############################################################################ # # LIGHT SHEET IMAGE PROCESSING # # version 1.0 - 01/03/13 # # Copyright 2013 Laboratoire Jean Perrin # Adress: CNRS / UPMC Univ Paris 06, FRE 3231, Laboratoire Jean Perrin LJP, F-75005, Paris, France # Contact: Raphaël Candelier (raphael.candelier@upmc.fr) # # This program is free software (see LICENSE). You can use it, modify it and # copy (distribute) it without any restriction. However, the following citation: # "Light sheet image processing, v1.0, Laboratoire Jean Perrin (CNRS/UPMC, France)" # should be present in each copy of this program, either it is modified or not, # and in copies of copies, recusively. # # If you plan to use this program or a modified copy in peer-reviewed # scientific journals, conferences, or in any other academic form, you are # kindly asked to include a reference to the following paper: # # Fast functional imaging of multiple brain regions in intact zebrafish larvae using Selective Plane Illumination Microscopy # Thomas Panier, Sebastian Romano, Raphaël Olive, Thomas Pietri, German Sumbre, Raphaël Candelier and Georges Debrégeas # Frontiers in Neural Circuits, March 2013. ISSN: 1662-5110 # # A set of images is provided with this software for testing only. These # images are the properties of the LJP and should not be copied, modified, # or used in any other way than for this original purpose. # ############################################################################ ---------------------------------------------------------------------------- LICENCE This program is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. The GNU General Public License can be found here: ---------------------------------------------------------------------------- PREREQUISITES This software has been designed to work under Matlab version R2012b or higher. In addition, the following Matlab toolboxes should be installed : - Image processing toolbox - Curve fitting toolbox ---------------------------------------------------------------------------- INSTRUCTIONS A reduced set of images is provided at: http://www.labos.upmc.fr/ljp/Documents/Tools/LSIP/Images.zip for test purpose. We recommended you to use these images for your first use. 0. Download the file: http://www.labos.upmc.fr/ljp/Documents/Tools/LSIP/Programs.zip and uncompress it. 1. Launch the program 'Main.m' in Matlab. If asked, add the path. A GUI window appear. 2. One need to define the directory in which the images are. Enter the full path of the image directory and press Enter. The path should end with a file separator ('\' on Windows and '/' on Linux and Mac). If the default path is correct, just press Enter. Normally, the first image appears in the GUI window. 3. The next step is to define manually a ROI. Click on the "define ROI" pushbutton and click on the image to define the vertices of a closed polygon. Once the ROI polygon is closed, you can adjust the position of the vertices by drag & drop with the mouse. When you have a satisfying ROI, double click on the image to validate. All the pixels ouside the ROI appear now in dark. 4. Before the segmentation of neurons, one needs to create an average image to reduce the noise. This can be done by clicking on the "GO" pushbutton in the "create time-averaged image" panel. Minute movements of the fish can be compensated during the computation by ticking the "correct for drift" option. The computed drift is shown (in pixel units) during the computation. This option slows down the computation time. Note: After each operation, you can save the current session in a file. Just click on the "SAVE" pushbutton to save the current data in a file called "D.mat" in the saving directory. If you close the program and start again, just click on the "LOAD DATA" pushbutton to load these data and get back in the same state. 5. Adapt the segmentation parameters (minimal and maximum area and maximal excenticity) in the "Segment image" panel, then click on the "GO" pushbutton. One segmentation is over, the number of neurons is displayed. 6. In the same panel, the button "delete region" allows you to define a polygon in which all neurons will be removed. This can be useful for neuropil regions, for instance. The button "delete/restore" allows you to delete and restore neurons by manual click on the image. Save. 7. To get the fluorescence signals, choose the quantities you want to compute in the "Fluorescence signal" panel and click on the "extract fluorescence signal" pushbutton. You may be asked to select a background region for the computation of DF/F. Save. After saving, the data will be stocked in the D.mat file in the saving directory.