Welcome

The Lilly Hall Zeiss LSM 710 microscope is a state-of-the-art laser scanning confocal microscope well-suited to imaging live cells as well as standard fixed immunofluorescence samples. It is equipped with laser lines at 405, 458, 488, 514, 561 and 633 nm and a 34 channel spectral detector that enables simultaneous imaging of multiple fluorescent proteins. The inverted platform is adapted for a variety of cell culture chambers, and includes a full incubator with CO2 and temperature control. It is outfitted with 40X and 63X apochromat water immersion lenses that are corrected for live cell imaging in culture medium, as well as a 63X planapochromat lens appropriate to fixed material in standard mounting medium. Zeiss Zen software enables advanced imaging including FRET, FRAP, fluorescent protein photoactivation, time lapse and 3-dimensional imaging. The microscope computer is on the university network and transferring image files to your lab computers is simple.

If you think the scope could be useful for your work, please contact Dr. Don Ready (dready@purdue.edu) and we’ll get you started.

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LSM710 Getting started

Getting Started

The Lilly Hall inverted Zeiss LSM 710, is a state-of-the-art confocal laser scanning microscope well suited to live cell imaging. Advanced methods available on the scope include spectral unmixing, time lapse, multi-target imaging, FRET, FRAP, photoactivation, and ratiometric imaging of ion indicator dyes. If you think the scope could be useful to your research, contact Dr. Don Ready (dready@purdue.edu). We have a reasonable amount of experience with the microscope and can advise you regarding the capabilities of the scope, compatible culture dishes and medium and LSM 710-friendly choices for live cell stains.

Some quick tips

  • The LSM710 does not have a UV excitation line suitable for DAPI. Invitrogen Hoechst 34580 can be excited by the 405 nm laser and is a suitable alternative nuclear dye. In general, the Invitrogen website is well worth browsing for live cell imaging reagents. Evrogen is also a source of advanced fluorescent protein reagents.
  • Plan to use phenol red free culture medium for imaging. Phenol red can interfere with fluorescence.
  • The LSM710 has an X-cite metal halide lamp and filters for FITC, TRITC and DAPI epifluorscence viewing. NOTE these are used for direct visual inspection but cannot be used for imaging.
  • The ‘Zeiss Campus’ website has very useful information on the LSM710; the interactive tutorial on the 34 channel Quasar detector is particularly informative.

Training

  • Each new user must be trained by Dr. Ready or Facility Administrator, Dr. Xia. (Users may not train other users). Training consists of an initial 2 hour session using prepared slides to orient users to the proper use of the inverted platform, basic light path configuration, imaging and care of objectives. A second session, in which these operations are applied to the users own sample will be scheduled soon after the first. You are strongly encouraged to use the equipment soon after training, so you do not forget what you have learned. (It’s not rocket science, but there are a lot of settings to remember.)
  • Solo use on evenings or weekends is permitted after training and introductory sessions are complete.
  • Orientation to advanced methods such as spectral imaging, FRET, FRAP and photoactivation will be provided as needed. If you plan to image multiple or ‘exotic’ fluorescent proteins, i.e., other than the usual CFP, GFP, YFP, RFP, mCherry, etc., plan ahead, some combinations work better than others.

Reservations

  • Reserve the microscope using the Resource Allocation Tool (RAT) website. https://engineering.purdue.edu/ECN/Resources/Tools/RAT/Entities/LSFIFL
  • Please do not reserve the equipment more than 2 weeks in advance. Be considerate of other users and do not reserve time that you ‘might’ use.
  • If possible, do not reserve the equipment for more than 3 hours during the day. Time-lapse experiments exceeding 3 hours should be scheduled after 6 pm or during the weekend.
  • Please start your session on time.

Cancellations

  • If you must cancel or change a session length, please email Dr. Xia and Dr. Ready and we’ll adjust the RAT calendar.
  • If you cancel your session, and the user before you would leave the scope on for you, you are responsible for ensuring the microscope is turned off. This is particularly important if you are booked to be the last user of the day. The phone number at the microscope is 44647 and you may be able to reach the user on the scope and ask them to shut it down. If you do not get confirmation that the scope will be shut down, you must go to the scope and turn it off.

Samples/immersion oil

  • Frames available for the microscope stage can carry standard slides, 96 well plates and 35mm culture dishes.
  • Do not use slides or dishes previously oiled at another microscope. (Immersion oils from different suppliers are often incompatible.)
  • Use only the Zeiss immersion oil kept at the microscope. Zeiss Immersol W should be used with the 40XW and 63XW lenses.
  • NB: The ‘water immersion’ lenses are NOT ‘dipping’ lenses and must not contact culture medium. A water immersion lens is optically corrected to view, through a coverslip, a sample in an aqueous medium. Use only Immersol W between the lens and coverslip.
  • Use the 63X lens and Zeiss immersion oil for high power observation of fixed samples mounted in glycerol-based mountant, e.g. Invitrogen ProLong Gold.
  • If the X-cite lamp is turned on, it must be kept on for a minimum of 30 min and needs to cool for 30 min before being reignited. (

