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 with Java, including MS-Windows, MACs & Linux.  Although it may not look 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 - Go google "imagej plugins desired task(s)"

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. 

		Copyright 2001-08  Dr. M. Chua, Office of Research, University of North Carolina, Chapel Hill, NC 27599
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