I am still new to ImageJ, so hopefully others can offer alternative suggestions.
*1) Set Global Scale
1a) Open an image with a scale bar, and draw a delection line over it (click
shift to create a perfectly level line).
1b) Go to Analyze --> Set Scale. In the Set Scale window the length of the
line, in pixels, will be displayed. Type the known distance and units of
measure.
Measurements will now be shown using these settings. If the pixel:length
relationship is known from a previous measurement you may directly type this
information in the Set Scale window.
1c) Alternatively, if one already knows the pixels/unit of length, such as
px/um, then one may directly enter that value in the "Distance in Pixels."
One also needs to enter 1 as the "Known Distance", and the desired "Unit of
Length" such as micron.
1d) Check 'global' to apply this scale to other image frames.
1e) When opening new images make sure to not have the global scale disabled!
Source:
http://rsbweb.nih.gov/ij/docs/menus/analyze.html#scale*2) Using the ROI (Region of Interest) Manager
2a) Open the ROI Manager: Analyze --> Tools --> ROI Manager
2b) Make a selection (explained below) and click "add" on the ROI Manager or
use the keyboard shortcut "t" to save the selection to the ROI Manager.
2c) To update a selection (if you made a mistake), simply select the
correct ROI in your image and then highlight the correct ROI you want to
update in the ROI Manger and select "update"
2d) To delete an ROI select it in the ROI Manager and select "delete".
2e) Select "show all" to view all the selections, select "show all"
again to hide the selections.
2f) Once all your selections are complete, click "deselect" in the ROI
Manager (to make sure no single ROI is selected in the manager such that
they are "all" selected) and select "save" and name the file with "-
Compressed - Selections" ending such as: 20090922 #1 60x - Compressed -
Selections.zip
2g) To show one's selections, simply open the corresponding image and the
ROI Manager and select "open" and navigate to the corresponding .zip file
containing the selection information.
Source:
http://rsbweb.nih.gov/ij/docs/menus/analyze.html#manager*3) Creating Selections
3a) Zoom into the selection area using the zoom tool (left click = zoom,
right click = zoom out). The zoom level is displayed in the image's title
bar. Double click the zoom icon to reset the image's zoom level.
There are multiple ways to select a region of interest, such as:
3b) Freehand Selection: Simply freehand a selection
3c) Segmented Line: Click to create a segmented line in which the
segments can be "pulled" or "pushed" to modify the selection.
3d) Freehand Line: Similar to the freehand selection tool, but works
well on more pixilated boarders.
3e) It is often helpful to view the composite image at the same time such
that you can move through the slices to clearly see cells and faint areas on
the compressed image.
Source:
http://rsbweb.nih.gov/ij/docs/tools.html*Note: Hold shift down while making your selection to form a perfectly
straight line.
*4) Creating Advanced Selections
4a) Advanced selections do not work well for these images since the
fluorescence covers many structures of non-interest and is not uniform.
See the following resources:
* Page 43 of "Image processing and analysis with ImageJ and MRI Cell Image
Analyzer.pdf" from Montpellier RIO Imaging by Volker Baecker on 13.05.2009
* Area Measurements and Particle Counting.pdf by Larry Reinking on August
30, 2001 -
http://rsbweb.nih.gov/ij/docs/pdfs/examples.pdf* Page 6 of "3D Processing and Analysis with ImageJ.pdf" by Thomas Boudier
*5) Set Measurements Properties
5a) To select what measurements will be analyze navigate to: Analyze --> Set
Measurements
5b) Uncheck all but "Feret's Diameter" and change "Decimal Places" to 2.
Source:
http://rsbweb.nih.gov/ij/docs/menus/analyze.html#setNote: Read the source link above and play with other measurement
possibilities such as bounding rectangle.
*6) Make Measurements
6a) Make sure the image and the selections are open (see step 4g above).
6b) In the ROI Manager select "Measure". Alternatively one could use Analyze
--> Measure
6c) To save the results to excel select File --> Save As or Edit --> Select
All and Copy.
6d) Perform your ratio measurements in excel.
Source:
http://rsbweb.nih.gov/ij/docs/menus/analyze.html#managerhttp://rsbweb.nih.gov/ij/docs/menus/analyze.html#measure-Kalen
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