Counting single RNA molecule probes
This software is intended to assist with the enumeration of single molecule RNA probes. It operates on microscope data files, eventually outputting an annotated image showing a projection of the data with suggested cell boundaries and probe counts for those cells.
Installation notes for Mac
Ensure that you have Xcode installed, for example by running
gcc in the
terminal. Alternatively, you can use the app store.
ruby -e "$(curl -fsSL https://raw.githubusercontent.com/Homebrew/install/master/install)"
Install freetype and libtiff using Homebrew.
brew install freetype brew install libtiff
easy_install (virtualenv allows you to create
a virtual Python environment).
sudo easy_install virtualenv virtualenv env
Source the virtual environment (note the
. at the start of the line).
Install Python dependencies into the virtual environment.
pip install numpy pip install pillow pip install scipy pip install six pip install "scikit-image==0.10.1" pip install freetype-py pip install libtiff
If you are working with czi file you may need BioFormats less than version 4.4.4, e.g. 4.4.2
The Python image analysis code works on Tiff files. We therefore need to
convert the microscopy data using the BioFormats
The BioFormats tools require Java, which needs to be installed. Download the installer from the Oracle website. https://www.java.com/en/download/mac_download.jsp?locale-=en
Install the Java JDK. Download the installer form the Oracle website. http://www.oracle.com/technetwork/java/javase/downloads/jdk8-downloads-2133151.html
Install both of them.
bftools.zip file from:
Place the downloaded
bftools directory where you want it. For example:
mkdir ~/tools mv ~/Downloads/bftools ~/tools/
bftools directory to your
$PATH environment variable.
Download FISHCount software
Download the FISHcount software zip file from GitHub.
Go into the FISHCount directory (e.g.
Convert microscopy images to tiff files
Make sure that the
bftools directory is in your path.
python scripts/protoimg/unpack.py lif_input_dir tiff_output_dir
lif_input_dir is the directory with the original lif files and
tiff_output_dir is the directory to where the tiff files will be written.
Do the image analysis
Make sure that the Python virtual environment has been sourced.
Run the analysis on the image directory of interest and specify an output directory to store any generated images.
python ./scripts/count_probes.py tiff_output_dir/image_of_interest_dir output_dir
Note that the file of most interest is