Advanced Tutorial

Cell Sampler

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• Cell Sampler
  • Advanced Tutorial
    • Prerequisites
    • Example Dataset
    • Opening a TIFF Movie
    • Movie Navigation and Zooming (Arrow Keys)
    • Applying Filters to Enhance Feature Contrast
    • Sampling a Moving Feature (Nudge Control)
    • Sampling a Moving Feature (Shortcut Keys)

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Advanced Tutorial

Prerequisites

• Install the Cell Sampler/ImageJ bundle (see [Installation]).
• Read the [Basic Tutorial].
• Download Example Dataset.

Example Dataset

Download Example Dataset

The example data set consists of a TIFF movie.
This movie has 172 noisy frames (512x512 px), each frame contains 8 fuzzy Regions-Of-Interest (ROI) whose intensity varies sinusoidally overtime. Each feature represents a cell and is ~6 pixels in diameter. Each frame is placed in a unique file ordered numerically by file name. An example of a frame from this example data set is shown below. Each feature in the movie performs a random-walk every other frame, moving towards the outer edge of the movie. This example data set was created with ImageJ.

A single frame from the example data set showing 8 Regions-Of-Interest.

Opening a TIFF Movie

1. Download the example dataset for this tutorial. Unzip the archive and place the images somewhere you can find them.
2. Create an directory to store output files created by the plugin.
3. Start ImageJ and from the main menu, select Plugins->Cell Sampler to display the plugin Start Window .
4. Click on the left on to open a File Dialogue window and Choose the directory containing the example dataset images (i.e. ~/test_data2).
5. The plugin will load the imaging data. Three windows will appear, the Cell Sampler Window and 2 Image windows.
6. Arrange these window on screen so that that don't obscure each other. Position the 2 image window centrally on screen as Image Windows will resize during zooming.

Movie Navigation and Zooming (Arrow Keys)

Frame navigation is keyed on the arrow keys.

1. Use the scroll bar at the bottom of the Cell Sampler Window to scroll forward and backward through the frames of the movie. Notice that the particles in the movie drift over time.
2. Return the movie back to frame one.
3. Click on the title of the Processed Image window and without clicking, move your mouse onto the movie surface.
4. Press Right Arrow to move forward through the movie frames.
5. Press Left Arrow to move backward through the movie frames.
6. When scrolling with the arrow keys, the scroll bar in the Cell Sampler Window will also move.

Zooming is also keyed on the arrow keys.

1. Click on the title of the Processed Image window and without clicking, move your mouse onto the movie surface, placing the cursor on the movie surface. The zoom is centered on the current position of the mouse cursor.
2. Press Up Arrow to zoom in.
3. Press Down Arrow to zoom out.
4. The Image Windows displayed by the plugin will resize during zooming.

Applying Filters to Enhance Feature Contrast

This plugin displays the imaging data in 2 windows, labelled Raw and Processed.
Greyscale images are displayed in the Raw window and it is from this window that pixel intensities for a time series are sampled.
Filters can be applied from the main ImageJ menu to enhance contrast.
The precise nature of the filter applied to a movie will depend on the source movie.
To avoid corrupting time series, filters are applied to the Processed window.

1. To apply a filter to the Processed window, put focus on that window by clicking on the windows title (i.e. Processed).
2. From the ImageJ main menu, select the appropriate to enhancement option.
3. From the ImageJ main menu, select Processed->Smooth to apply a low pass filter to the movie.
4. From the ImageJ main menu, select Image->Lookup Tables->Fire to apply an LUT to the image pixel intensities.

Sampling a Moving Feature (Nudge Control)

1. With the example data set open, zoom in on the 2 top left features in the movie.
2. Scroll through the image and note that these features drift during the movie.
3. Return the movie to frame 1.
4. Clicking on the Add ROI On button.
5. Click on the feature in the movie that you wish to track, a numbered ROI circle will appear on both Image windows.
6. Click on the Show Plot button to display the time series of the feature. This time series should be sinusoidally oscillating. As the feature is drifting the sampling fra,e (ROI) loses the feature and the time series contains the random background pixel intensities from the later frames of the movie.
7. Return the movie to frame 1.
8. Click on the Show Cell Nudger button to display the Nudge Control. The Nudge Control is used to re-position an ROI placed over a feature.
9. Click on the green arrows to move the yellow ROI circle up/down/left/right. The aim is to position the circle over the feature being sampled.
10. Click on the black arrows to move, the next frame and adjust the ROI position over the feature.
11. Continue tracking the features path until the end of the movie.
12. Click on the Hide Cell Nudger to close the Nudge Control.
13. Click on the Hide Plot and Show Plot buttons to re-plot the time series. It now shows a noisy sinusoidal trace as the ROI is tracking the feature movement.
14. Scroll through the movie, you will see the ROI circle move between frames.
15. Return the movie to frame 1.
16. Click on the Show Paths button and scroll through the movie. The path of the ROI up to the current frame is displayed on the Image Windows.
17. Click on the No Paths button to hide the ROI path superimposed on the movie.

Sampling a Moving Feature (Shortcut Keys)

The functions of the Nudge Control and the Cell Sampler Window are also mapped to shortcuts accessed via the keyboard. By default these shortcut keys are inactive. The text below shows how to use the shortcut keys to re-position an ROI for sampling.

1. With the example data set open, zoom in on the 2 top left features in the movie.
2. Return the movie to frame 1 and click on the Add ROI On button.
3. Click on the movie, placing a second ROI on the second feature. Click on the Add ROI Off button.
4. The second ROI is now the active ROI.
5. Click on the Show Cell Nudger button. When the Nudger Control is displayed, click on the Shorts On button. This will make the shortcuts active in the Image windows.
6. You will notice that the arrows of the Nudge Control are grayed out as the plugin is now expecting input for ROI positioning via the shortcut keys.
7. Click on the Hide Cell Nudger button to hide the Nudge Control.
8. Double click on a Image Window, switching the window focus to the movie images. If the window focus is not on the movie images, the shortcuts will not work.
9. Press the W key, this moves the active ROI up.
10. Press the S key, this moves the active ROI down.
11. Press the A key, this moves the active ROI to the left.
12. Press the D key, this moves the active ROI to the right.
13. W/S/A/D are referred as the direction shortcuts in the remainder of the document.
14. To move navigate through the frames of the video, use the Left and Right arrow keys.
15. Return the movie to frame 1.
16. Use the short direction shortcuts and the arrow keys to track the feature through the rest of the movie.
17. You will now have tracked 2 ROI features in the example data set.
18. To plot the time series of the active ROI, press P to plot the graph.
19. When plotting the graph, focus shifts to the graph window. Double click on the movie images to switch focus back to the Image Window.
20. Press H to display on the active.