Fiji is an open source program for image processing. This package is based on ImageJ and is very powerful. You can enhance the images, perform background correction, colorize, produce a montage, segment objects, track cells, and do a million other things. Let’s take a look:
The Enhancement Overlay will allow you to turn on / off each image in an overlay. That way you could count the number of cells that aren’t in the other 2 images.
A convenient feature of Fiji is to load a sequence of images, File>Import>Image Sequence, then just save as an AVI. You can save it uncompressed or compressed, a convenient way to send files to a collaborator. You can always reload it back into Fiji or ImageJ and view or save individual images. The compressed version is nice to send in an e-mail.
One nice Plug-in to start out with is Cell Counter. After downloading the cell_counter .jar to the Plugins folder, the added feature is available the next time you start Fiji. After starting Cell Counter, and selecting a sequence of images, you can start to count your cells. Select a counter type (a color) and just click on the cells. The program counts the clicks and annotates the image. You have the option of counting any number of different types of cells. You can add or subtract, to improve your accuracy. You can do this for each image or only one. It’s possible to get growth curves or just a final end point. Another possibility is to count divisions. The output can be saved to Excel, with coordinates, so you can plot the positions and the counts. You can also save the images showing the counts. Very nice for a quick count of cells.
A second Plug-in that is very useful is Manual Tracker. Download the Manual_Tracking.class to the Plugins folder. This Plugin lets you manually track individual cells over a series of images. Select Add track, then click through the series of images. Every time you add a track, a different color is selected. You can edit each track and change the graphics. Very convenient for quick analysis of motion. The track coordinates can be saved to Excel, for further analysis and the image sequence with tracks can be saved as an AVI for later review and presentation.
To enhance the options for time-lapse and live cell image analysis, we have begun supporting a freely available open source package called CellProfiler. It’s modular and flexible design eliminates the tedium of the many steps typically involved in image processing. Additionally, it is capable of handling hundreds of thousands of images, making use of clusters of computers when available. Ready-made algorithms are available and new methods can be added and shared. Kairos expert Harry Athanassou has contributed a phase contrast cell counting routine and has added enhancements to simplify automation for cell counting.
CellProfiler is an open source program for image processing a series of images. Here is a quick overview of how to set up and run the program. Methods are linked together to produce a Pipeline that can then be run on a series of images. There are a number of available modules for loading images, correcting an uneven background, counting cells based on intensity and outputs to Excel.
Here is a ‘Pipeline’ in CellProfiler for counting cells. This method works best with fluorescent images. The images are first corrected for background noise, then a threshold range determined. Next the objects (cells) are selected, based on a range of sizes. This method also works on visible cells if they are phase images and rounder type cells. The more elongated the less accurate the results. It’s a good idea to use a manual method, to check your results. This simple Pipeline only takes 4 minutes to process 40 visible phase images and output them to an Excel file for plotting the results.
CellProfiler can process individual images or a series of images. Here are some tips for processing certain types of images and every 10th as well as processing sub folders of images.
This version of CellProfiler includes the Module KICorrectIllumination_modelFit. Use this in place of the Module CorrectIllumination_Apply. The Module will fit a function to the background illumination and along with CorrectIllumination_Apply will flatten the background. Refer to CellProfiler_Background Correct Cell Counting To use this file, unzip and create a new folder for this version, then run the CellProfiler.exe. If you have a shortcut, redirect it to this new executable. Don’t copy and paste this executable into your current version of CellProfiler. The program uses additional files; modules, instructions etc. in the folders included with this version.
BioImageXD – FREE OPEN SOURCE SOFTWARE FOR ANALYSIS AND VISUALIZATION OF MULTIDIMENSIONAL BIOMEDICAL IMAGES
BioImageXD is another powerful bioimage processing and analysis program.
The Getting Started Manual by Pasi Kankaanpää.
An abbreviated tutorial showing steps in tracking cells using BioImageXD