WinSIM Manual

WinSIM
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. Dialogue buttons are noted as enclosed in brackets, for instance
[OK] indicates the OK button.

● Introduction, requirements, installation

● Setting system preferences

● Opening and saving data files

● Checking spectrum data file information
● Printing spectra and parameters
● Copying spectra to the Windows clipboard

● Controlling the spectrum display

● Filtering noise from the spectrum
● Speeding simulations by combining FFT Real & Imaginary
● Accessing FFT data directly

● Introduction to EPR simulations

● Setting instrument and global spectral parameters
● Setting hyper fine coupling, relative area, and line shape values
● Computing the correlation value and residual spectrum

● Introduction to Optimizations
● Setting options
● Starting an interactive or batch optimization
● Reading optimization results

● Accessing on line help functions

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 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.

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Requirements

PEST WinSIM requires one of the following systems:

● Windows NT (intel only)

● Windows 95
● Windows 3.11 + Win32s libraries (not included with distribution)

No additional memory or hard disk requirement is anticipated.

Installation

Check the main PEST documentation

Preferences

On 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:

Defines default spectra and/or parameters to be opened when WinSIM is started if these
start up
options are selected. Click on [Save_now] to save the current spectra and/or parameters
project
as defaults and [Open_now] to reload the default data.
The
Is the location for default data files for import/export exchange with the EPR.EXE program.
Temp
This is normally C:\LAB\EPRDATA.
directory
Help Refers to your WWW viewer, such as Netscape. Enter the full path so that WinSIM can start
Browser the browser for viewing help files and the Spin Trap Database.
Refers to the HTML format help files that are part of the PEST distribution. A user may
access them either over Internet directly from the NIEHS or install them onto her own hard
disk. The current Internet address for these files is
Help File
http://alfred.niehs.nih.gov/LMB/pest/manual. A typical hard disk address might be
c:\lab\docs\manual. Since the first document is named home.html, a user only needs to
enter the path of the directory.

Opening and saving data files

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

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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.

● NIEHS binary from the PEST programs

● Bruker spc/par from the Bruker OS/9 system
● ASCII text columns

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:

.lmb ---- experimental spectrum file (NIEHS binary format only)

.sim ---- simulated spectrum file (NIEHS binary format only)
.out ---- ASCII version of parameters file, calculated parameters only
.epr ---- ASCII printout file of simulation parameters
.sdx ---- parameters file including all parameters

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.

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Printing

PEST 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.

Spectrum information

Once a spectrum is opened, choose Edit:Information to check the number of data

points, scan range and field center, and other instrumental parameters.

Copying data to the Windows clipboard

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.

Controlling the display

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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.

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 Manipulations

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 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

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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:

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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 spectra
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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:

● Computes up to 10 species of free radicals.

● Computes up to 16 hyperfine nuclei sites per species. Each site may have an
unlimited number of equivalent nuclei.
● First order FFT based computations.
● Approximates the M(I) dependent linewidths of motionally restricted nitroxide
radicals, assigning independent linewidths to the -1,0,+1 line positions.
● Approximates the effects of Time Constant and Modulation Amplitude.
● Adjustable mix of Lorenztian and Gaussian lineshapes.
● Optimizes any/all simulation parameters via:
❍ LMB1 algorithm (published in J.M.R.)

❍ Simplex algorithm

● Any parameter may be excluded from optimization.

● Variable limits on parameter adjustment during optimization.
● Graphically interactive and batch mode Optimization.
● Optimization reports generated.

Choose Simulation:Scan_info to enter consequential instrumental parameters :

● Field Center (G)

● Scan Range (G)
● Modulation amplitude (G)
● Time Constant (TC)
● Harmonic ( 0th, 1st, 2nd )
● CW or FFT
● Simple isotropic or M(I) dependent nitroxide

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:

● Previous] [Next] : use these buttons to skip to another radical species.

● Calculate this species : choose whether or not to include each species in the
simulation. Note that if no species are computed, a single line should result.
● Area : the relative spectral area of each species. For single species this data is not
used. All simulations are computed with the same maximum peak height as the
experimental spectrum if one is present.
● g-shift : the relative shift in gauss of the center of the spectrum (in G) from field

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center. This is used to move the spectrum in the magnetic field domain. I used this
method since this value is more straightforward to estimate than g-value.
● Lineshape : the percentage of the spectrum that has Lorentzian shape. A pure
lorentzian line would be 100%. A pure Gaussian line would be 0%.
● Simple LW : the linewidth used when computing single linewidth spectra. If
nitroxide simulation is chosen, then this value is not used.
● Nitroxide : If nitroxide simulation is chosen, then these values are used in the
computation:

Hyperfine: coupling from the principal nitrogen nuclei.

