Virtual Frap User Guide

Transcription

1 Virtual Frap User Guide Center for Cell Analysis and Modeling University of Connecticut Health Center

2 1 Introduction Flourescence Photobleaching Modern confocal microscopes provide the ability to perform a variety of fluorescence photobleaching and photoactivation experiments that were previously limited to biophysicists with highly specialized equipment. These experiments allow researchers to explore numerous questions about the localization and dynamic behavior of cellular constituents including Diffusive behavior and mobile fraction Flow On rates and off rates for binding interactions Although confocal microscopes have made the experimental method accessible, interpreting and analyzing the data obtained from these experiments remains a difficult undertaking due to the complexity of the cellular milieu and the nature of diffusion itself. Because the exact geometry of the cell and the bleaching experiment dramatically affect the expected time constant obtained from the experiment, there are few analytical expressions that can be easily utilized for fitting experimental data from photobleaching experiments. Thus, for each experimental protocol new equations must be derived to appropriately fit the experimental data. Furthermore, in many cases, analytical expressions are only poor approximations of the expected redistributions, and thus are inadequate to obtain accurate quantitative data. Spatial simulations provide the means to analyze the redistribution of species within any given geometry, and have been used to analyze photobleaching and photoactivation experiments. The VirtualFRAP tool was designed to take advantage of the power of the Virtual Cell modeling environment to use spatial modeling and simulation within realistic experimental geometries to extract diffusion coefficients and fractional recoveries from photobleaching experiments. In a photobleaching experiment, the basic experimental design is to perturb the original distribution of a molecular species, and then to assess three basic parameters: 1. The extent to which molecules return to the original distribution (mobile fraction, %R). 2. The time constant(s) for the return to the original distribution, from which the diffusion coefficient (D) can be obtained. 3. The extent to which flow, or directional movement, is involved in the redistribution of the molecules. Photobleaching is generally currently accomplished using a laser scanning confocal microscope, or custom widefield fluorescence systems. A basic assumption (but not necessarily an accurate assumption) is that the fluorescent version of the protein faithfully mirrors the behavior of the unlabeled protein. The basic experimental protocol is as follows: 1. Images of the cells are collected (of the initial distribution of the molecule) with a highly attenuated laser beam. This defines the initial distribution of the molecule. (The Virtual FRAP tool requires you to have at least one prebleach image.) 2. A region of the cell is selected for bleaching, generally by selecting a particular region of interest (ROI) using the software. This region is subjected to a high intensity laser pulse, which photobleaches a significant fraction of the fluorescent molecules within the region of interest. True photobleaching is an irreversible process, so once bleached the molecules do not recover the ability to fluoresce. (Note that some fluorophores can undergo other forms of light induced changes that are reversible; for example light induced transitions of GFP between fluorescent and non-fluorescent states. Proper controls need to be included in the experiment to determine if this occurs.) 3. After photobleaching, a time series is collected to determine the mechanism, time constant, and extent of redistribution of the molecules to the original state. When collecting the data, it is important to collect immediately after the bleach pulse, with a high enough time resolution (short - 2 -

3 enough time step) to ensure that the initial regions of the redistribution curve are sufficiently sampled, and long enough that the new steady-state distribution has been reached. Because bleaching during monitoring can become a problem, in many cases it may be necessary to change to a longer timestep in the middle of the time series. Virtual FRAP Virtual FRAP is designed to analyze FRAP experiments that collect all of the fluorescence associated with the cell, and where the bleach region does not vary through the Z dimension. In order to achieve this, certain experimental conditions must be met. 1. The fluorescence of the entire depth (z-dimension) of the cell must be collected in the image. This is accomplished by working with a low enough numerical aperture and, if you are using a confocal microscope, opening the pinhole aperture in the confocal system such that the full width at half maximal intensity (FWHM) of the collection system is larger than the depth of the cell. If the cell is 10 µm thick at its highest point, then the FWHM must be at least 10 µm. 2. The geometry of the bleach in Z needs to approximate a column throughout the depth of the specimen. This is controlled solely by the numerical aperture of the objective lens; it is independent of the confocal aperture. 3. In order to correct for bleaching during monitoring, you should collect the fluorescence from the entire cell during monitoring. Regions outside of the bleach area serve as the baseline for changes due to bleaching as long as we assume that changes due to redistribution are negligible. 4. Virtual FRAP requires you to provide at least 1 prebleach image. Currently, Virtual FRAP can be used to fit D and %R for either one or two diffusing components of cytosolic (soluble) proteins. It does not analyze lateral diffusion within the plasma membrane

