Documentation

Introduction

This document describes the installation and use of a custom plugin written for the image analysis program ImageJ. While this plugin was originally designed for extracting quantitative information from FISH images of telomeric DNA in interphase nuclei, it has also been applied to FISH images using a pan-centromeric probe, and should be generally applicable to FISH images where the amount of target is of interest and is related either to the signal intensity or the number of signals.

Installation

  1. Install the latest version of the Java Runtime Environment (JRE) if you haven't already.
  2. Go to the Telometer downloads page and download the installable (not the source code). You may also wish to download the sample images.
  3. Unzip the installable.

Starting the Telometer Plugin and Logging in

  1. In the unzipped directory, double click ImageJ.exe. If you're running ImageJ for the first time, imageJ will create a config file for you, and you may be asked to locate a JRE (javaw.exe). If successful, ImageJ will be displayed.
  2. In the ImageJ window, go to 'plugins' then 'Telometer' then 'Run Telometer'
  3. Click the login button, and then enter your username and password. If you do not have a username and password, contact the system administrator. If you're using the JHU server, the system administrator is ameeker1@jhmi.edu.Main Telometer Window

Scoring Images : Selecting a Sub Project

  1. Follow the previous step to start the plugin and login.
  2. Click the score button.
  3. Select a project
  4. Click the Ok buttonSelect Project Dialog
  5. Select a sub project. You can create a new sub-project by clicking the new button.
  6. Click the Ok button.Select Sub-Project Dialog

Scoring Images: Image Analysis

  1. Follow the steps before to select a sub-project
  2. At this point the Telometer Counter Toolbar is displayed. The Telomere Counter Toolbar is designed to walk you through the standard analysis process. In general, you will be performing some requested action(s), then pressing "Next" to continue, as instructed. The Telomere Counter Toolbar has 5 controls and a text area which displays context help.

    Telomere Counter Toolbar

    • Magnification: Select the appropriate magnification corresponding to that used when collecting the images. Selections here result in changes in the various presets (see below) to values optimized for images taken at that particular magnification. Note that these presets usually work well, but were originally selected for optimal use with our own particular microscope/camera combination. Different set-ups may require adjustments of the presets for optimal performance, even if the objective magnification is the same value.
    • Overide: Opens the Override Presets combo box: this lists various preset modes available. The current values are populated based upon the selection of the magnification value (e.g. 40X, 100X, etc.) These modes will change aspects of the program for optimal performance based on image attributes, such as the size of objects, etc. The user can change these values if required for custom optimization.
    • Reset button: press this button at any point during the analysis process to close all the images Telomere Counter has opened and to clear the list of cells selected. This is useful after finishing analysis of an image pair, to continue analyzing more images without having to restart the program.
    • Next button: when you are done with a stage of analysis press this button to bring up the instructions for and change the available options for the next step.
    • Action button: at the start of analysis this button, located just to the right of the "Next" button, is blank, the label will change for each step. This button will give you quick access to the tools you need at each stage.
    • Subtract Cy Noise checkbox: If this option is selected then during the final stage of analysis, the nuclear CY3 background noise is subtracted from each pixel of that cell’s telomeres. The background noise is the average intensity of all non-telomeric nuclear pixels in a cell. This option is only available to change at the beginning of analysis.

      Presets Combo Box

  3. Opening paired TIFF images for processing

    Click "Next" to open your images. You will be prompted with a File Open dialog for the CY3 image first and then the DAPI image. After you have selected your images the "Image Info" dialog will appear. This screen allows you to store information about the source of an image pair in one of three ways. The first option is to fill in a JHH TMA number and the tissue array coordinates of the image, the second option is to give a TissueDiagnosisID from TMAJ, and the third option is to give an outside TMA number and source. At this point you should also select if you want to subtract CY3 noise and select the appropriate preset mode from the Telomere Counter toolbar.

    Image Information Dialog

  4. Background subtraction in CY3 image Select an area of the of the CY3 image that is devoid of features and press the action button which should now be labeled "Normalize". If background subtraction is not desired then press "Next" without pressing "Normalize" and proceed to the next step. In some cases the changes in intensity may not be apparent.

    CY3 Before Normalization

    CY3 After Normalization

    In some cases the changes in intensity may not be apparent.

  5. Background subtraction in DAPI image

    Select an area of the of the DAPI image that is devoid of features and press the action button which should now be labeled "Normalize". In some cases the changes in intensity may not be apparent. If background subtraction is not desired then press "Next" without pressing "Normalize" and proceed to the next step.

    DAPI Before Normalization

    DAPI After Normalization

  6. Segmentation of FISH objects

    This stage will make a bitmask from the objects in the CY3 image.

    The first step is to run a rolling ball "background subtractor". The rolling ball radius is automatically selected from the preset mode you have chosen. It can be changed manually as well.

