We also briefly show how to define the mobile and immobile fractions of a fluorescence protein, using imagej and graphpad prism software. Frap measurements of egfp fluorescence in a spine from a cultured hippocampal neuron. Department of biotechnology engineering, ben gurion university of the negev, israel. Given that the plugin is looking for the barycenter of the hit spot, it can only be applied for frap experiments with a single spot.
It has proven to be a highly versatile technique, allowing for the characterization of a wide variety of phenomena. Please refer to the short tutorial contained in the archive for basic usage. We have analyzed published data on dynamics of various nuclear proteins and note that frap protocols and methods of data analysis vary between laboratories. Supports all modern cpus pentium 4 and above with sse2 captures fastest with an nvidia geforce or amd radeon graphics card. It was developed during the image processing school pilsen 2009, and updated to modern fiji. Obtaining the raw data to determine the raw frap data the total or average pixel values in the bleached roi has to be determined for each timepoint. The program has tools for stitching and alignment to create large multidimensional image stacks and offers a powerful analysis. Nevertheless, the range of flexible and powerful open source software and resources for bioimage analysis continues to grow. A very powerful java image processing program with plugins and macros. Analysis of frap data provides information on the kinetic behavior of the studied molecules, such as diffusion and macromolecular interactions sprague and mcnally, 2005. A quicktime movie being played in the qt4d player plugin within imagej. Frap can also be used to study protein dynamics outside the membrane.
A typical frap data set consists of a number of noisy images acquired before, during and after photobleaching. Fluorescence image visualization and analysis software, only for the mac not tested. Fraps is a universal windows application that can be used with games using directx or opengl graphic technology. Fluorescence recovery after photobleaching frap is a fluorescencebased biophysical tool often utilized to investigate the dynamics of molecules within live cells. Imagej is a free nih program for macs, pcs and linux. Frap analysis is a plugin that can be used through the imagej software. This technique commonly utilizes gfp fusion proteins, although it could also use red alga fusion proteins 3. It has great potential for researchers to acquire images, analyze data in batches for colocalization, frap, fret, etc. Fluorescence recovery after photobleaching frap is a fluorescence microscopy method that enables the quantification of protein movement over time in living cells.
It uses the first postbleach image as initial condition, making assumptions about the underlying initial. Fluorescence recovery after photobleaching frap and its. Advanced light microscopy facility services downloads. Frap data analysis software tools advanced fluorescence. Finally, we estimate the percentage of mobile and immobile fractions of the gfp in spines, by analyzing the imaging data using imagej and graphpad softwares.
In this imagej tutorial basic analysis of any image like length and area measurement are demonstrated both by manual and thresholding process. An opensource application for frap data robin kohze,1 cindy e. For now, i ve just been pulling roi intensity data from fiji to analyze in. This frustrates some users, but i feel that it is better to understand the memory limitations of a particular task rather than lock up the computer at some unknown point during analysis. Acquisition analysis collections color filters segmentation graphics inputoutput. Benchmarking software show how many frames per second fps you are getting in a corner of your screen. For now, ive just been pulling roi intensity data from fiji to analyze in. Adds a new menu option under the hyperstacks submenu. It also allows to determine the intensity of the rest of the cell in order to normalize the frap measurements. Analysis of a large frap data set is therefore time.
The general method is to label a specific cell component with a fluorescent molecule, image that cell, photobleach a small portion of the cell, then image the recovery of fluorescence over time. Analysis of protein and lipid dynamics using confocal. This plugin operates on a frap dataset open in imagej as a tiff stack. Digital image analysis with imagej microscope core facility. Pyfrap is a novel simulationbased analysis software that makes use of pde simulations to analyze frap experiments in 3d geometries. The frap module is not included in the base zen 2009 software and is available for purchase as an addon.
Fluorescence recovery after photobleaching frap is a common technique to assess molecular diffusion. Download sample data and unzip it in a location where you have write permissions. Easyfrapweb is compatible with all main frap data formats. Sony jumps on the pc train with its playstation now program. Allows users to analyze fluorescence recovery after photobleaching frap experiments. Comments on the content of this tutorial are welcome in the disscussion page. Fluorescence recovery after photobleaching an overview. Software for frap analysis download table researchgate.