LW -1: the low field component linewidth
LW 0 : the middle field component linewidth
LW +1 : the high field component linewidth

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 :

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
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.

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Optimization

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.

Select Simulations:Optimize to initiate the process. Choose the algorithm you will use
and [Setup] to change the default optimization. Choose Contraints to set boundaries on
how far certain parameters my deviate from their initial values.

Optimization options

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● LMB1 setup

Two options are available:

❍ Non-parametric : hyperfine values are adjusted independently

Hyperfine ❍ Parametric : hyperfine values are adjusted such that the sum of splitting

analysis: values multiplied by twice the spin multiplied by the number of nuclei is
constant. The keeps the total spectral width constant. Use this when you
are certain of the end lines.
Initially, the simulation parameters are adjusted in large increments and then
increment size is decreased to digital resolution in finite steps. This value controls
Parameter
the curvature of the descent. The default value is binary which is the steepest
descent
descent. Quadratic and linear descents are much less steep and thus consume
gradient:
much more time but are somewhat less likely to miss important minima in the
error domain.
This controls the number of steps in the parameter descent curve. The steeper the
Levels of
descent, the smaller this value should be. For instance, the default value for binary
granularity:
gradient is 7, while corresponding value for linear would be 2^7=128.

● Simplex setup

Number of controls the number of times the algorithm will restart once it has reached an
restarts: error minimum.
Max. this puts a limit on the number of trial simulations that will be computed,
iterations per keeping the program from wandering endlessly when it has an obviously wrong
restart: set of parameters.
Simplex stops when the slope of the imaginary line connecting the lowest errors
Fractional
thus far achieved is below a certain value. This tells the algorithm when to
tolerance:
terminate within a minima.

● Constraints

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.

● Data point range

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.

Starting the optimization

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WinSIM 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:

● Algorithm used: LMB1 or Simplex

● Starting error ( sum of squared residuals )
● Current error
● Number of simulations ( n )
- and -
● Level : LMB1 parameter for the current step, see levels of granularity above; this
value will decrement to one. If this value seems to never change, it may indicate
that the initial parameters are in error, which would be obvious by the simulation
plot.
● Species, Par : LMB1 parameters for the current species and parameter being
optimized. This is mostly for internal use as a programmer's diagnostic.
- or -
● Restart : SIMPLEX restart value, this value will increment to the number of simplex
restarts, see above.

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:

choose NIEHS binary or ASCII format data files. If the TUNE will be run on your Windows
Format:
PC computer, choose binary. If you intend to run TUNE on a UNIX computer, choose ASCII.
TUNE requires input files to execute; WinSIM will write out the required TUNE files
automatically. By default, the current filename with an underscore prepended will be used.
Filename:
The user may enter a different filename if needed. The files will be written into the current
directory.
Choose this to initiate the TUNE process. First the necessary files will be written, then TUNE
[ Start
will be started as a separate process. The directory containing the TUNE binary (tune.exe)
Now ]:
must be in the current system PATH (this is usually c:\lab).
[ Save Choose this to only save the necessary TUNE files but not start the TUNE process, for
files ]: instance, when the TUNE is going to be run on a UNIX computer.
Choose this to read in the TUNE results once a TUNE has completed. The files associated
[ Read
with the above displayed filename will be read. The simulation spectrum is not read
results ]:
automatically, but can be easily calculated with a Simulation:Simulate selection.

Optimization Results

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The optimization results are written in three places, depending on whether the process is
run interactively or in batch mode.

A printout file is created containing the final parameters and error statistics in a readable
and printable ASCII form. For interactive Optimization, the Notepad program is launched
Printout
with this information. For batch Optimization, a file is written with a .epr extension, if you
file:
read in the TUNE results through the TUNE dialogue box, this file will be loaded into a new
instance of Notepad.
A parameter file is created containing the final parameters in the TUNE input format which
Parameter can be read by TUNE or WinSIM. This file has the .par extension. Only the batch mode
file: TUNE program produces this file at the end of an optimization; however, WinSIM can
produce this file through the File:Save:Project options.
A simulated spectrum file is created by the batch mode TUNE program; however, WinSIM
Simulated
can produce this file through the File:Save:Project options. This file has the .sim
spectrum:
extension if it is a NIEHS binary file or the .dat extension if it is an ASCII file.

Online help
Select Help:

To view this help file.

Index
The Edit:Preferences must be correctly entered and saved.
To access the online Spin Trap Data Base (STDB).
Database
The Edit:Preferences must be correctly entered and saved.
About To view information about the author of this program and the revision being used.

Top of page

last updated on Sept 10, 1997

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