Sample Visualizer - LeonieZ/ACCEPT GitHub Wiki

After processing, the results can be visualized in the Sample Visualizer.

The Visualizer has the following components and functions:

Sample Information: In the Sample Information box all sample information retrieved from the data files accompanying the images of a sample is contained. Which information can be read and displayed here depends on the type of sample shown.

Overview Image: Next to the extracted information, an overview of all the images in the sample is shown. Below the display is a box indicating the channel that is illustrated. By clicking on the box one can alter the fluorescent channel. The overview image can be used to assess the number of cells and the quality of the sample. If you see a systematic background error in all images (half of the image is yellow, half blue) or if you see that the sample is very full, the segmentation quality might suffer and you have to be careful when analyzing the results.

Thumbnail Gallery: In the Thumbnail Gallery all objects that were evaluated are shown and the images are linked to the scatter plots on the right. The left thumbnail is an overlay of the first three fluorescent channels. Red is used for the first fluorescent channel (in this case CD45), blue for the second channel here DNA and green for the third channel here CK (cytokeratin). The white scale bar in the overlay represents 10 pixels. In this example the pixel size is 0.64 μm (retrieved from the sample information) and thus the bar has a size of 6.4 μm. The red lines in the thumbnails show the contour of the object as identified by the image analysis algorithm. One can visualize all objects by using the scroll function at the right of the thumbnails or use the spacebar on your keyboard to make jumps of five. To change the overlay from a three channel overlay to a two channel overlay deactivate the "3 Channel Overlay" box. Note that the overlay image is scaled from the minimum to the maximum value within the evaluated signal (indicated by red contour) while the other thumbnails that show only one fluorescent channel show the full intensity range and therefore often appear dimmer. If you make a right-click on the images an enlarged version of the image pops up and here the signal is always scaled. If you want to add a thumbnail to your selection, left-click on the overlay image and a blue frame appears and the corresponding point in the scatter plot is highlighted as well.

Scatter Plots: On the right are three scatter plots. One can change the displayed features of all parameters by clicking on the box next to the x or y-axis. For each channel you can select the Area, Eccentricity, Perimeter, Mean Intensity, Median Intensity, Max Intensity, Standard Deviation of the Intensity, Mass (Sum of the Intensity), Perimeter2Area (a circularity measurement) and the relative overlay with the second fluorescent channel (nucleus). The dots in the scatter plot represent all objects identified in the sample. By clicking on the + and – buttons next to the x or y-axis one can increase or decrease the scale at which the objects are displayed. By changing the number 10 in the top right corner of the top scatter plot one can alter the size of the dots. Above the three scatter plots you see the total number of events found (in this example 9426 objects) and how many of them are currently selected.

Selection of an Event: By pushing the "Select Event" button under each scatter plot one can click on an object that turns blue and the thumbnail associated with this object appears in the thumbnail gallery and is highlighted by a blue contour drawn around the overlay thumbnail. An example in which an object is identified in the top scatter plot (position indicated with a blue arrow) is shown below. The object also appears blue in the other scatter plots and is identified by the black arrows.

Gating in the Scatterplots: Next to the "Select Event" button is the "Gate" button. Pushing this button allows one to indicate a region in the scatter plot by selection the corners of the desired region and closing the region by returning to the starting point (a blue open circle appears when reaching the starting point). The figure below shows a gate set on the scatter plot.

Once a gate is specified, all objects that fall into the specified gate are depicted in blue. In this case 1643 out of 9426 events in total are selected (see figure below). All thumbnails associated to a point in the selected region are highlighted by a blue contour around the overlay thumbnail.

Gating with a Gate Set: For the simultaneous use of more features from the measured parameters one can use the “Multiple Gates” feature. After a click on the button the display shown in the figure below shows up and one can select a new gate (“Specify Gates.”) or use an already existing gate (“Load Existing Gates.”).

Resorting of the Gallery: By clicking on the "Selected cells to top" button the thumbnail gallery is resorted so that all selected thumbnails are shown at the top of the thumbnail gallery followed by all other thumbnails. To return to the original sorting click the "Original sorting" button. If you update your selection (by deselecting or adding events), update the thumbnail gallery by clicking the "Selected cells to top" button again.

Exportation and Loading of a Selection: If you want to save a made selection, you can click on the “Export Selection button” and specify a name for your selection.

Previously stored selections or gates can be loaded by the “Load Selection” button. If multiple selections are stored for the sample that is shown, you can select which one you want to visualize. The figure below shows an example; in this case three selections (named "Prior Scores", “Manual Gates” and “Dapi_pos_CK_pos”) were stored.

Exportation of Thumbnails: In case you want to store all or only the selected thumbnails as .tiff files on your hard drive, click “Export Thumbnails”. The saved thumbnails can be found in the results folder specified in the main user interface. Note that the thumbnails are optimized for further processing with the toolbox, not for presentations or publications.