electron Microscopy for 38 years, beginning at the Yale School of Medicine and ending up on the commercial side with several key EM companies. His specialty is Cryo-techniques and Thin Film Technology: i.e. Freeze Fracture/Rotary Shadowing, High Pressure Freezing, and more. He is currently with Electron Microscopy Sciences where he has been the Technical Director for over 20 years.

Michael Kostrna
Michael was the program director of the Electron Microscopy Technician program at Madison Area Technical College and has more than more than 35 years in EM technical education and research experience. He has been training EM students for 29 years and has developed curricula and lab exercises for TEM, SEM, OLM, lab safety, introductory and advanced biological EM, EM, maintenance, and x-Ray microanalysis. He has worked with companies such as SC Johnson Polymer, Dow Chemicals, Io Genetics, Virent Technologies, ABS Global, NanoOnocology, and Microscopy Inovations, and in the process gained insight to the various applications of EM.

View our schedule to see if this course is currently offered.

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