This document's organization
This 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.
Dialog buttons are noted as enclosed in brackes, for instance [OK]
indicates the OK button.
Introduction
PEST 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 hyperfine 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.
Requirements
PEST WinSIM requires one of the following systems:
Installation
Check the main PEST documentation
Preferences
On startup, 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 files
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.
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:
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 distrubed.
Picture: The File:Spectrum open / save dialog box
Picture: The Project open / save dialog box
Printing
Spectrum information Copying data to the Windows clipboard
Controlling the display
Select Display:Display to further control the spectrum screen plotting.
A dialog 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).
Picture: Display controls dialog
Spectrum Manipulations The following display shows a spectrum before and after filtering:
Picture: FFT filter example
Simulation speed is improved by calculating only the FFT signal and
thus not computing the inverse FFT. To compare wtih 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 Nyquist theorem. Therefore, we can display the FFT signal as a combination
of the Real and Imaginaries 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:
Picture: FFT combined data example
The forward and inverse FFT of the experimental can
be computed using Manipulations:exp_>>_FFT and Manipulations:exp_<<_FFT
respectively. The display will swith 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 dialog box with controls for transferring datasets
and executing forward and inverse FFT computations directly.
Picture: FFT manipulation controls
Simulating spectra
Choose Simulation:Scan_info to enter consequential instrumental
parameters :
Choose Simulation:Parameters to edit the hyperfine, relative area,
and lineshape parameters. A LARGE dialog box appears with several input fields:
Then enter the hyperfine coupling parameters as in this example :
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 dialog 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.
Picture: Simulation parameters dialog
Optimizations
Select Simulations:Optimize to initiate the process. Choose the algorithm
you will use and [Setup] to change the default optimization. Choose Contraints
to set boundries on how far certain parameters my deviate from their initial values.
Optimization options
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 optimizations concurrently to test different hypothesis or to test one hypothesis
on several sets of data. WinSIM will present a dialog box with these options:
Optimization Results Online help
Select Help:Index to view this help file.
The Edit:Preferences must be corectly entered and saved.
Select Help:Database to access the online Spin Trap Data Base (STDB).
The Edit:Preferences must be corectly entered and saved.
Select Help:About to view information about the author of this program and
the revision being used.
The 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.
A dialog 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
dialog box to load the spectrum as either FT Real, FT Imaginary, or
CW simulation
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.
PEST WinSIM will print spectra to any Windows compatible printer.
Select File:Print:Spectrum and the standard Windows dialog
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.
Once a spectrum is opened, choose Edit:Information to check
the number of data points, scan range and field center, and other
instrumental parameters.
PEST 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.
The 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.
Spectra 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 inforamtion 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 dialog 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 signicant 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 most definitive means of identifying a free radical species is to
compute a successful simulation and assign the hypefine 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:
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)
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.
Set Hyperfine Spin Number Opt
-------------------------------------------
[x] 1 14.0 1.0 1 [x]
[x] 2 16.2 0.5 1 [x]
[x] 3 2.2 0.5 3 [ ]
[ ] 4 2.2 1.0 2 [ ]
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
optimzation, 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.
PEST 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.
Starting the optimization
Used to limit the range of parameter deviation. To simplify
the selection process, WinSIM implements constraints for the major classes of parameters:
Hyperfines, g-positions, Linewidths, and relative Intensity. Enter a percentage value
for the amount of deviation from the original value and use the checkbox [x] to
enable or disable these constraints. Constraints are only enabled for the LMB1 algorithm.
Use this to limit the range of the scan that is used in
the error computation, for instance when an unidentified signal grows during the scan to
dominate the high field area ; when the main signal just decays very rapidly ; or when
a bad baseline effect is visible in the wings.
WinSIM may start either a interactive or batch mode optimization. Interactive
optimizations run in the WinSIM window and present continuous updates of the simulation plot
and error statistics. Batch mode optimizations 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.
- and -
- or -
The optimization results are written in three places, depending on whethe the process is
run interactively or in batch mode.
DRD, April 1996