37° Operation/CO2

  • The Pecon heating insert and CO2 cover are best suited to 35 mm coverglass bottom dishes. These are available from MatTek, BD and others. Specify No. 1.5 coverglass thickness. (Note that the central wells of LabTek chambered coverglass can be viewed, but the microscope is not currently equipped with the Pecon LabTek heating insert.
  • The microscope takes approximately 1 hour to stabilize at 37° and an equal time to return to room temperature. (Although the heating insert comes to temperature quickly and observations can begin within a few minutes, time lapse imaging requires the microscope stand reach thermal equilibrium for stable focus. In order minimize downtime as the scope moves between temperatures, reservations for 37° operation have priority for Thursdays. If not reserved for 37° use by Thursday morning, other reservations are welcome.
  • A special, non-billed ‘Warmup’ login is used to bring the scope up to temperature.
  • CO2 should be turned on and off using the top valve on the tank, not the regulator. CO2 users are responsible for determining that there is sufficient CO2 available for their experiment. Notify Dr. Ready or Dr. Xia if the tank needs replacement.

Problems during a session

  • If there is a software malfunction, you may reboot the system. To reboot the real time computer at the left of the anti-vibration table, open the front panel and depress the large button for ca. 30 seconds, until the red LED flashes. Lasers and X-cite lamp can remain on (don’t turn off Switch 1).  Any unsaved files will be lost, so save important image files as they are obtained.
  • If there is a problem not resolved by rebooting the system, IMMEDIATELY contact Dr. Ready (44975) and/or Dr. Xia (44933/44963). If neither is available, shut the scope down completely, post a note on the door alerting the next user not to use the scope and email Drs. Ready (dready@purdue.edu) and Xia (xiah@purdue.edu) regarding the problem.
  • DO NOT attempt any hardware fixes or adjustments (e.g., changing lenses) or make adjustments under the Zen Maintenance tab.

Logging out/Shutting down

  • Use the server in the outer room to check the RAT to determine if no one will use the scope for more than one hour. If so, follow the shut down procedure.  Log off your session, but do not turn the computer off, but WAIT 5 minutes for the lasers to cool before turning off main power (Switch 1).
  • If you finish more than one hour early you may contact the next user to alert them the microscope is available.
  • Clean oil from objectives. Your last (gentle!) swipe across the lens, including the metal nosepiece, should show no sign of oil.
  • Logging out signifies that the scope was operating normally at the end of your session and you’ve left the scope clean for the next user.
  • If the next user is scheduled to start within 30 min, you may log out, but leave the scope on. Turn switch #4, the HeNe laser to standby, leave switches #1, #2 and #3 on.

Data management

  • Files on the microscope computer are automatically transferred to the server in the outer room and must be accessed for transfer there. In most instances, is easiest to transfer files from the server to the net drive, but a USB drive may be used here. (But NOT on the microscope computer.)
  • There have been some issues with the automated transfer system. Please alert us to any problems so Bio IT staff can resolve them.
  • Small files (e.g. useful for reloading settings) may be kept on the microscope computer, but should be clearly labeled Do Not Delete.
  • Don’t accumulate copies of your files on the microscope computer. The size and volume of image files produced on the scope can quickly tax the capacity of the microscope computer.
  • Files on the microscope computer are unsafe and may be deleted without warning.
  • Use the microscope computer ONLY for operation of the microscope. Do not access the web. (Zen is readily compromised by viruses and malware rendering the scope inoperable and is a major production to reload, requiring a service call by Zeiss.)
  • Free image processing software, Zen Lite, is available for download from Zeiss after registration (PC only). NIH ImageJ also supports .lsm files. Keep at least copy as a .lsm file; if you convert to TIFF, you will lose image acquisition information attached to the .lsm file.

Session

  • At the beginning of each session, visually inspect the microscope platform and lenses to make sure they are clean and free of oil. (While Zen is booting up, use the touchpad to step through the lenses and confirm they are clean.) Logging in indicates you found the scope in good order. You may clean the lenses if you find them oily, but please note to Dr. Ready and Dr. Xia so we can remind users of microscope etiquette.
  • Follow the posted start up procedure.
  • Turn on only those components you will use. E.g., don’t turn on the X-cite lamp if you will not use it. (Note that an error message stating the lamp is not on will be displayed during Zen startup. This is not a concern and does not affect Zen operation.)

Fees

  • Weekdays, 8 AM-6 PM                        $30.00/hr.
  • Nights and weekends                         $15.00/hr.
  • Usage will be determined from scope log and billed monthly. Users are asked to provide an account number to the Biology business office. There is an annual $5K ceiling for charges on the scope. Time exceeding this is not billed. NOTE in order to qualify for the ceiling, charges must be against a single account. Plan accordingly!

NIH Acknowledgement

  • Funding for the LSM710 was provided by NIH NCRR Shared instrumentation Grant 1 S10 RR023734-01A1, which should be acknowledged in any publications using data collected on the scope. (Obviously important!)

Location/Keys

  •   The inverted LSM 710 is housed in Lilly G403C. The key code to the microscope room will be provided to users at the completion of training. To obtain a G403 key (and Lilly Hall key, if needed), get a key card from the Biology Office, have it signed by Dr. Ready and give it to Biology Stores who will process the request and provide a key.

 

 

 

 

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