4 2 Getting Started This Chapter covers the following: What are the basic configurations to run Virtual FRAP How to download and install Virtual FRAP 2.1 System Requirements In order to run Virtual FRAP successfully, your computer must meet the following minimum system requirements Windows 1GHz Intel/AMD CPU Microsoft Windows 2000/XP/Vista/7 1GB RAM 10GB Free Disk Space Monitor resolution 1024 by 768 pixels Macintosh 1GHz Intel CPU Mac OS X 10.x 1GB RAM 10GB Free Disk Space Monitor resolution 1024 by 768 pixels NOTE that the current version of Virtual FRAP requires Java Runtime Environment (J2SE Runtime Environment 5.0 or later). 2.2 Installation Instructions There are two ways to install and start Virtual FRAP. 1. Install Virtual Cell ( From the Virtual Cell main menu, select Tools-> launch Virtual FRAP to start the Virtual FRAP. 2. Install the Virtual FRAP as a standalone application in your operating system. Please read the instruction below. (If you have an older version of Virtual FRAP already installed, you can install the current version to the same directory and the new version will overwrite the previous version) For Windows User 1. Download the Virtual FRAP installer for Windows (VMSetup_win32.exe) from web address To check that you have downloaded the - 4 -

5 full, uncorrupted software file be sure that the size of the file you have downloaded matches the byte size shown in the directory. 2. Execute the VMSetup_win32.exe and follow the instructions. Choose one of three installation methods: typical, custom or complete. Typical installation is recommended. 3. The installer will automatically install the software files into a directory UCHC/Virtual Microscopy under the default parent directory C:\Program Files. If desired, users may specify a different parent directory during installation. The sample data is located in My Documents\VirtualMicroscopy\Sample data and the spatial simulation result is located in My Documents\VirtualMicroscopy\SimulationData. 4. Launch from windows start menu Virtual Microscopy Virtual FRAP For Macintosh User 1. Download the Virtual FRAP installer for Macintosh (VMSetup_macosx.zip) from web address To check that you have downloaded the full, uncorrupted software file be sure that the size of the file you have downloaded matches the byte size shown in the directory. 2. Extract the VMSetup_macosx.zip into a user specified directory. A directory named Virtual Microscopy will be created in the specified directory. Under Virtual Microscopy, there are Sample Data, SimulationData and application VFRAP.app folders. SimulationData directory will be created upon first spatial simulation run. 3. Launch by double clicking on VFRAP.app

6 3 Quick Tour of Virtual FRAP Virtual FRAP estimates and evaluates diffusion rates from FRAP experimental data in four steps. The four steps are intuitively guided through a wizard-based work flow: Loading FRAP experimental data. Defining regions of interest (ROI). Selecting diffusion model which can either be a single diffusing component model or two diffusing components model. Estimation of a primary diffusion rate and an optional secondary diffusion rate. 3.1 Virtual FRAP Main Interface Figure 3-1 illustrates the Virtual FRAP 1.0 main user interface. It contains the raw FRAP data, the data analysis workflow, the best results and the spatial simulation all in one frame. 1. Data Analysis Work Flow Wizard based work flow guides users through the whole process from loading raw data to getting estimation results. 2. Data Analysis Results Panel Shows the best model and the model parameter values that fits the FRAP dataset. 3. Spatial Simulation Toolbox Runs a 2D spatial simulation based on the FRAP image with the estimated parameters and shows the simulation results together with the original data. 4. FRAP Image Viewer Displays the FRAP image based on the time series Figure 3-1 The Virtual Frap User Interface - 6 -

7 3.2 Loading FRAP Experimental Data The first step of FRAP data analysis is to load raw experimental data. The data can be loaded either from a single file or a file series. It is displayed in the image viewer after loading successfully. Loading data from a single file Click on the Load Frap Data button, a "Load Frap Data" wizard is popped up. In the wizard page, choose "From a single file". In the next page, click on the open button to start an open file dialog to load data from a single file. Choose the file path and select the appropriate format. FRAP data can be loaded from different sources. Figure 3-2 shows how to load a single file from "Load Frap Data" wizard and Figure 3-3 shows the open file dialog. Virtual FRAP is able to load the following file types: 1. Images - More than 50 image file types are supported in Virtual Frap by using Bio-Formats library (detailed information about supported image file types can be found in Zeiss lsm and TIFF image formats are both available in Files of Type. To load image types other than.lsm and.tif, users need to choose All Files as file type. 2. Virtual Cell Simulation Log files - VCell simulation result data are retrievable from a.log file. By opening the VCell log file and selecting a specific variable, the spatial results will be loaded as initial FRAP data. To see the log files, users need to choose All Files as file type. Figure 3-2 Loading a Single File in "Load FRAP Data" Wizard - 7 -

