Normal Procedures for AMT CCD Imaging on Morgagni

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The AMT (Advanced Microscopy Techiques Corp., Danvers, MA) CCD system has been installed on FEI Morgagni 268 electron microscope since March 03, 2004. It is a bottom mounted CCD camera. Here is some basic information about three major parts of this system:

The camera: It is a nominal 1k X 1k camera (the display size of the installed chip is set to 1280x1024, actual chip size is a little bigger than the above dimensions). The pixel size @ 1280x1024 is 24 microns. It is a lens-coupled optical system with a very thin layer of phosphor on the top facing electrons. This is a 12-bit camera.

The Controller: The controller box has two cables: one is the controlling cable that connects to the camera, the other cable is a firewire (IEEE 1394) cable that transfers data to the computer.

The computer: An ATX PC computer with a P4 2.40 GHz processor and a CD burner. Installed software includes the AMT software and a photo editing software, PhotoImpact. It is on rose2 network and has the latest patches for Microsoft Windows 2000 Professional operating system.

This document gives you a brief procedures of everyday operation of this AMT CCD camera. For details, please refer to the series of the original documents that comes with the equipment.

You can also get pdf version of this document.


Table of Contents
1 Get Morgagni Ready
2 Turn On the AMT CCD camera
3 Background Collection
4 Set Up a "Case" for saving Images
5 "Click for Live Image" to View Specimen
6 "Click For Final Image" to acquire a Quality, Full Resolution Image
7 Saving Images
8 Manage Your Image Files - Locally and Remotely
9 Convert to MRC format
10 Closing Up
11 Pixelsize and Magnification Calibration

1 Get Morgagni Ready

Before collecting any CCD images, it is needs to have microscope ready for the operation.


2 Turn On the AMT CCD camera

Usually, the CCD controller box is left ON after use, and sometimes is also computer.

Note: In case the you have to switch ON the controller, wait for about 10 seconds before starting the software.


3 Background Collection

You can skip this step if just want a quick checking without too much worry about the quality of your images. In that case, you are using the latest acquired backgroud.

Background correction is important to compensates for the phosphor inhomogeneites and optical imperfection. Background images need to be collected each day or whenever there is a change in the gun high tension.


4 Set Up a "Case" for saving Images

After a "case" is set up, the images saved will have filename reflecting the case name and of incrementing if multiple images are taken.


5 "Click for Live Image" to View Specimen


6 "Click For Final Image" to acquire a Quality, Full Resolution Image


7 Saving Images

With AMT software, there are four image format options: 8-bit TIFF, 16-bit TIFF, Lossless JEPG (100% compress) and Normal JPEG (50% compress).

There are two ways to save an image:

Note: In either of the two ways above, one has choice to save the TIFF image with or without captions. If "Save with Captions" is clicked, the information is not only stored in the image Tag, but also made into an extra TIFF image which is appended on the real electron image to form final image with captions.


8 Manage Your Image Files - Locally and Remotely

There are ways to manage your data saved on AMT computer - locally or remotely. You can transfer them away from the AMT computer using SSH/SFTP. Or you can get them remotely from any other computer on compus after "mounting" C:\Images. Even you can burn a CD on AMT computer.

Note: Any user data on AMT CCD computer should be transfered/saved somewhere else. The local hard drive is not for long term storage purpose. Any data left on the local drive will eventually be deleted.


8.1 Locally

From AMT computer, you can either transfer your data to any of the UNIX or Unix-like machine on which you have an account or burn it to a CD.

In case you did not know, users who have Brandeis ID can always remotely access to one of several Linux computers. Talk to ITS people for details.

  • Using SSH: double click the "SSH" or "SFTP" icons on the desktop, and then follow the instructions.

  • Using CD recording software: double click the "B's Record" icon on the desktop and follow the instructions. Most likely you will use "data" format to burn a CD. The default location for AMT-CCD image will be at C:\images.

Note: Despite one might think to use USB jump drive to transfer your data, as the easiest way, it is not recommended. USB jump drive could contain viruses to affect the CCD computer.


8.2 Remotely

All the data should be directly saved in local computer's hard drive. It is recommended to put them into "C:\Images". For convenience, this folder is now configured as shared and can be accessed from other computers remotely. Thanks to Lynn for setting this up. This folder can now be mounted on various platforms as the detailed below.

  • LINUX

    To start up the ftp-like smbclient and connect to the share from the command line, enter:

     % smbclient //amt.rose2.brandeis.edu/Images -W EMLAB -U emlab
    

    The user will be prompted for the password for the emlab account. Enter help <return> for information on using smbclient.

  • WINDOWS

    To mount the share on windows, Start->Run, then enter the following in the "Open:" field:

    \\amt.rose2.brandeis.edu\Images 
    

    When prompted, the user should authenticate using User name emlab and the password for the emlab account. A window into the share should open on the desktop.

    Note: On Windows 7 computer, "RUN" might have not been configured available as default. However, you can directly put the line

    \\amt.rose2.brandeis.edu\Images 
    

    into "Search":field, it should also work.

  • MAC OS X

    Use CMD-K to pull up the Connect to Server dialog box, then enter the following in the Server Address field:

    smb://amt.rose2.brandeis.edu/Images  
    

    The user will be prompted for authentication information and should enter:

    Workgroup or Domain:  EMLAB [if not already filled in]
    Name:  emlab
    Password:  password for emlab account 
    

    An icon for the share should appear on the desktop.


9 Convert to MRC format

If the image saved from AMT interface is in TIFF format, it can then be directly opened by Photoshop, IMOD etc.. In case you really want to convert it to MRC format, the program tif2mrc from IMOD package can do so.

	
		% tif2mrc <image.tif> <image.mrc>
		

10 Closing Up


11 Pixelsize and Magnification Calibration

The default factory magnification calibration might not be good enough if you need a accurate value for size measurements. Finally, I did a mag and pixel size calibration for each mag. The specimen used for this calibration are 463nm grating replica grid for mag of 8900x and lower, and homemade Tropomysin para-crystals in negative stain for 8900x and higher.

Mag_ScreenDown(x) Mag_ScreenUp(x) Mag_OnCCD(x) Pixelsize@1280x1024(nm)
880 1000 1,188.47 21.19
1100 1300 1,519.85 15.79
1400 1600 2,029.25 11.83
1800 2000 2,400.90 10.00
2200 2500 3,229.22 7.43
2800 3200 3,796.33 6.32
3500 4000 4,896.05 4.90
4400 5000 5,982.03 4.01
5600 6300 9,940.55 2.41
7100 8000 10,971.83 2.19
8900 10000 12,895.61 1.87
11000 13000 16,204.35 1.48
14000 16000 20,102.28 1.19
18000 20000 25,108.86 0.956
22000 25000 30,571.14 0.785
28000 32000 40,205.91 0.597
36000 40000 51,524.05 0.466
44000 50000 61,768.10 0.389
56000 63000 83,240.51 0.288
71000 80000 100,392.04 0.239
89000 100000 124,046.58 0.194
110000 125000 154,389.87 0.156
140000 160000 197,574.68 0.122
180000 200000 232,283.54 0.103