Michael Hooker Microscopy Facility (MHMF.ORG)

Viewing Confocal Images with ImageJ

Introduction:

ImageJ is a public domain image processing program from the Research Services Branch of the NIH institutes of NINDS & NIMH, which runs on diverse operating systems using Java, including MS-Windows, Mac OSs & Linux.  Although it may not look too slick, it is simple, effective and handles 8, 16 & 32 bit images and there are many plugins which enhance its capabilities.

• Getting ImageJ
• Official Plugins (optional)
• Collected Plugins (optional)
• More plugins - Try google search of "imagej plugin desired task(s)"
• Memory considerations - what can be done when you get the <out of memory> message
• A simple sample plugin to convert all the tiff files in a directory to 16 bit and how to install the macro into ImageJ on your computer

Viewing & Analysis with Zeiss Confocal .lsm Stacks

ImageJ

• Installing (First time use only)
  • Down load the ImageJ setup program for your operating system
  • Run the setup executable (e.g. ij140-nojre-setup.exe)
  • Note: It may be necessary to download and install the bundle with Java
  • Double click on the setup.exe and follow the instructions
  • Note: No special plugins are required to view Zeiss .lsm files with ImageJ v 1.36 or later.
• Running

Start ImageJ

• Opening an Image 

File ---> Open    (or control-O) and choose an .lsm file (Tip: Have the Zeiss Image Browser running with the image database open.  This will help with the determination of the image file name of the image thumbnail.  Sometimes the Zeiss software will create ambiguous or confusing file names, which do not exactly match the name in the image browser   Also the calibration information can be read from the Zeiss mage Browser, which may be required later in order to calibrate ImageJ measurements.)

Hit the "Open" button or do File --> Open

Data scan will open with one window.  Channels can be selected with this scroll bar.  Section/z-position with this scroll bar.  (Note: RGB overlays are not made with the base configuration of ImageJ)

Click on scroll bar in order to se the other channel

• Measurements

Making linear measurements in the x-y plane is simple.  ImageJ v 1.37 or later will import the correct calibrations from the .lsm file.

For measuring select the ImageJ line tool

Select the desired z-section and left click and hold on the start position and drag to the end position

Distance is displayed in the main ImageJ window.

The measurement can be logged into an Excel format window and saved as an Excel file for later processing by pressing ^M (control-M). 

Multiple measurements will accumulate into this windows.

Use File ---> Save as  to put the data as an Excel file onto a disk drive.

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Note: Plugins are available to enable measurements in 3D space. 

Memory considerations: ImageJ uses a predefined block of memory on startup which it requests from Windows.  The size of this request is set in a configuration file (imagej.cfg) which can be edited directly or changed in ImageJ by doing Edit ---> Options ---> Memory & Threads.   The amount of the allocated memory granted and the amount used can be found in Help ---> About ImageJ...  If the amount used leaves insufficient memory to load images etc. then an "<out of memory message>" will appear in the log window.  The computer called \\Schott in the facility has 8 GBytes of physical RAM.

• WindowsXP, Vista & 7 (32bit) maximum physical data memory that can be used is 3.2 ~GBytes so ImageJ can only allocate 1.6 GByes (half the physical memory)
• Windows 7 (64bit) & WindowsXP (64 bit) maximum memory should be set to half the physical memory installed on the computer.  Note that we have noted odd things happen if this number is larger than half the available physical memory.  Just be aware that setting the Memory & Threads option to a large value may not provide the expected amount of memory to ImageJ.  

Making Measurments:

• Measuring DAB faction of area labeled

Macro Examples:

Installing a macro to convert all the .tif images in a specified directory into 16 bit tiffs:

• Open file containing dv-convert16bit.txt
• Select all and press ^C to copy all the lines

• Run ImageJ
• Click on Plugins --->  Macros ---> Startup Macros...
• Go to end of StartupMacros.txt file opened, add a blank line and click at end of the blank line
• Press ^V in order to paste the macro lines copied above
• Save file  (^s usually will do that)
• Exit ImageJ
• Restart ImageJ
• Just pressing the u key should start the conversion macro
• Alternatively the macro can be run from: Plugins --->  Macros ---> DV  convert to 16 bit

		Copyright 2001-2014  Dr. M. Chua, Program in Molecular Biology & Biotechnology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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