This document's organizationThis help file is organized in a tutorial manner to guide users through specific tasks, and does not specifically explain every detail of the program. Menu items are noted as choice:choice:choice; for instance, File:Open:Spectrum will indicate choose File then Open then Spectrum. Dialogue buttons are noted as enclosed in brackets, for instance [OK] indicates the OK button.
Introduction, requirements, installationPEST WinSIM is designed to compute the simulations of multiple species of isotropic EPR spectra. Up to 10 independent EPR signals each with up to 16 sets of hyper fine couplings can be calculated. Experimental spectra may be loaded for on-screen comparisons. A custom algorithm (LMB1) and the Simplex algorithm have been implemented to optimize the simulation to achieve the best possible fit with the experimental signal. RequirementsPEST WinSIM requires one of the following systems:
No additional memory or hard disk requirement is anticipated. InstallationCheck the main PEST documentation PreferencesOn start up, PEST WinSIM reads preferences from the niehs.ini file located in the Windows root directory. Other programs from the NIEHS may also access this file. Use Edit:Preferences to customize the program for your needs:
Opening and saving data filesThe File:Open:Import function allows quick interchange with the DOS mode EPR.EXE program. The EPR.EXE program contains many more spectrum processing, measurement, and output options. Spectrum files are written to a temporary directory and located by number. For instance, in the DOS program, export the spectrum as #1, and in the WinSIM program import the same spectrum as #1. the user can also export from WinSIM and import into EPR.EXE. The File:Open:Spectrum function is the primary means for loading new spectrum files into the program. The following types of files can be loaded.
A Dialogue box will prompt the user for the filename. By default, the spectrum will be loaded as the experimental continuous wave spectrum. However, the user may choose in the filename Dialogue box to load the spectrum as either FT Real, FT Imaginary, or CW simulation The File:Open:Project function groups spectrum and parameter files together with a single base filename. This allows the user to save and recall an experimental spectrum, the simulated spectrum, and the parameters at once. Filename extensions are used to denote the different types of data:
By default, only the .lmb, .sim, and .sdx files are saved and opened. The user can select any set of these files to open and save. Therefore, the user can choose to load only the parameters from a project, for instance. Note that the extensions cannot be changed. Chose File:New to erase the current spectrum data. This will not affect the current simulation parameters. File saving works the same as file opening. If a spectrum file is opened, the current simulation parameters are not disturbed. If simulation parameters are opened, the current spectrum data is not disturbed. Spectrum informationOnce a spectrum is opened, choose Edit:Information to check the number of data points, scan range and field center, and other instrumental parameters. PrintingPEST WinSIM will print spectra to any Windows compatible printer. Select File:Print:Spectrum and the standard Windows dialogue box will appear. The spectrum plot screen will be printed in a fixed aspect ratio and size; therefore, independent prints can easily be compared. When the user chooses File:Print:Parameters, the simulation parameters will be written to a temporary file and will be loaded into the notepad program in a separate window. The user may then add her own notes and print or save the information. PEST WinSIM does not print the spectrum and parameters on the same page. Copying data to the Windows clipboardPEST WinSIM will copy data to the Windows clipboard, which the user may employ to paste the data into a spreadsheet style program such as Microcal Origin or Microsoft Excel for custom manipulations. Choose Edit:Copy_data and the current spectra displayed will be formatted into multiple data columns for pasting into a spreadsheet control. Pasting from the clipboard into the spectra arrays is not supported. Controlling the displayThe user may refresh the screen display with the Display:Plot command. Use Display:Rescale to automatically scale a spectrum plot which is too large or too small for the display area. Use Display:Overlay to switch between plotting multiple spectra as overlapped or separate lines. Select Display:Display to further control the spectrum screen plotting. A dialogue box appears with entries for the left, right, bottom, and top values of the plotting area ; selections for which spectrum data to plot ; whether or not to overlay the plots ; and to show the x and y axis. To zoom into or out-of the plot, click on the arrow buttons [<][>]and switch between scale movements or position movements. The X scale is shown as datapoints and the Y scale is shown as program plotting units; the default Y scale is the same as the DOS EPR.EXE program (+/- 500). Spectrum ManipulationsSpectra may be filtered by selecting Manipulations:Filter. The experimental spectrum will undergo an FFT to separate its frequency components and the Real and Imaginary domains will be displayed. The FFT is a display of the magnitudes of the fourier frequencies in the data. Therefore, if you zero the lowest FFT data points, you will filter out low frequency information such as a slowly rolling baseline. If you zero the highest FFT data points, you will filter out high frequency noise in your spectrum. A dialogue box prompts the user for the starting and stopping filter points. The suggested High Filter value is the point at which the FFT signal has decreased to 0.025 (1/40) of its highest value. The FFT spectra are displayed with this value as the middle point of the X domain. For instance, if the 0.025 point occurs at FFT datapoint 98, then the X domain of the display will be from 0 to 196. This value is only a guess at a good point for filtering, the user may want to change it based on the display. If it appears that significant signals exist beyond that point, the user should enter a higher value. FFT datapoints above (to the right) of the High_Filter will be zeroed, and FFT datapoints below (to the left) of the Low_filter will be zeroed. Once you have verified the filter points, click on the High_Filter and/or Low_Filter buttons to actually zero out those FFT datapoints. Then select Keep compute the inverse FFT and receive the filtered spectrum; or select Discard to return to the un-filtered spectrum. Once a spectrum has been filtered, it