Authors: Olivier Marchal (marchal_olivier at hotmail.fr) for programmation
and No�l Converset (noel_converset at hotmail.com) for concept.
History: 14 feb 2006 : First version. Limitations: Works with 8-bit images. Source: Fret_Analyzer.jar Installation: Download Fret_Analyzer.jar to the plugins folder.
Restart ImageJ to add the "Fret Analyzer" command to the Plugins menu.
Description: This plugin's allows a supervized computation of FRET index by a pixel by pixel method.
It requires sets of images acquired according to the methodology of FRET computation by the sensitized emission of fluorescence method.
During analysis with this plugin, lot of controls were proposed to the user in order to confort her results:
* Bleed Through control : show artefacts that could lead to false FRET.
* FRET calculation min and max control: show interval of confidence of FRET index calculation.
* FRET and colocalization diagram control: allow users to associate FRET with colocalization information and so to eliminate false FRET.
Plugin overview Step 1 - Bleed Through evaluation -
This first part of the plugin allows users to evaluate Bleed Through first call Donor BT between the donor channel and the fret channel image and second call acceptor BT beetween acceptor channel and fret channel. Up to 10 controls can be compute by the user for each BT evaluation. Selecting the Show controls option allows user to supervise BT evaluation with two additional windows Regression graph and Regression Control for an informative Bleed Through control. The Regression graph inspired by Color Comparison (Bill O'Connell) plugin, allows user to appreciate regression quality and interestingly to visualize the possible presence of pixels out of the regression (#) and corresponding pixels localisation in the Regression Control image in order to visualize presence of artefacts that could lead to false FRET.
Step 2 - FRET computation -
This second part of the plugin proposes to display non-normalized FRET index images in a novel and interactive way to correlate FRET and colocalization between the two fluorophores. Selecting the FRET min and max option allows user to visualize to new other non-normalized FRET index images processed with min and max values obtained during BT evaluation. The second control derives from Colocalization_Finder (Christophe Laummonerie, Jerome Mutterer) plugin and consist in a interesting an interactive 3D diagram plot called Colocalization diagram and FRET link with a new FRET index image called Colocalized FRET index. In the diagram plot Colocalization diagram and FRET an interactive square invites the user of the plugin to select zone in which colocalization occur. This selection directly update the Colocalized FRET index image and allows user to eliminate false FRET corresponding to non-colocalized areas of donor and acceptor fluorophores.
You can download a user guide and visit us at http://recepteurs.u-strasbg.fr ...