- Installation of CASP
- Quick start of CASP
- Initial adjustments
- Opening images for analysis
- Setting thresholds
- Analysing comets
- The image view and profile view options
- Saving and exporting results to MS Excel or OpenOffice Calc
- Starting a new series
- Adding results to an existing file
- The measurements and result parameters
K. Końca, A. Lankoff, A. Banasik, H. Lisowska, T. Kuszewski, S. Góźdź, Z. Koza, A. Wojcik. A cross platform public domain PC image analysis program for the comet assay. Mutation Research 534:15-20, 2003.
Installation of CASP
Under Windows systems
Unzip the CASP.ZIP file into a choosen directory on your hard disk. Once this is done, the program does not require further installation. Splitting of the unzipped files into different directories should be avoided. The program can be moved to a different directory but please make sure to include all files. DLL files can be moved to the SYSTEM directory in windows main directory, usually it is C:\WINDOWS\SYSTEM. Run the program by clicking on the CASP.EXE file.
Under other sytems (from source package)
Unzip casp_src.zip or untar casp_src.tar.gz, casp_src.tar.bz2 files with command “unzip casp_src.zip” or “tar -zxf casp_src.tar.gz” or “tar -jxf casp_src.tar.bz2”. Next go to “./casp_src” directory and type “./make”. That is all. If there occur some problems try to edit “Makefile” or “Makefile.cygwin”. Sorry but Ican’t write “configure” scripts and you should make nessesary changes yourself.
Quick start of CASP
When CASP is installed, it is ready to use without the necessity to make any adjustments of the measurement settings. If you want to change the settings see the chapter Description of CASP/Setting thresholds.
- Choose the TIF image files which you want to anal yse by selecting the Files/Select Files menu or pressing the SELECT FILE button.
- Select the measurement area by dragging the mouse arrow anywhere in the image window with the left mouse button. A measurement frame will appear. Y ou can change its size or draw a new frame.
- Press the START button to freeze the size of meas urement frame and disable the adjust button. This step is important, because you can not store any resu lts unless the START button is activated.
- Move the measurement frame onto a comet image and press the ASSAY button. The measurement results will appear in the Profiles window.
- Press the STORE button to store the measurem ent result in a temporary result spreadsheet.
- Move the frame onto a new comet or pr ess the NEXT IMAGE to load a new image.
- Measure the image by pressing the ASSAY button and st ore the results by pressing the STORE button.
- Continue with next images.
- Save the results by choosing File /Save results from the File menu.
- Export the results as a txt file by choosi ng the File/Export Results from the File menu.
- For statistical analysis import the results into a spreadsheet software such as Open/LibreOffice or Excel.
IMPORTANT: The description of the shortcut buttons w ill appear when you hold the mouse arrow above the button. The buttons are also described in the Assay menu.
CASP starts with the image window and profile window opened (see screenshot). Under a 600×800 resolution the program will fill up the whole screen. When you work with other resolutions, the sizes of windows can be adjusted by dragging the window borders with the left mouse button.
Opening images for analysis
Images can be opened by clicking on the MARK button or through the File/Mark Files menu (short key = CTRL F). The Mark Image Files window opens. If all files in a directory should be analysed mark them by holding the Shift button and clicking on the first and last file name. Selected images can be marked by holding the CTRL button and clicking on selected file names. After clicking on the OK button the first image is loaded into the image window. The next image is loaded by clicking on the NEXT button. Press the PREV button to go back one image. File names and numbers appear on the window bar. When you have opened first or last image and you try open previous or next image then you will see warning that there is already opened first or last image.
Before analysing comet images the sensitivity thresholds should be set. They will influence the measurements. Open the Setup window by clicking on the ADJUST button or the Option/Adjust menu (short key = CTRL J). The following parameters can be adjusted:
- Head center threshold (HCT): regulates the detection of the head center. Reducing the threshold will move the head center towards the tail. Optimal value assessed by us = 0.8. HCT determines points to detection of the head center which is the center of gravity of found points. Value 0.8 means that all points of intensity greater then 80% of maximal intensity will be used to calculate the head center. Tail threshold (TT): reducing the threshold will reduce the tail size. Optimal value assessed by us: 0.05. Value 0 gives the background intensity and 1 gives maximum intensity.
- Head threshold (HT): reducing the threshold will reduce the head size. Optimal values assessed by us: 0.05. Value 0 gives the background intensity and 1 gives maximum intensity.
- Comet thresholds (CT): combined head and tail thresholds. This can be activated if both thresholds have the same value. Reducing it will reduce both the head and tail size. Optimal value assessed by us = 0.05. Value 0 gives the background intensity and 1 gives maximum intensity.
Thresholds TT, HT and CT are not used in the same time. If you have chacked the Use Comet Threshold checkbox, CASP will use only CT as the tail thershold and the head threshold. Otherwise TT and HT will be used to determine the tail and the head and CT will not be used.
- Profiles: This option allows to chose between two methods of image analysis: profile 1 and profile 2. Differences between these two profiles applay only to detection of the tail.