Software facilitating the analysis of frap datasets have previously been developed. In its current form fraps performs many tasks and can best be described as. This plugin is for the analysis of frap experiments. The frap method and analysis we show here can be broadly used in neuroscience, cell biology and other studies. Fiji based on imagej open source image analysis software with large user and developer community. Once the user has loaded a good frap movie, well aligned with no drift, and has specified the roi for the frap zone and another for the control zone. Zeiss lsm, leica lcs or with other image processing software which can handle time series e. It was developed during the image processing school pilsen 2009, and.
Fraps show fps, record video game movies, screen capture. Hi, ive been using frap tests on a confocal microscope to test local. Fluorescence recovery after photobleaching frap of. In the frap calculator macro, the interval in milliseconds between images must be entered 100 is the default. The advantage of this software suite is that it is free. Firewire and gige vision camera control software windows only. In addition, many software packages originally developed to control microscopes now include powerful image analysis features e. With the lsm frap module, frap data generated using the lsm systems can be displayed and analyzed. Fluorescence recovery after photobleaching frap is a tool widely used in studies of dynamic behavior of fluorescentlytagged proteins in live cells. Frap calculator macro microscopy services laboratory msl. Imagej manages this process in a relatively transparent fashion. Automated online analysis by an imagej macro selects an eres suited for a frap experiment arrow and reports the xy coordinates back to the microscope, which centers the eres and zooms in dotted rectangle to start the frap experiment. Download table software for frap analysis from publication.
What exact plugins are you using for frap alignment and intensity versus time. Analyzing fluorescence microscopy images with imagej. In the nic we mainly use it to process images acquired by our new spim microscope, the lavision ultramicroscope ii. Currently, frap data processing and analysis are often performed using commercial software packages. To decide which diffusion model describes best a set of frap data, repeated curve fitting and data analysis are required. Obviously the intensity part is relatively simple in imagej fiji. No programming is required, but some knowledge of the basic physics of diffusion is required to use it sensibly, and to interpret the results. Fluorescence recovery after photo bleaching frap the frap technique was first used to analyze the mobility of individual lipid molecules within a cell membrane. Imagej is a cost effective and versatile tool for faculty and staff. Frap analysis has been broadly used in vivo and in vitro 12 2studies. These files are provided for use under open software license v.
Cellprofiler open source image analysis for large datasets highthroughput cellcognition open source software to analyze timelapse data on a large scale highthroughput. Here is a jython script that does the analysis of a frap movie. For the past 25 years nih image and imagej software have been pioneers as open tools for the analysis of scientific images. Adaptive fluorescence microscopy by online feedback image. Diffusion measurements inside biofilms by imagebased. Hi im looking for software to analyse frap experiments both intensity measurement and the subsequent model based curve. The user is requested to click on the prebleach and stable postrecovery parts of the corrected frap curve. The zip file contains a frap time series acquired in our lab by daniel blumenthal of fibroblast cells stained with dii a lipid analog. Koopman,1 roland brock,1 samuel schmidt1,2 abstract we introduce frapbot, a freeofcharge open source software web application written in. The original approach to calculating diffusion coefficients of a fluorescent probe from fluorescence recovery after photobleaching frap measurements assumes bleaching with a.
Im looking for software to analyse frap experiments both intensity measurement and the subsequent model based curve fitting. Dear all, what would be your tool recommendation to perform frap analysis. This tutorial is brought to you by joao firmino, knust lab, mpicbg. This analysis is sensitive and can be used to characterize the mobility of gfptagged proteins. This can be done with the most confocal operating software e. It relates how to correct for the drift of your biological samples during longterm timelapse imaging for subsequent analysis using fiji. This manual illustrates how to setup and acquire frap data on your zeiss lsm using zen 2009. Fluorescence recovery after photobleaching frap is a method of determining the kinetics of diffusion in living cells usually using fluorescence microscopy. Imagej is written in the java programming language, so limitations in java apply to imagej. This book is based primarily on the wayne rasbands fantastic imagej. This chapter discusses the potential of frap to address biochemical and functional questions about proteins in their native environment. The app allows the user to fit a model for 1d diffusion with a constant diffusion constant, d, moreorless directly to the data output by a confocal microscope. Once the user has loaded a good frap movie, well aligned with no drift, and has specified the roi for the frap.
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