Frequently Asked Questions for Brandeis CryoEM Facility

The BrandeisEM Documentation Project

This is the FAQ for The Brandeis CryoEM Facility. All entries are assumed to be relevant to Brandeis CryoEM Facility, hardware and software. Some entries might not be directly relevant to specific hardware or software of the facility, but related to electron microscopy in general.

PDF version of this document is also available.


Table of Contents
1 In General
2 Morgagni 268
3 Philips CM12
4 Tecnai F20 & Tecnai F30
5 SerialEM

Chapter 1 In General

Welcome to the Brandeis CryoEM Facility FAQ!

This document aims to cover the most frequently asked questions concerning Brandeis CryoEM Facility (and of course answer them!). Although originally intended to avoid the same old questions being asked over and over again, it can, as we hope, also server as valuable information resources, for our own users and for users of other labs, specially new users and fresh student to CryoEM community.

Every effort has been made to make this FAQ as informative as possible; if you have any suggestions as to how it may be improved, please feel free to mail them to xuchen@brandeis.edu.

1.1. What is sign-up policy?
1.2. Are outside users allowed to access your facility?
1.3. How can I sign up the instruments?
1.4. Is there a mailing list for Brandeis CryoEM?
1.5. How to log my session?
1.6. Can I log my session from Tecnai F20 and Tecnai F30 computer?
1.7. How do I transfer my data collected on Tecnai F20 and Tecnai F30?
1.8. I think something is not working right, what should I do to report it?

1.1. What is sign-up policy?

Qualified user can sign-up instrument following EM Lab Policy.

1.2. Are outside users allowed to access your facility?

In general, No. Our EM facility is not a core facility so doesn't open for public service. However, it is possible for outside user to access our facility through collaboration. If you are interested, you can send inquiry to xuchen@brandeis.edu .

1.3. How can I sign up the instruments?

Send email to xuchen@brandeis.edu to ask being added on BrandeisEM sign-up calendars. We use Google Calender for sign up purpose. If you don't have a valid Brandeis Unet ID, talk to Chen.

You can take a look at our Master Calenders.

1.4. Is there a mailing list for Brandeis CryoEM?

Yes, there is. You can find some basic information regarding the list here. This mailing list mainly serves as information exchange chennel among our users. The address is this mailing list is emuser@lists.brandeis.edu.

If you like to join the mailing list, please send an email to xuchen@brandeis.edu.

1.5. How to log my session?

We use electronic logbook for all of our electron microscopes - Tecnai F30, Tecnai F20, Philips CM12 and Morgagni 268 and some other smaller instruments as well. The program we use for this task is called ELOG. It is free and extremely configurable. It has served us very well. If you want to try it and like to have our configuration file, please feel free to talk to Chen, xuchen@brandeis.edu.

A new user to Brandeis CryoEM Facility should ask Chen for username and passcode, before you can input your own log entry. However, you don't need to login if you just want to take look at our e-logbooks.

1.6. Can I log my session from Tecnai F20 and Tecnai F30 computer?

Yes. Point the web-browser to http://192.168.1.1:8080 and log your session the normal way.

1.7. How do I transfer my data collected on Tecnai F20 and Tecnai F30?

Two ways.

  • From Tecnai F20 and Tecnai F30 computers. Using SFTP to connect to any computer that runs SSH or OpenSSH server program and transfer your data file.

  • From your computer to connect to data fileserver tf30.rose2 via SFTP and transfer your data. If you don't have an account on tf30.rose2, ask Chen for it.

1.8. I think something is not working right, what should I do to report it?

First, we want to thank you for your help, as it is important to have feedback from users when there is a problem with a system. Based on your feedback, we could take action in an efficient way.

Modern electron microscope is an extremely complex system. Many things can go wrong. Sometimes it is a piece of hardware, sometimes it is software. And there is time we are not sure what exactly causes the trouble. When you report it, it is important and more useful to be scientific. That means, you try to record the symptoms and conditions as detailed as possible, rather than jumping into your own conclusion without detailed information describing the problem. For example, when you report a problem of a CCD camera, try to record the symptom, error message, mode, exposure time, binning, which software is used, if the problem can be reproduced etc.. This will be more useful than simply saying "it doesn't work". When we report an issue or a problem to manufacture, we need to provide a detailed description and sample bad image as well if possible. Otherwise, it is not only not helping to have the problem resoled promptly, but can also hurt our credit so that our future report and complain will be more likely less weighted and even ignored.

