Microscopy Core: User Guidelines
New investigators should first contact Yan Deng to discuss the projects and imaging techniques that are required for the experiments.
We encourage users to run the microscopes independently. All users must get trained. No one may use any equipment unassisted without first being trained and checked out by core staff.
Electron microscopy is generally performed as a drop-off service. The core will also provide training for new users and common, necessary materials for experienced users. Please contact Alice Liang first to arrange a consultation and to decide the best ways to approach your project.
Specimen Preparation for Electron Microscopy
The core staff offers full service for sample preparation of many standard EM techniques, include:
- Embedding using either epoxy or acrylate-based embedding resins at ambient or low temperatures
- Thin sectioning with Leica UCT or Leica EM UC6
- Cryoultramicrotomy using Leica EM UC6/EM FC6 cryomicrotome
- Immunolabeling following pre or post embedding protocols
- Tokyasu immunogold labeling of ultrathin cryo sections
- High-Pressure Freezing using a Bal-Tec HPM-010 (NYSBC), and following Freeze-Substitution by Leica EM AFS2 automatic freeze substitution system
- Negative Staining of virus, bacteria and macromolecules
- Rotary shadowing by BOC Edward Auto 306 Evaporator for DNA, RNA and macromolecules
- Electron tomography data collected by either Philips CM200 or Tecnai F20 (NYSBC)
- 3D EM using FEI Helios 650 (NYSBC)
Service is provided on a first-come first-served basis. Top priority is given to scientists from NYU Medical Center. In general, the turnover time for negative staining and rotary shadowing is within a week; routine morphology analysis is within a month and immuno EM projects is between one to three months depending on the workload.
Hours of Operation
Although we try to help all users as quickly as possible, both staff and microscopes may be booked in advance. Therefore, please try to consult with us at least one week before you plan your experiment, and book the microscopes and microtomes in advance on the Sign-Up page! Also, please fill out the Usage log after each use and inform us immediately about any problems you have had with the microscopes so that we can arrange service as quickly as possible.
Schedule and Sign-up
Contact core staff first before training or assistance.
All users and their lab information should be set up on LIMS system. Please contact your lab manager to create a project for you on LIMS.
Light microscope users who have been trained are required to book microscopes in advance through LIMS system (except Zeiss LSM710 and Leica SP5).
All trained users are required to sign in on the schedule calendar at least 24 hours before their planned session to use any piece of equipment, even just a five minute look. Every user must fill in his/her name, time range, special mode, and camera requirement. Cancellation should be made on the schedule calendar as soon as the user decides to cancel a scheduled session.
We reserve the right to limit the time of the reservation during daytime (9AM to 6PM) on weekdays (Monday to Friday). This regulation won't apply to night time and weekends.
For each session, EM users should record in the
users log the name for the session, date and exposure numbers of the films they have taken. Write down also in the comments area any malfunction, warning messages on the EM screen, abnormal vacuum readings, and other relevant information.
In addition to keeping a record in the user's log, a user should report immediately to the lab manager any problems that prevent normal operation or require prompt service.
Operation of Light Microscopes
When an emergency service, maintenance or administrative issue arises, the core staff has the right to cancel and reschedule.
Users must sign the log book for their usage. The last user of the day must make sure the microscope is properly shut down.
No food or drink is permitted in the vicinity of microscopes.
Users may not change default settings on any microscope. When work is done, equipment should be left clean and in default configuration.
User's data must be saved in the designated User Files directory. Any data that are not in the right place will be deleted without warning.
Users must transfer data to their own media as soon as possible. Old data will be cleaned periodically by core staff. The core is not responsible for any data loss. Users must not delete any files from the computer (including their own data) or install any software on the computer.
Any material that may be hazardous must be pre-approved by Environmental Health and Core Director. No radioactive materials are allowed in the core.
Failure to comply with core regulations and policies will result in termination of privileges.
Operation of Electron Microscopes
Always follow instruction manuals. If one is not sure about a certain operation, consult the core staff or an experienced user.
The cold finger is used to reduce contamination in the microscope's column. Users are recommended to use it whenever possible. Check and fill up liquid nitrogen in the cold finger dewar before starting a session. Upon finishing a session at the end of a day, the user should pour out the remaining liquid nitrogen and put the dewar back on the stage to let the cold finger warm up slowly, avoiding a sudden contamination of the column.
Before powering up the filament, one should always check the vacuum, especially the IGP reading. Turn up the filament current slowly. It is acceptable to leave the high tension (HT) either on or off, but one should turn off the filament unless there is another user after one's session.
Do not turn the HT off when the filament is on. For CM200/FEG, some tips on how to increase the lifetime of the emitter are posted on the bookshelf inside EM Room 1. Below are a few most critical tips: 1. ALWAYS close Valve V7 when away from the scope or changing samples. 2. STANDBY mode should be used as much as possible. 3. DO NOT switch on HT BEFORE at least 1 kV of EXTRACTION is reached.
Each EM room is facilitated with a Magnetic Field Compensation System. One should see only green lights when the system is running properly. A momentary field surge may result in a warning beeper without interrupting the system. An intensive field change may trip off the system, and one in this case will see a red light on the DISABLE push button. Then the user can enable the system by pushing the reset button. No one is supposed to adjust the three (gain, warning, and trip-off) level-control potentiometers. If one has problem with the system, please contact the lab manager.
EM Camera and Darkroom
Taking pictures with CCD Camera
User should transfer images to his/her own disk or to a computer elsewhere via internet for data storage immediately after finishing the scheduled session. One should never store the data in the core hard drive. All data should be saved in the designated User Files directory. Any data on the desktop or other system folder directories will be deleted without warning.
Taking pictures with film
Always check the film stock number in the microscope before starting a session. If one needs a full camera (36 exposures for CM12 and 56 exposures for CM200) or a certain number of films in the camera, please make a note on the schedule calendar (which can be found where) so that the user before the session can leave the camera in an appropriate condition.
If one takes exposures that require special treatment/development, it is his/her responsibility to change and develop the camera immediately. As a corollary, sign up for sufficient time to accommodate changing the camera.
Before venting the camera, make sure the dry nitrogen gas is turned on and the pressure is set at about 2 psi. Normally, the gas regulator is set on the right pressure, and the user only needs to open the gas valve.
A user should change the camera and develop the films in any of the following cases: (a) There are less than 12 films (2/3 of full load) left in the camera. (b) More than 12 pictures have been taken. (c) Pictures have been taken in the low-dose mode. (d) The next user has requested a full camera or more films than what are left in the camera.
Always wear gloves when you touch films, film holders, and cameras.
Make sure to reset the camera after changing it.
Reload the camera and place it in the vacuum desiccator after film development. Make sure there are enough films in the desiccator for the next load. Be sure to check, and replace if necessary, the phosphorous pentoxide (P2O5).
After using the darkroom, inspect and be sure to leave it in a clean state. Clean up developer/fixer spills on the floor and/or bench top.
Remember to turn off the safe lights after using the darkroom; otherwise other users will be confused.
Safety and Security
All NYULMC safety codes regarding radioactive materials and chemicals should be followed in the core.
We encourage unassisted usage of the equipment in the core at any time, however the last person leaving the EM suite after 6:00 p.m. on weekdays or any time on weekends must make sure the main gate of the EM suite is locked.