8 Loading data from file series Figure 3-3 Opening a Single File Dialog in Virtual FRAP Click on the Load Frap Data button to open the "Load Frap Data" wizard. On the first wizard page choose "From Multiple File Series ". In the next page, a file series input dialog is displayed to load file series (e.g. a time series of FRAP experiment images). Users have to specify the time interval between images for the time series data. The Choose Files button will lead users to an open file dialog which allows users to select multiple files. The file types are the same image file types supported by Bio-Formats as mentioned above. Figure 3-4 shows how to load multiple files from the "Load Frap Data" wizard and Figure 3-5 shows the dialog to open multiple files

9 Figure 3-4 Loading Multiple Files in "Load FRAP Data" Wizard Figure 3-5 Opening Multiple Files Dialog in Virtual FRAP - 9 -

10 Adjusting FRAP image data In the Virtual FRAP image viewer, there are buttons that provide basic image processing functions, which enable users to have a detailed look at the data. The functions are described below. Zoom in Clicking on the zoom in button Zoom out Clicking on the zoom out button will make the image one size bigger with each click. will make the image one size smaller with each click. Increase contrast Clicking on the increase contrast button higher with each click. Reduce contrast Clicking on the reduce contrast button with each click. will make the image contrast one unit will make the image contrast one unit lower 3.3 Defining Regions of Interest In Virtual FRAP, spatial data are summarized under regions of interest in order to speed up the parameter estimation process. Regions of interest are generated based on three primary regions of interest. They are Cell ROI, Bleached ROI and Background ROI. The Define ROI wizard is designed to lead users through the process of creating primary regions of interest, which includes cropping images, defining cell ROI, defining bleached ROI, defining background ROI and selecting regions of interest for error calculation in optimization. Each step is highlighted on top of the wizard page. To start the Define ROIs wizard, users just need to click on the define ROI button in the Data Analysis Work Flow. Cropping FRAP image data It is highly recommended to apply cropping to any raw image data to remove unnecessary information in order to speed up the performance. Figure 3-6 shows cropping of a FRAP image. Custom Crop Clicking on the crop button to draw the desired rectangular crop window. allows the user to click on the image and drag the mouse Automatic Crop Clicking on the automatic crop button default crop window. creates a crop window that can be used as a Figure 3-6 Cropping a FRAP Image Data

11 Defining regions of interest The toolbar functions in the image viewer are all enabled when the wizard page navigates to the defining cell/bleached/background ROIs steps. Users are able to manually draw the ROIs on FRAP images by using Drawing Toolbox or define ROIs automatically by using ROI Toolbox. Once the primary ROIs are defined, Virutal FRAP will automatically generate eight ROI rings to summarize spatial data. Figure 3-7 shows creating a cell ROI automatically with ROI Assistant in the Define ROI wizard. 1. Defining ROIs manually Draw ROI area Once the paintbrush button is pressed, the paint mode will be on until another button is selected. When users drag the mouse over the image in the paint mode, the area the mouse has passed will be highlighted and saved as the specified ROI. The files open with brush as the default selection. Erase ROI area Select the erase mode by clicking on the erase button painted area will erase the part that the mouse has passed.. Dragging the mouse over a Fill ROI area Select the fill mode by clicking on the fill button closed area, the area will be automatically filled.. If users click the mouse again in a 2. Defining ROIs automatically Clear ROI Clicking on the clear ROI button will clear the selected area for the current ROI. ROI time course Clicking on the time plot ROI button will pop up a dialog showing the plot of the average intensity of all the pixels in the area of the current ROI against time. Import ROI mask Clicking on the import ROI mask button will pop up an open file dialog. Users can select an image file to import the ROIs that are defined in the file. The imported file must have the same size as the current image data. The primary ROIs defined in the imported file will be copied to the current file. ROI assistant tool Clicking on the ROI assist node on Define ROIs wizard pages will expand the ROI Assist panel. Users have to make decisions on selecting ROI threshold source and spatial enhance threshold. Users can also play with the slider bar to shrink or grow ROI size. The FRAP image will be changed simultaneously when users are tuning the thresholds. Once the automatically generated ROI is accepted, users can click on the resolve button to have the image resolved. The fill voids button will be enabled if any void pixels exist in the ROI area that are required to be filled. Once the created ROI is ready, the apply ROI button will be enabled. Users can click on that button to apply the current ROI and the ROI assist panel will then collapse

