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prolifToolbox


Toolbox interface

interface

The proliferation Toolbox can be used to (I) detect nuclei within images, (II) determine the proliferation/cell cycle length within a tissue or cell type, and (III) to visualize the results.


Detect nuclei

Detect nuclei

To detect the nuclei one should choose a label-file, nuclei images and proliferation images. Datasets should be aligned. Greyscale tiffs (8,12 or 16 bit), either as multi-page or as tiffs series are supported. Note when using tiff series the last three characters of the filename (excluding ".tif"), are used as file number. When using a label file which is generated using Amira pixel sizes are automatically determined.

The label file is a segmented set of images where each tissue or region of interest has its own unique pixel value. Background is zero. Only nuclei within the labels will be detected.

The nuclei images are the images where the nuclei of interest (all nuclei or a subpopulation of nuclei) are stained. The proliferation images are images wherein nuclei are stained during a phase within the cell cycle. It is possible to use 2 sets of proliferation images, using different exposure times. When using one proliferation dataset it is possible to compute the fraction of nuclei labelled. On the other hand when using two sets, the cell cycle time can be computed. Only nuclei stained in the nuclei images will be processed and checked whether they are positively stained. After image selection a result directory can be chosen. Default the resulting files will be placed in the sub-directory "celldetection" of the directory where the label file is placed. Optionally control images can be created; In the control images detected nuclei are outlined in red and tissue blue. Binary files (_bin.tif) are created in which the position of the nuclei is stored.


Advanced settings

advanced settings

In the advanced settings one can change the different settings. First a threshold can be set for intensity nuclei should be above background. The second setting is how many pixels a nucleus should consist of before considered a nucleus. The third option is filter size which is used to determine a local maxima image on which the nucleus threshold is based on. The fourth option is the number of standard deviations a nucleus should be above background to be considered positively stained for a proliferation marker.


Compute proliferation

Choose proliferation images

The necessary input fields are already filled in when in the same session nuclei are detected. Else the label file and binarized nuclei and proliferation images needs to selected. The second proliferation file is optional and needed for the computation of cell cycle lengths.

This step can produce the following files:
label file name + label +

  • _i (labelling index)
  • _i(1) (labelling index first proliferation file)
  • _i(2) (labelling index second proliferation file)
  • _e (average cell size)
  • _tg (cell cycle length)
  • _ts (length of s-phase)

  • excel file of summary


Paramaters labelling index

paramaters labelling index

The size of the projection cube and sapling volume need to be filled in. The sampling volume which is used is important for the reliability of the measurements. The optimal size of the sampling volume is depend on the tissue density. A size should be choosen that at least 171 nuclei are present in a majority of sampling volumes.


Paramaters cell cycle length

paramaters cell cycle length

In addition to the parameters that are used for the labbeling index it is also neccasarry to provide the exposure time to the Thymidine analogues


Visualize

Visualization

Labelling indices and cell cycle length data can be mapped on the label images. One can chose to map on a single label or on a combination of all labels


Visualition in Amira

In Amira a surface file needs to be created (how to do this can be found in the amira documention). The computed labelling indices or cell cycle length can be open. When opening those files, a colormap will be available automatically. A surfaceview should than be added to the surface. When the white square in the surfaceview is clicked, the colorfield option should be connected to the Tiff-file with the quantitative data and the colormap to the colormap.

Visualization in Amira

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prolifToolbox (automated content)