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Invasion assay

42 bytes added, 09:08, 15 December 2014
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SPIM allows for three-dimensional imaging of thick specimens. Among other approaches this can be exploited to analyse invasion of different cell types deep into tissues. Due to their potential role in cancer treatment the invasion of mesenchymal stem cells (MSC) into tumors is a medically relevant issue. Therefore, we  We developed an ''in-vitro'' screening tool to analyse the invasion potential of MSC under different conditions . Therefore, tumor model spheroids with multicellular spheroids as tumor modelsinvaded MSC are imaged and their invasion depths are quantified. We present here the computational approach to cope with the amount of data our high-throughput invasion assay produces.
=Image processing=
<source
lang="bash">
# Commands toBasic commands:/* # change directory: */
cd /path/to/folder
/* # show content of current directory: */
ls
/* # create subfolders: */
mkdir converter fuse register
/* # delete all data within current directory: */
rm *
</source>
A prerequisite for effectively using these scripts is the following data organisation:
Parent folder (e.g. one folder for each measuring day) containing several subfolders each containing original spim data of only one measurement (e.g. control1, condition1, …). These subfolders should be all on the same level (not further nested). In order not to mix original data with computed ones one has to start a shell script first that creates for each measurement a subfolder named „convert” (see below). This folder is located below the folder of the original spim data.
<source
lang="bash">
/* #Example commands: */
./1generate_convert_dir /path/to/data
--- macros
| --- converter
| --- ...1.js | --- ...2.js
| --- …
<source
lang="bash">
/* #Example command:*/
./3execute_convert_macro_js
</source>
2convert_TIFFF_TIF_js
Shell script generates fiji macros doing the pre-processing of spim data ([http://openspim.org/Pre-processing)pre-processing] of spim data. Specify complete path to data and an integer for each data directory as second parameter (cf. Usage). These macros are stored in the folder „../macros/converter“.
3execute_convert_macro_js
4generate_register_macros
Shell script that generates fiji macros doing registration of spim data ([http://openspim.org/Registration)registration] of spim data. Before using this script you have to adopt the registration parameters to the needs of the respective experiment. Specify complete path to data and an integer for each data directory as second parameter (cf. Usage). These macros are stored in the folder „./macros/register“.
5execute_register_macros
6generate_fusion_macros
Shell script that generates fiji macros doing fusion of spim data ([http://openspim.org/Fusion)fusion] of spim data. Before using this script you have to adopt the fusion parameters to the needs of the respective experiment. Specify complete path to data and an integer for each data directory as second parameter (cf. Usage). These macros are stored in the folder „../macros/fuse“.
7execute_fuse_macros
InvasionAnalysis_macro
Fiji macros macro doing analysis of cell invasion depths. Both spheroids and invaded cells are segmented and the shortest distance of each invaded cell to the surface of the spheroid is displayed. Analysis is based on the 3D object counter and 3D manager fiji plugins. Before using this script you have to adopt the threshold parameters for segmentation to the needs of the respective experiment.
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