must be re-opened from disk to go back to the original data. The following display shows a spectrum before and after filtering: Simulation speed is improved by calculating only the FFT signal and thus not computing the inverse FFT. To compare with the experimental, we must first create the FFT of the experimental spectrum. Because the FFT has both Real and Imaginary domains, we then have two signals to display. However, only the first half of the FFT datapoints are meaningful, the Nicest theorem. Therefore, we can display the FFT signal as a combination of the Real and Imagines by just tacking on the Real component onto the end of the Imaginary. As with the filtering, we can discard frequencies that have insignificant intensity. When a user chooses Manipulations:Combine_FFT, the FFT of the experimental spectrum is computed and the Real and Imaginary domains are displayed. As with the filter function, the 0.025 (1/40th) intensity is chosen as a like combination point; although, the user may input another value. Once the user accepts a value, the experimental spectrum will be represented by the significant portion of the Imaginary domain on the left and the same size portion of the Real domain on the right: The forward and inverse FFT of the experimental can be computed using Manipulations:exp_>>_FFT and Manipulations:exp_<<_FFT respectively. The display will switch appropriately. If you use external programs to process FFT data you can save and load those datasets independently using the Manipulations:Controls selection which brings up a dialogue box with controls for transferring datasets and executing forward and inverse FFT computations directly. Simulating spectraThe most definitive means of identifying a free radical species is to compute a successful simulation and assign the hyperfine parameters to structural members. The most important function of this program is to assist the user in that process. This program is specialized to compute the simulations of multiple species of freely rotating isotropic free radicals, such as found in complex biochemical spin trap systems. PEST WinSIM has the following capabilities:
Choose Simulation:Scan_info to enter consequential instrumental parameters :
To skip the Mod.Amp. and Time Constant computations, enter zero for their values. Note that that the Time Constant is expressed as the ratio which is generally in the range of 0.1 to 1.0 for most CW scans : TC = Time Constant (ms) / Scan Time (s) Choose Simulation:Parameters to edit the hyperfine, relative area, and lineshape parameters. A LARGE dialogue box appears with several input fields:
Each of these fields has a checkbox [x] in the Opt column for selecting if that parameter is to be adjusted during optimization. To exclude a parameter from optimization ( ie.. fix the value as constant ), make this selection unchecked. Then enter the hyperfine coupling parameters as in this example :
The simulation will be computed with a spin=1 and coupling=14.0G nucleus ; a spin=1/2 and coupling=16.2 G nucleus ; and a spin=1/2 and coupling=2.2 G on three nuclei ( producing the 1-3-3-1 pattern of a methyl group ). Set #4, with a spin=1.0 and coupling=2.2 on two nuclei is not computed since the leading checkbox is not selected. The Opt field is used to indicate if that parameter should be adjusted during the optimization, in this example only the first two hyperfine will be adjusted during optimization. The user could easily switch off Set #3 and switch on Set #4 to alternate hypothesis about the radical structure. Now select [Simulate] (or press Enter) to compute the simulation. The simulated spectrum will be plotted on the screen along with the experimental. (if there is an experimental spectrum loaded) The Simulation Parameters dialogue box will stay on the screen, the user can close the box or just move it to the side of the screen. This allows quick cycling of changing parameters - computing spectrum - viewing results. Choose Simulation:Correlation to compute the Spearman's Rank Correlation Coefficient between the current experimental and simulated spectra. The value shows up on the status bar at the bottom of the PEST WinSIM window. Choose Simulation:Residual to subtract the simulated spectrum from the experimental spectrum. Choose it again to quickly return to the simulated spectrum. OptimizationPEST WinSIM will adjust the values of the simulation parameters to create the best fit between simulated and experimental spectra. This is an iterative process consisting of guess adjustments, trial computations, and error computation. In general, EPR spectra are overdetermined, that is, more than one set of simulation parameters will produce an apparently good fit to the experimental spectrum. Therefore, any optimization algorithm must rigorously check the error space for local minima in the error estimation to produce a robust result. Two algorithms have been implemented: LMB1 was developed by Dave Duling as an extension and derivation of the TUNE algorithm of Ann Motten and Joerg Schreiber and was published in J.M.R. The well known Simplex algorithm is also available. References for both methods may be found in the PEST manual reference section. Select Simulations:Optimize to initiate the process. Choose the algorithm you will use and [Setup] to change the default optimization. Choose Constraints to set boundaries on how far certain parameters my deviate from their initial values. Optimization options
Starting the optimizationWinSIM may start either a interactive or batch mode optimization. Interactive Optimization run in the WinSIM window and present continuous updates of the simulation plot and error statistics. Batch mode Optimization run as a separate TUNE process whose results can only be viewed after the process has completed. However, several TUNE processes can be run concurrently and these processes may be run on other computers on the network. Select [ GO ] to start the optimization as an interactive process. The plot of the simulation and experimental spectra will update on a timely basis and the status bar will show the current status of the optimization with the following information:
Select [ TUNE ] to start a batch mode optimization. This will run the Tune program as a separate process; therefore, you may run several TUNE program Optimization concurrently to test different hypothesis or to test one hypothesis on several sets of data. WinSIM will present a dialogue box with these options:
Optimization ResultsThe optimization results are written in three places, depending on whether the process is run interactively or in batch mode.
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