It is very true that an experienced user at expert level might spot a problem quickly and even make correct conclusion, but including details of the problem will not hurt anything but to make situation more clearly.

It is best to use E-logbook to record all the details. E-logbook has attachment feature so you can upload your problem images to the E-logbook server easily. If a screen shot is sufficient to show it clearly, use screen snapshot rather than MRC or dm3(4) images. You can capture a screen snapshot by press Alt+PrintScreen on a Windows XP computer.

And, there is nothing wrong to send a warning email to Chen and emuser mailing list that something is not working. You are encouraged to do so.


Chapter 2 Morgagni 268

2.1. High Tension goes off and there is error message saying “Leakage! HT allowed after 1 minute”. What can I do?

2.1. High Tension goes off and there is error message saying “Leakage! HT allowed after 1 minute”. What can I do?

You can simply click on button Empty Buffer on Vacuum module. This will empty buffer tank and keep pressure in the tank low, which helps to maintain sufficient pressure difference between front and back of ODP pump. The temperature of ODP pump will increase after emptying the buffer tank.

Empty Buffer manually once a while, e.g. every 2 hours, will avoid the leakage problem.


Chapter 3 Philips CM12

3.1. How to normalize lenses on CM12?
3.2. How to reset objective lense?

3.1. How to normalize lenses on CM12?

While the large fluorescent screen is DOWN, press the Exposure button on left hand panel.

3.2. How to reset objective lense?

Press the Auto button under Focus knob on the right hand panel. This is to set the objective lens current to standard value so that the focus value is at Eucentric Focus.

Note: This is different from pressing the "reset focus" key on interface screen. That only changes relative display value, while the actual objective lens current is not modified.


Chapter 4 Tecnai F20 & Tecnai F30

4.1. I don't see beam on large screen, what can be wrong?
4.2. When I put Objective Aperture in, the beam disappears, what should I do?
4.3. Neither of two Gatan CCDs are available, what's wrong.

4.1. I don't see beam on large screen, what can be wrong?

There could be a number of causes that you don't see beam on large screen. Check the followings.

  • On Tecnai F30 make sure there is a specimen rod inserted in or the beam will be blanked for safety concerning X-ray.

  • Column valves are NOT closed.

  • Vacuum status is ready.

    H.T. and Extracting Valtage is at set points

  • Beam blanker is not ON. You can check this from CCD Camera module in Tecnai interface where the Blank should not be in Yellow.

  • If SerialEM is launched and in Low-Dose mode, then you should either un-check "blank beam when large screen is down" in Low Dose control panel or simply turn off Low Dose mode.

    Note: SerialEM uses scripting beam blanker to control beam shutter, and it is independent of beam blanker from Tecnai interface.

  • Grid bar or edge is not blocking the beam. You can change magnification to lower numbers.

  • Objective aperture is not blocking the beam. You can take it out if it is at IN positions.

  • CryoBox is not blocking the beam. Check it by first going to very low magnification like 120X or so and then moving CryoBox position mechanically.

4.2. When I put Objective Aperture in, the beam disappears, what should I do?

The Objective Aperture is blocking the beam. The correct procedure to insert and center Objective Aperture is as following.

  • Make sure there is sample at current stage location.

  • Put DOWN the large screen.

    Warning: It is very important to do this before continuing to next step, as focused diffraction beam can damage CCD or other digital cameras.

  • Switch to diffraction mode, and adjust camera length to about 1 meter.

  • Put in Objective Aperture. If the edge of the aperture is not sharp, try to adjust intensity or/and defocus until it becomes clear. Now center it.

  • Switch back to image mode.

4.3. Neither of two Gatan CCDs are available, what's wrong.

Be patient! When Gatan DigitalMicrogaph starts up, it needs time to communicate with each of the camera controllers. Allow 10 seconds or so for both Gatan cameras to complete registration with DigitalMicrogaph, before starting up TIA.


Chapter 5 SerialEM

5.1. How to setup SerialEM to run different executable or at different kV?
5.2. When I collect montage with SerialEM, I can see clear boundary between pieces in overview, what's wrong?
5.3. Why I can not change camera setup parameters for Record, the options gray out?
5.4. How to collect polygon montage maps at multiple position?
5.5. There is beam with Focus parameter in Low Dose mode, but when I do Autofocus I get no beam, why?