- Profile 1 is based on the method of percolation. This means that only those pixels are considered as belonging to the tail which form an uninterrupted “carpet” of pixels touching the head region. In other words this is the solid area and there is a connection between any two points in the tail. This process eliminates pixel debris not belonging to the tail. In addition, only pixels right of the head center are considered. According to us, this is the optimal profile setting.
- In profile 2, all pixels which are outside the head region and are located between the head center and end of the tail and are enough intensive are identified as belonging to the tail.
By pressing the Set default button the settings will be saved as default. If different adjustments are used for measurements of different experiments it may be convenient to save the adjustments in a setup file which can then be loaded. Saving is performed through the menu Options/Save settings. The default file extension is *.csp. The settings can be loaded through the menu Options/Load settings.
Once the adjustments are set, the program is ready to start analysing comets. Draw the measurement frame by dragging the mouse arrow anywhere in the image window with the left mouse button. The size of the frame can be modified by pulling the edges or border lines. The top window of the frame is used to measure the background intensity (figure 1). The position of the background frame can be flipped by clicking on the Bkg button or through the menu Options/Move background area (short key = CTRL W). When the frame size has been adjusted click the START function button. It changes into STOP button. Pressing START freezes the frame size, but not the option to flip the background frame. If the frame size needs readjustment, click on the STOP button (it will change to STOP). Pressing START also enables the option to store the measurement result (important: see Saving and exporting results for further details!). The measurement frame can be moved around the image window and positioned on a comet. The background frame can be flipped if several comets lie next to each other on the image. To analyse the comet press the ASSAY button or enter the menu Assay/Assay (short key = CTRL A). The STORE button lights up. Pressing it will save the result in the spreadsheet (see Initial adjustments for visualizing the result spreadsheet). Now the frame can be moved onto another comet on the same image or the next image can be loaded by pressing the NEXT button.
The image view and profile view options
Right of the profile window are two fields with check boxes (figure 1): the upper field sets the options of the profile window, the lower field sets the options of the comet image window. Checking the appropriate box in the profile window options turns the individual profiles on/off. In the default setting all profiles are turned on. By checking the boxes of the comet image window the corresponding features can be turned on/off. In the default setting the head contour, head center and tail length are turned on. Checking the Tail and Head boxes activates “painting” of the corresponding comet features with false colors. Both fields can be hidden by clicking on the shaded bar in the upper part of the top field.
Saving and exporting results to Excel or OpenOffice Calc
When CASP is started it automatically opens a first non existing file named _casp0.res or _casp1.res (or _casp2.res and so on) in which the results are temporarily saved. Each start of program creats its own temporary file named as above. When the analysis of images is finished you should save the results under a different file name beacause in one session of CASP you can make few different series (see Starting a new series) of measurements. CASP will use one temporary file to save all resutls of all series but you need to save each series in different file. To save the results choose File/Save results (short key = CTRL V). The results are saved in a special CASP format. Therefore, before importing results into a spreadsheet calculation program, they should be saved in form of a text file. To export the results into a text file choose File/Export Results (short key = CTRL P). A special note on the START/STOP button. As long as the START button is up, the thresholds and frame size can be adjusted. However, the results of measurements can not be stored (and in consequence of it saved). The idea is to avoid accidental changes in adjustments during the analysis of an experiment. Therefore, saving of results is only possible when the START button is pressed and the STOP button shows up. Consequently, when the STOP button is up and results can be saved – adjustments can not be modified.
Download step by step manual how to export results and import it to MS Excell or OpenOffice Calc.
Starting a new series
If you want to start analysing a new set of images and save the results in a new file, choose the command File/New series (short key CTRL N). This command will clear the image window screen of previously chosen images and the spreadsheet of previously obtained results.
Adding results to an existing file
You cannot simply add an existing results to an existing result file. For that purpose you should first load results (File/Load Results or CTRL-L) and then you can make new measurements and add results of it to memory by pressing Store button or CTRL-Z. Now you can save all of it to a one file (File/Save Results or CTRL-V) as one series.
The measurements and result parameters
The table contains explanations of the result parameters and modes of measurement.
|Name of parameter||Explanation|
|Name||Name of image file|
|HeadArea||Area of the comet head in pixels (sum of pixels in the head)|
|TailArea||Area of the comet tail in pixels (sum of pixels in the head)|
|HeadDNA||Amount of DNA in the comet head (sum of intensities of pixels in the head)|
|TailDNA||Amount of DNA in the comet tail (sum of intensities of pixels in the tail)|
|HeadDNA%||Percent of DNA in the comet head|
|TailDNA%||Percent of DNA in the comet tail|
|HeadRadius||Radius of the comet head (in pixels)|
|TailLength||Length of the comet tail measured from right border of head area to end of tail (in pixels)|
|CometLength||Length of the entire comet from left border of head area to end of tail (in pixels)|
|HeadMeanX||Center of gravity of DNA in the head (x coordinate)|
|TailMeanX||Center of gravity of DNA in the tail (x coordinate)|
|TailMoment||TailDNA% x TailLength ([percent of DNA in the tail] x [tail length])|
|OliveTailMoment||TailDNA% x (TailMeanX-HeadMeanX) ( [percent of DNA in the tail] x [distance between the center of gravity of DNA in the tail and the of center of gravity of DNA in the head in x-direction] )|