    "Implements a rolling-ball algorithm for the removal of smooth continuous background from a two-dimensional gel image. It rolls the ball (actually a square patch on the top of a sphere) on a low-resolution (by a factor of 'shrinkfactor' times) copy of the original image in order to increase speed with little loss in accuracy. It uses interpolation and extrapolation to blow the shrunk image to full size." --Description of the Rolling Ball algorithm from the ImageJ source code

    Rolling Ball Dialog

    CY3 Before Rolling Ball

    CY3 After Rolling Ball

    The next step is a manual thresholding. The goal of thresholding is to find a level that maximizes the size of the spots but does not introduce too much noise. A small amount of noise is OK as this will be automatically removed.

    Thresholding - Too Much Noise

    Thresholding - Too Little Noise

    Thresholding - Just Right

    After the proper threshold is determined press "Apply" in the "Threshold" dialog box to perform the actual thresholding operation. A despeckle filter will be run immediately after the thresholding operation and will clean up most of the leftover noise. This despeckle filter counts the number of white pixels that are immediate neighbors to an origin pixel. If the number of positive pixels / 8 is greater than the despeckle ratio specified by the preset mode then the pixel will be white in the resultant image. This operation is run on every pixel in the image and depending on the despeckle ratio will reduce the amount of "salt and pepper" noise in the image and will make solid positive areas slightly larger, both effects are desirable for the next stage. NOTE: if you do adjust the Threshold, you should use the same value (or number of mouse clicks) for each image you process in the same image set. Once this is finished press "Next" to move on to the next step.

  7. Selecting Nuclei for Analysis

    This stage will have the user determine, by hand, which nuclei will be analyzed.

    Draw a freehand ROI around a nucleus of interest in the DAPI image and click the "Add Cell" button on the toolbar. When outlining the nuclei, draw the outline closely to the DAPI stained nuclear outline, avoiding dark pixels outside of the nucleus as much as possible. Inclusion of too much black pixel area at this stage can impact the calculated average nuclear Cy3 noise level. A cell information dialog will appear asking to name the cell and choose the tissue, lesion, and cell types either by selecting from a drop down or by typing the name directly into the drop down. Any user entered values will appear in the proper drop down as an additional option after saving. Press the "OK" button to add this nucleus to the list of nuclei to be analyzed, or press "Cancel" to discard this nucles and choose another. Once a nucles is selected it will be circled and labeled in red with the cell name provided. Repeat this process until all of the desired nuclei have been selected. Press "Next" when finished.

    Entering First Nuclei

    Entering Second Nuclei

  8. Removing Halos and Other Artifacts.

    This optional stage will allow hand modification of the FISH objects selected.

    The program will automatically be in magnification mode. Zoom in on one of the chosen nuclei until the FISH objects are easily recognized. Often, some objects (telomeres) will have merged together, while others may display visual artifacts such as halos. Press the "Activate Draw" button to go into drawing mode, then click and drag to erase halos and to split merged objects. For images taken at high magnification, this may be necessary, as a group of merged objects creating a new large object may be greater than the "Max Spot Size" for the preset mode, and thus would not be included in the final analysis. Pressing Ctrl + z will undo the last line drawn in case of mistakes. Large areas of spurious signal (e.g. autofluorescence noise) can also be "erased" in this step, although the later MAX size exclusion filter will likely effectively exclude these objects. It should also be noted that objects smaller than the "Min Spot Size" preset will be ignored. Finally, for images captured with lower magnification, this type of editing should not be used due to the relatively small size of the objects compared to the size of the eraser – in this case careful trimming will not be possible, as attempts to cut apart objscts will result in severe signal losses. In these cases trimming should not be attempted. Press "Deactivate Draw" to stop drawing and go back to magnification mode.

    Repeat this process for each chosen nucleus and press "Next" when finished.

    Before Cutting

    After Cutting

  9. Analyzing and Saving FISH Object Information

    This stage will present you with the data derived from the chosen nuclei, and will give you the option to save this data to the database.

    The "Nuclei Data" dialog should appear after a short wait. To review the data, click on the name of a nucleus in the list on the left (leftmost column) to display data for that particular cell. Press the "Save to Server" button to save the data for all cells currently displayed to the database. After a few seconds, a message will appear in the lower left confirming that "x cell(s) saved to the database". Saving your data to the database is typically the best option, as it allows for querying later.

    Alternatively, instead of saving to the database it is possible to select the entire visible data table with Ctrl + a, and then copy it to the clipboard with Ctrl + c. The data can then be pasted into a spread sheet or tab delimited text file. Saving to file is another available option.

    When finished viewing and saving data, close the "Nuclei Data" dialog and then press either "Next" or "Reset" to analyze another image. There will be prompts to save the resultant images from the analysis, saving these is optional.

    Nuclei Data Dialog. Here, data for cell named "Lum A" is being displayed

Contact

For any questions about Telometer, please contact Dr. Alan Meeker at ameeker1@jhmi.edu