Difference: AllHoursAccessCryoEM (17 vs. 18)

How to acquire authorization for independent use of microscopes at any hour of the day

The electron microscopes are in principle available for ~20 hours per day for data collection. However, several requirements need to be met before independent use of the instruments is approved.

1). Meet requirements for 24 hour access to the NYSBC .

2). Special training on EM operation must be completed. Training will only apply to individual instruments, as each has slightly different requirements. The topics to be covered are listed below.

3). User will be given an opportunity to practice independent instrument use during normal working hours. When ready, user will then take an operational test with EM staff to establish final authorization for independent use.

Session 1: Absolute Basics of operation

1. How to get an electron beam
2. How to adjust the eucentric height of the specimen
3. How to adjust the intensity of the beam
4. How to focus the image
5. How to change magnification
6. How to introduce and center a condenser aperture

Session 2: General Setup of microscope

1. How to align the optical axis of the instrument
   1. gun tilt, gun shift, voltage center
2. How to align the spot sizes
3. How to align the pivot points
4. How to correct for condenser astigmatism
5. How to introduce an objective aperture.
6. How to correct for objective astigmatism

Session 3: Advanced Setup of Microscope

1. How to set up the low dose kit of the microscope.
   1. search mode using the fast scan camera and/or binoculars i focus mode at a suitably high magnification
   2. imaging on CCD or film
2. How to select appropriate electron dose and exposure time
3. SerialEM (optional)
   1. set up SerialEM in low-dose mode.
   2. set up and collect a tilt series.
   3. Microscope alignments to be particularly careful about for proper operation of SerialEM.
4. Slow Scan CCD camera operation
   1. Flatfield correction
   2. Gain adjustments and features EM menu
   3. Binning of images
5. Refresh rate of the fast scan camera.
6. How user Film (optional)
7. How to view and record electron diffraction patterns (e.g. for checking for vitreous ice)
8. how to safely leave the microscope at the end of the day

Session 4: Sample Insertion and Removal

1. Sample insertion and removal with the room temperature holder. Loading of negative stained grids, and their removal.
2. Use of the cryoholder at room temperature. Loading of specimen and procedure for insertion of cryoholder into the mocroscope.
3. Loading of frozen hydrated sample in the cryoholder and insertion into the microscope. Monitoring of temperature stability.
4. Vacuum state of the microscope. What values should be reading in the different gauges and what to do when vacuum is poor.

Session 5: Emergency Procedures

1. What to do when software crashes
2. What to do in case the vacuum breaks
3. What to do if the beam shuts down
4. What to do if the lenses are not responding
5. What to do if there is water leaking
6. Precautions to be followed with liquid nitrogen
7. Precautions to be followed with liquid ethane
8. Etiquette of the electron microscopy facility

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-- RubenDiaz - 12 Mar 2008