Advanced Cell Classifier

Version: 1.1 (12/08/2009) (Matlab version)

System requirements
Windows and Mac OS (Unix and Linux is not yet tested) Matlab 7.5 (R2007) or newer o Image processing toolbox o Neural Network toolbox (optional for NN classifier, recommended) o Matlab report generator (optional for report generation)

Installation
1. Copy necessary files a. Copy the installation file (accx_x.zip) content to an arbitrary directory ($ACC_PATH). b. Add to the $MATLAB\toolbox\local\startup.m file the following line: javaclasspath('$ACC_PATH\weka.jar'); (Windows) javaclasspath('$ACC_PATH/weka.jar'); (MAC) eg.: javaclasspath('E:\projects\software\ACC1_1\weka.jar); c. Check the success of the installation: Start MatLab -> Go to $ACC_PATH and type: ACC_main_GUI (ACC main window should appear) 2. Create phenotype icons and description a. ACC supports major and sub-phenotype classification. It is possible to assign for each class an icon. These icons are bmp files with the size of 60x60 pixels for major classes and 40x40 pixels for subclasses. b. ACC has the following icon name convention: major types has the name of typexx.bmp, xx are increasing numbers from 01 and subtypes named as typexx_yy.bmp, yy starts also from 01. Icons should located in the $ACC_PATH\img folder. Example:

c. It is possible to give a descriptive name for the phenotypes. Create a file called type.txt into to same $ACC_PATH\img folder (see the above image) and write the phenotype names such that each of them are in a new line. 3. For libSVM users: for Win32 the installation folder contains the required files, for Mac, Linux, and Win64 download the source files and create mex files as described. (http://www.csie.ntu.edu.tw/~cjlin/libsvm/).

Creating new project

To create a new project click File->New project or the new project icon . Data structure should follow the SSS format (described below). The Load data set window will appear (see figure). The following settings required: - The main path of the data set, here type or set the folder which contains your plates.

After defining the path, ACC will show available plates, you can select those you are interested in. (Hint: use CTRL and SHIFT buttons for individual and multiple selections). Define subfolders which contain your segmented images, metadata and original images. Define plate type (96 and 384) are available.

After your settings are done click OK button and the first image of the first plate should appear on the main window and the Image selector window should show the selected plates and their contents.

Setting up the main view

Different visualization opportunities are possible in ACC. switch on and off cell numbering, this has also effect on showing quick classification results (Settings -> Show cell numbers) switching on and off different color channels Show images in color or grayscale show the segmentation result (overlaid images) or the original images ACC by default stretches images to the optimal view, but it is possible to manually set the lower and upper bounds of the intensities. For this, an image histogram is shown at the bottom right corner. First the Stretch image checkbox needs to be unchecked and than clicking with the left mouse button the lower and with the right button the upper intensity limit can be set (white and yellow horizontal lines).

Change, modify, extend existing project

The following project modifications are available: - Add/Remove plates: Project -> Add plate(s)/Remove plate(s)

Import trained data: it is possible to copy already labeled data from existing project. Project -> Import trained data Change project folder: it is important that a project is linked to the raw (processed) data statically and if the location of the data changes ACC cannot find it, therefore it is possible to change the main folder. Project -> Change data folder

Training
To label cells, ACC offers a variety of opportunities. Three different cell selection methods exist: 1. Free-hand selection: By clicking any arbitrary cell on the loaded image the current cell will be highlighted 2. Sequential training: By default ACC jumps to the next cell on the image after labeling 3. Random training: ACC randomly jumps to another cell. It chooses a random number of cells (with an average 5 cells/image) and jumps to a random image in a random well. This method is perfect to get an objective human independent trained set. Choosing class labels for cells is done by clicking the phenotype buttons on the bottom part of the window. For subtypes one first choose the major type and than the subtype.

Teaching the classifier

The current version of ACC has ~20 different classifiers. To choose and teach a method click or Classification -> Training properties

Choose the desired classifier and click the Train button.

Prediction
After a classifier is trained, it is possible to start the real analysis. Two types of predictions are possible in ACC: Prediction on the current image: This method labels each individual cell on the current image by its predicted label. The labels are numbers representing the phenotypes. Switching on/off the cell numbers also applies when they are labeled. Prediction of selected plates or the entire data set. (see the picture below) It is possible to predict bigger set of data. Choose the phonotype(s) you are interested in as hit and as normalization and select the desired plates. ACC classifies each cell on the selected folder and creates the following statistics: o An html report with the details of the experiment and hit maps

o Into each metadata folder CSV and MatLab files with the cell number and desired analysis

Standard Screening Structure (SSS) format specification

General description - For practical reasons, since all microscope providers use different file formats we use a data structure that can be easily extracted from the most popular screening microscopes output. This structure is:
Experiment\ PlateName_1\ AnalysisName_1\ AnalysisFiles AnalysisName_i\ AnalysisFiles AcquiredImagesFiles PlateName_i\ AnalysisName_1\ AnalysisFiles AnalysisName_i\ AnalysisFiles AcquiredImagesFiles

Names ending with \ are folders. Experiment folder can be freely chosen. PlateName_i is optional as well but to have unique file structure the image and analysis result names are derived from this folder name. AcquiredImagesFiles are the images with names following the rule: Platename_%Row%Column_%Num*. Where %Row is a digit from A to H or from A to P in case of 96 or 384 wells. %Column is a number from 01 to 12 or 24. Be careful with digits, 1 digit numbers are completed with 0. %Num identifies the individual images in the plate, the order how the montage and detailed views are created depends on the alphabetical order of %Num. * is the remaining part of the name, this should contain the channel information and the file extension. An example file name: 2008-06-28_001_B01_00_dapi.tif where 2008-06-28-001 is the plate name, B is the row 01 is the column of the well. 00 is the number and dapi is the channel. In this case the folder name of this image should be 2008-06-28-001. AnalysisName_i is an arbitrary folder name. AnalysisFiles are files containing any kind of metadata or analyzed images. These files can be attached to the detailed view of wells. The filenames follows a similar naming as the acquired

images: Platename_%Row%Column*, every file fitting this scheme will be added to the detailed view of the well identified by %Row and %Column. ACC specific settings The following files are required: - One of the analysis folders contains the segmented images in jpg format. - The second folder contains the metadata, these are text files. Each line belongs to one cell describing its features, but the first two numbers are the location of the cell on the image. - The third folder contains the original images in jpg format.