12 The region is being defined Drawing Toolbox ROI Toolbox ROI assistant panel Figure 3-7 Creating a Cell ROI using ROI Assist Selecting regions of interest for error calculation The last page of the Define ROIs wizard shows the generated ROI rings based on the primary ROIs. Users can select any number of ROIs by clicking on the checkbox which specifies the different rings. The generated ROI rings are displayed in different colors. Figure 3-8 shows the ROI selection page. Figure 3-8 Selecting ROIs for Error Calculation

13 3.4 Selecting FRAP Model After loading the FRAP image and setting up all the required ROIs, users can proceed to select possible models that may fit the data. VFrap 1.1 supports two types of models: diffusion dominant and reaction dominant. There are two available models for diffusion dominant type: diffusion with one or with two diffusing components. The only available model of reaction dominant type is the reaction off-rate model. Users can choose one or more of these model types. The Choose Model Type wizard has only one page. To select the model, users just need to check the checkbox related to the specific model type. Figure 3-9 shows the wizard. Figure 3-9 Choose Models Wizard in Virtual FRAP 3.5 Estimating Parameters The traditional FRAP fitting is usually done based on the bleached area for a single diffusion rate. The Virtual FRAP is able to fit more accurately by taking the spatial data into account. The fitting is conducted over all the selected ROI rings instead of only bleached area. Moreover, the Virtual FRAP is able to deal with a secondary diffusion rate. The estimated parameters for one diffusing component are primary diffusion rate, primary mobile fraction and bleaching while monitoring rate. The estimated parameters for two diffusing components are primary diffusion rate, primary mobile fraction, secondary diffusion rate, secondary mobile fraction and bleaching while monitoring rate. The last step of data analysis work flow is to estimate diffusion parameters. To start the estimation wizard, users need to click on the Estimate Parameters button. A spatial reference simulation is then started which is followed by optimization. The best estimates are acquired by minimizing the errors between the simulated data and the experimental data. To understand if the best parameters are identifiable, confidence intervals (based on different confidence levels) of each parameter are calculated after optimization. Confidence intervals are basically acquired by evaluating the profile likelihood curve of a parameter

14 The Estimate Parameters wizard contains the best estimates for each selected model and a summary page which displays the statistics for the selected models and allows users to choose the best fit model. Understanding the best estimates Once the simulation and optimization is completed, the results for selected models will be shown in the different wizard pages. Figure 3-10 shows the estimate results page for two diffusing components. The top half of the page displays the best estimates for such a model. The bottom half is a real-time plot which illustrates the difference between simulated data and experimental data according to the parameter changes. In this page, users are able to tune a primary diffusion rate together with a secondary diffusion rate and the bleach while monitoring rate. Users can set values either by typing into the text fields or tuning the slider bars. The different colors of the plots represent how the diffusion works in different ROIs of the cell. Clicking on the show ROIs button will show the ROIs distinguished by different colors. Users can click on the best fit button to get the best fit parameters based on the current initial parameters in the text fields. In addition, by clicking the show confidence intervals button, the parameter confidence intervals will be shown in the confidence interval panel in Figure The confidence interval panel contains several plot panels, one for each parameter. All the plot panels are able to be expanded or collapsed and the default plot is for primary diffusion rate. Users can click on to expand a specific plot panel or to collapse a specific plot panel. The x-axis in the plot is the log base 10 ratio of parameter value and the y-axis is the likelihood of the simulated results. The Virtual FRAP has built in confidence levels which are 80%, 90%, 95% and 99%. Horizontal lines with different colors denote different confidence levels in the plot. At the bottom of the confidence plot, a table lists the confidence intervals according to the confidence levels. Figure 3-10 Parameter Estimation Results for Two Diffusing Components

15 Profile likelihood plot with lines denoting confidence levels Confidence interval table Figure 3-11 The Confidence Interval Panel Model parameter comparison and best model selection The last page of Estimate Parameters wizard is a summary page. The page has four parts. Firstly, a table lists all estimates for one or two models according to the model selection. Model identifiability is shown in the last row of the table. This is visible by default. Secondly, a plot contains all the plots for experimental data and simulated selected model data, which is invisible by default. The third part is a table showing errors between experimental data and simulated data under each ROI for selected models. The last part allows users to choose the best model based on their judgement, however, a machine made decision will be given by default. All the table data are able to be partially or wholly copied to clipboard. The summary page is illustrated in Figure Figure 3-12 The Summary Page in Estimate Parameters Wizard

