This is a simple program for handling (FL)uorescence (im)ages of cells
which have been tagged with some kind of fluorescence probe. Its
primary function is to perform image enhancement, and to allow the
relative fluorescence intensity between images to be quickly
compared. Relative intensities are computed using a pixel
sampling method, in which the weighted average of the most intense
pixels (based on the red, green, or blue value) within a specific range.
So far, it seems to work pretty well for the images I have tried, but
if you are interested in doing something fancier with your images, you
would be better of using Photoshop, Gimp,
or one of the other professional photo editing programs.
Being a Java based program, it should be able to run on any OS that has
the latest JRE installed. You
can download the most current version of the program as a 38 kb *.jar
file
here. To run, simple save to desktop, and double click.
The source code can be downloaded here.
User manual? Unfortunately I
have not had time to write one yet, but here are a few pointers for
using the
program. You can also just email me, and I would be happy to answer any
other questions.
It will only
open images in jpg, png, or gif format, and only saves in jpg
format. Images are opened by using the "Open" button, either
individually or as a set. If images are open as a set then press
"Image List" button to select between the individual images.
Opening additional images would also add them to this list. An
"Out Of Memory" error may occur, however, if too many are loaded at
once. The list
can be cleared by pressing the "Clear List" button. Individual
images can be removed from the list using the Image List Dialog.
Background subtraction is done by first selecting a
rectangular region, then pressing "BG Color" to set it. After
that, press the "Run" next to the "Subtract Background" label.
Image enhancement is done by first setting the background color,
then
chosen a color to enhance (Red, Green, or Blue). Once a color has
been chosen, click on a point in the image where fluorescence is
clearly visible, to set the range (e.g.. 5-90). The range can
also be set manually. Now set the scale factor (any positive
number) and hit "Run". It is not necessary to first subtract the
background, as this is also done during the image enhancement
process. Save the image at this point if desired.
To calculate the Relative Intensity, set the number of pixels to
sample (10,000 by default), select the color to track, then select a
point in the image that looks close to the most intense color.
The
last step sets the range of the chosen color to average over.
This range can also be set manually. Now hit "Run" and wait for
the results to be displayed. If the "Display" option is
selected then , the pixels that were sampled will be displayed in the
images as white cross marks (these marks are not saved if the image is
saved). If no errors occurred during the calculation process,
then the error label will display "none", however, if it says "under
sampled" then not enough pixels were sampled. Either reduce the
pixels to sample, or adjust the range. The distribution can
also be viewed by pressing the "Distribution" button. A good
distribution is one in which the minimum intensity is greater than the
lower limit of the range, and the maximum is less than the upper limit
of the range.
Using "Image List" dialog allows notes about the list and images
to be entered. This list can then be saved (press "Save List"
button) in the native format of the program (*.fli), and it will
contain all the parameters used to perform image enhancement, and
intensity calculations. A link to the image is also saved, so
when the list file (*.fli) is opened, it will automatically try to load
the processed images as well, if they were saved. So make certain
the processed images are saved before saving the list. Once the
file name of the list has been entered, further use if the "Save List"
button will save any changes to this file.
Please note:
Permission to use, copy and modify this software and its documentation
for NON-COMMERCIAL
purposes is granted, without fee, provided that an acknowledgment to
the author, Nathan Stevens at casilab10.sci.ccny.cuny.edu/~nathan,
appears in all copies. Nathan Stevens makes no
representations about the suitability or fitness of the software for
any or for a particular purpose. Nathan Stevens shall not
be liable for any damages suffered as a result of using, modifying or
distributing this software or its derivatives.
Screenshot of the program running on a
Linux machine with JRE
1.5. The white crosses indicate the points that were sampled to
calculate the intensity.
