Electron Microscopy Workshops
EMi is pleased to offer workshops in many areas of biological electron microscopy. Workshops are offered on a quarterly or monthly basis depending on demand and EMi staff availability. EMi has provided training to the UCLA community for more than 10 years, and has the experience necessary to effectively teach even the most difficult EM subjects. EMi workshops are designed for quick learning and include theory and practice (or demonstration); the maximum group size is nine participants. Training is always tailored to trainees particular needs. Every student is trained on his/her own samples and receives EMi proprietary training materials and customized protocol for his/her particular sample.
List of scheduled EMi workshops for 2011:
(please contact EMi for exact dates, fees, etc.)
This technique has produced more publications than any other single technique. It allows visualization of the fine structure of tissue, cells and organelles. The same approach may be used in material-science applications. This is a 3-day workshop starting in the afternoon and continuing for 3-6 hours each day. During this intense workshop, students learn how to fix, process and embed in plastic their samples for further sectioning and EM imaging. Plastic-embedding requires dangerous chemicals including osmium tetroxide and uranyl acetate, therefore proper training is essential and highly recommended. Please note that this workshop does not include sectioning and imaging.
- Ultrathin sectioning of plastic-embedded material.
This is the next step after plastic-embedding but may be used alone. Electrons do not travel through very thick samples, therefore, the sample must normally be thinner than 200 nm. Participants of this highly individualized workshop learn how to operate $65K machine (the ultramicrotome) to produce 50 nm thick slices (sections)! Training begins with a two-hour lecture with video demonstration and instructions. Then, individual two-hour training sessions are scheduled to work on the state of the art modern ultramicrotome. At the end of the workshop, it is expected that every participant will have at least 5 grids with his/her 50-65 nm sections.
- Plastic-embedded sample analysis.
This is the next and final workshop in the plastic-embedding series. Participants learn how to make proper staining solutions and stain their own sections. EMi then demonstrates analysis of the sample sections in the electron microscope using a digital camera and Digital Micrograph software. This primarily hands-on workshop lasts three-hours. Individual image analysis session(s) may be scheduled in addition.
- Negative staining.
This simple technique allows visualization of individual macromolecules and their complexes, frequently with spectacular results. The four-hour workshop includes a lecture, sample preparation demonstration, imaging and image analysis. Please note that due to the irregular nature of biological samples, this training uses samples provided by EMi. Individual image analysis session(s) may be scheduled in addition.
- Double-carbon (sandwich) negative staining.
This technique is an advanced version of the standard “negative staining.” Sandwiching samples between two extremely thin layers of carbon provides better staining and may help when used in conjunction with a 3D approach. This is a three-hour workshop with lecture and live demonstration of negative staining basics, and demonstration of imaging.
- How to make good carbon film for EM?
In order to see anything in transmission electron microscope (TEM) ,the sample needs to be suspended so electrons may pass through and create an image. Usually “support film” has been used to provide support to the sample. Carbon is a great material for “support film.” During this two-hour workshop, participants learn all tricks how to make really good carbon film and why this carbon may not be called “perfect.” Training includes informal discussion on how laws of physics and chemistry work in EM applications and a live demonstration on carbon evaporation with DV502A Denton Vacuum evaporator.
- Cryo-immuno localization.
Protein or other antigens may be localized with EM precision within the cell and/or organelle using gold-labeled antibodies (immuno-gold). This approach works in both: immuno-EM on plastic-embedded samples and cryo-immuno. This elaborate 5-day workshop is designed particularly to show that a cryo-immuno approach is actually easier than classic immuno-EM. Participants receive detailed instructions and protocols and practical experience in all steps of the procedure from fixation to sectioning, labeling and imaging. Please note that this workshop is designed for participants with previous experience in plastic-embedding and room-temperature sectioning. Each participant processes his or her own sample and obtains images. Note that due to the unpredictable nature of “immuno-approach” (some antibodies just do not work), EMi does not warrantee the successful labeling of all samples. Nevertheless, EMi does guarantee that each participant will leave the workshop with great experience in cryo-immuno EM.
- 3D reconstruction with EMAN on negatively stained samples.
With development of free software and easy access to computing resources, techniques available only to very advanced privileged individuals just 10 years ago are now widely available. Negatively stained samples may be used in 3D reconstruction single particle approach. This 5-day workshop is designed to be practical. Participants are introduced to the world of free Linux and EMAN software. In a small group, we will walk together through a “command line” and other Linux obstacles. Participants will have detailed instructions for each step of the 3D reconstruction process, which they may use in further reconstructions. Please note that this workshop requires participants to have previous experience in negative staining and recording of the images; users provide their own digital images. Sample must be a “single particle” (no fibers, cells etc.) with molecular mass from 50 to 500 kDa negatively stained, magnification x40-80K measured at the image plane. EMi can provide sample images as well. Participants are required to have their own laptops and to run virtual Linux on their machines (it does not require installation of Linux).
April 21, 2011