5.1. How to setup SerialEM to run different executable or at different kV?

UPATE: With the later version of SerialEM, setting up it for different kV or for different set of property and calibration files to consult becomes even easier. Now it works with argument in "Target" area. For example, if there a folder called "80kV" existed under C:\Program Files\SerialEM, we can simply put "/80kV" in the as argument. This way, the setting file can be shared and doesn't need to be edited.


C:\Program Files\SerialEM\Develop\SerialEM.exe /80kV

To run a different executable, e.g., develop version or release version, go through following steps.

  1. Right click on the SerialEM shortcut icon on your desktop and select "Properties".

  2. For develop version, fill in "Target" area with

    
C:\Program Files\SerialEM\Develop\SerialEM.exe
    
  3. Or for stable release version, fill in "Target" area with

    
C:\Program Files\SerialEM\SerialEM.exe
    

To run SerialEM at a different kV, say 80, do the following steps.

  1. Make sure a new folder exist, if not, make one. Here is an example.

    
Z:\xuchen\SerialEM-settings\80kV
    
  2. Right click on SerialEM shortcut, select "Property" and put the above folder path into "Start in" area.

    
Z:\xuchen\SerialEM-settings\80kV 
    
  3. Start SerialEM and exit. This will create a new setting file in the above folder.

  4. Edit the newly generated setting file SerialEMSettings.txt in above folder, and modify the "System Path" entry to reflect where the Property and Calibration files locate. In this case of 80kV, input the line.

    
SystemPath      C:\Program Files\SerialEM\80kV
    

5.2. When I collect montage with SerialEM, I can see clear boundary between pieces in overview, what's wrong?

This is due to intensity gradient across the beam. Usually, it is caused by gun tilt alignment being off. Performing gun alignment procedure should resolve this issue, you will have to re-take the gain reference file afterwards though.

5.3. Why I can not change camera setup parameters for Record, the options gray out?

You have a file opened and this file contains image(s) collected with Record parameter. For example, after acquiring low mag montage (LMM) map, the montage file is opened until you close it. The program expects new image taken with Record parameters will be appandable to the current stack file. This it is as designed, for convenience. You can either open a new file or close the current file. After that, Record parameters can be changed again.

5.4. How to collect polygon montage maps at multiple position?

Once you know, it makes perfect sense. But before that, it can be confusing and even frustrating.

This is a very common task. One finds multiple promising grid meshes and wants to collect polygon montage maps for all of them. Naturally, one would think to draw multiple polygons and then ask SerialEM to collect montage for each one of them. However, if you think about it, it makes sense to only draw one polygon and collect the same kind of polygon montage maps at each of the locations, e.g., the center of the good meshes picked, because most likely all the meshes will share the same or very similar orientation and dimensions. This is how SerialEM does it, very clever indeed.

The key point is that once a montage file is opened, it contains all the information needed to collect a montage map images, at current stage location. The only left task is to go to a location.

Lets take a look at the procedure for a single location.

  1. Define a polygon. This can be done by adding polygon from Navigator window. This is usually done at lower mag where entire mesh can be viewed, e.g. on a Low Mag Montage(LMM) map. Alternatively, you can also do it by (1) manually driving stage to each of the 4 corners of a single mesh and (2) adding current stage position and (3) define it as corner point. Then you can define the polygon from NavigatorMontage & GridsPolygon from Corners.

  2. While the polygon item is highlighted in Navigator window, NavigatorMontage & GridsSetup Polygon Montage.

  3. Respond to Montage Setup Window.

  4. Define the filename for montage stack. As soon as you click on "OK", the file is opened and the stage will move to the X, Y of the drawn polygon.

  5. However, you can but you don't have to collect on the drawn polygon location. If you move stage to any position, and then click "Start" button on Montage control panel, the montage map will be collected at this very last location.

With this in mind, collecting polygon montage at multiple location becomes very simple.

  1. Add point item on Navigator window and turn flag "A" on.

  2. Do the steps 1-4 as above for single point.

  3. NavigatorAcquire at Points ... → select Acquire Map Image as primary task and click "OK".

5.5. There is beam with Focus parameter in Low Dose mode, but when I do Autofocus I get no beam, why?

It is very likely that Objective Aperture blocks the beam. Autofocus uses tilt beam pairs. If the aperture size is too small, or if the set beam tilt angle is too large, e.g. 5 degree, then beam could get blocked when tilted. Usually, for 40 - 50 μm objective aperture, 1.6 degree tilt beam works fine.