16 3.6 Spatial Simulation and Results As shown in Figure 3-1, the results of the selected model after parameter estimation will be shown in data analysis results panel. Once the analysis results are available, the run simulation button is enabled. Clicking on the button will run the spatial simulation with the selected model parameter set. A spatial viewer will then pop up to show the results. The default view contains only normalized experimental data and normalized simulation data, which is convenient for a comparison between them. However, users can choose other views by selecting other items in the combo-box. Figure 3-13 shows the spatial viewer. To understand and access spatial simulation results and export the results. Please refer to Virtual Cell documentation at for chapter 8 and chapter 9. Figure 3-13 The Spatial Data Viewer of Virtual FRAP There is one feature that is worth highlighting in the spatial viewer. Just by a button clicking on, the Virtual FRAP can generate a movie using normalized experimental data and simulation data to illustrate how these two sets of data change against time. The movie viewer is shown in Figure

17 Figure 3-14 The Movie of Experimental and Simulation Data Comparison

18 3.7 The Virtual FRAP Batch Run Mode The virtual FRAP batch run mode is created for users, who have conducted same experiments multiple times and want an average result over all experiments Starting the Virtual FRAP Batch Run Mode Click on menu Tools -> Batch Run in the main user interface to start the Virtual FRAP batch run mode. Figure 3-15 shows the batch run mode user interface. The batch run interface has four components. They are: 1. The Virtual FRAP batch run navigator The tree structure lists all the FRAP documents in a batch run and also provide a link to the results display when results are available. A toolbar at the end of the navigator window provides functions such as Add a document, Delete a document and Delete all. 2. FRAP Image Viewer It displays the FRAP image based on the time series. 3. Results Panel It shows the average parameter values over all the FRAP documents in a batch run. 4. Job Status Panel It shows job status while batch run is executing Figure 3-15 The Virtual FRAP Batch Run Mode Adding a Document to Virtual FRAP Batch Run Click on the Add a New Document button, a wizard is popped up. This wizard basically is a combination of the Load FRAP data wizard and Define ROI wizard in the main interface. The only difference is that the Add a New Document wizard in batch run allows loading a.vfrap document. To understand how to load a FRAP experimental data and how to Define ROI, please refer to sections 3.2 and 3.3. After adding a new document into the batch run, the new document s name will appear in the batch run navigator tree. Upon selection of a document tree node, the image data related to the document will appear in the image viewer

19 3.7.3 Deleting Document(s) from Batch Run To delete a document from the batch run, two steps have to be taken. Firstly, select the document from the batch run navigator tree. Then, click on the Delete a Document button. The selected document will be deleted from the batch run. If there are batch run results available, the results will be updated automatically. The results will be unavailable if there is no document left in the batch run. If users want to delete all the documents in the batch run, they just need to click on Delete All Documents button. All the documents and available results will be removed from the batch run Executing Batch Run When all the documents for a batch run are added, users can start executing a batch run by clicking the batch run button in the toolbar of the batch run interface. A select model type wizard will be popped up before proceeding to batch run. Users are required to select only one possible FRAP model for the batch run. To understand choosing a FRAP model, please refer to section 3.4. The batch run basically does estimation of parameters of selected model for each document involved and summarized over all the documents. The running job statuses are updated in the job status viewer simultaneously. Once the results are available, the results node will be updated in the batch run navigator Understanding Batch Run Results When the results are ready, a Results Available node will be added to the navigator tree. Clicking on the tree node will open the results display panel on top of the image viewer. Figure 3-16 shows the results display panel. Results Display Panel Statistics over all documents Estimates for each document Errors for each document Figure 3-16 Virtual FRAP Batch Run Results Panel

20 The batch run results panel has three tables. The first table shows statistics over all the documents in a batch run. The statistics are mean, standard deviation, median, minimum and maximum of different parameters in a model. The table in the middle lists all the estimated parameter values for each document. Clicking on the details button will lead users to an estimate parameters panel, which users may refer to section 3.5. The table at the bottom shows errors for each document calculated under selected ROIs. All the tables are able to be partially or wholly copied to clipboard to paste into other applications