1.0 Help Manual
FRETView is an attemp to develop a program to
reduce much of the complexity involved in obtaining FRET
parameters from absorption and fluorescence data. It integrates
many of the equations used for doing FRET calculations  with a
database of commercially available and user defined chromophores.
program modules and a description of their function is as follows:
- Spectral Database Modules - Stores the emission and absorption
spectra of various fluorophores and quenchers. Pertinent information
such as extinction coefficient, quantum yield, and radiative lifetime
is also stored along with the spectra.
- User Interface Module - Provides an
user interface (GUI) which allows the inputting of experimental
viewing of the resulting FRET parameters both in a spreadsheet format
as well as graphical plots. A GUI for the management of the
spectral database is also provided by this module.
- Math Module - Provides functions for calculating the FRET
parameters which include Förster distance (R0), efficiency (E), transfer rate (kt),
donor intensity (Fd), acceptor intensity (Fa), total intensity (Ft),
and the acceptor's lifetime as a result of the energy transfer process.
The Strickler-Berg formula  is also implemented to allow lifetimes to be
estimated from the spectroscopic data and quantum yield of a chromophore.
- Network Module - Allows the loading of a spectral database
file from a centralized webserver over the internet, thereby making it relatively easy for a
large number of users to gain access to a single database file.
Usage The user
interface is designed to be very intuitive. On the left hand side
is the spectral database which contains the list of chromophores,
and on the right hand side are the "FRET Parameters", "Manage Spectral
Database", and "Help" tabs. Please
that even though spectral data of the various chromophores in the
database should be
correct, the quantum yield (QY), extinction coefficient (EC), and
lifetime (Tau) values need to be verified. In a lot of cases, the QY
lifetime values could not be found so dummy values are used instead.
Also the intensity of all data is normalized on a scale of 100.
Prior to any calculation being performed the donor and acceptor must
first be selected from the list using the "Set Donor" and "Set
Acceptor" buttons. The "Plot" button in that panel can be
used to plot their respective absorption and fluorescence spectra.
Once a donor and acceptor have been seleted, the next step is
to input or calculate the parameters (R0 and Tau only) needed to obtain the FRET parameters
by the following process:
Calculate (R0) by simple inputting, or verifying the correct
values for EC (acceptor extinction coefficient at the absorption
maximum), QY (quantum yield of the donor), n (refractive index), and
K^2 (orientation factor). Once these values have been verified
press the "Ro" button. This calculated R0 and the result is displayed
in the "Ro" textfield: R0 can also be entered directly into this text
field if the value is known.
Next verify, or input the correct lifetime in the "Tau" textfield
for the donor. This is used to obtain the energy transfer rate
and to model the decay profiles that results when a long-lived donor
undergoes energy transfer with a short-lived acceptor. For
convenience, the Strickler-Berg formula  is also implemented and
allows the lifetime to be estimated from the EC, QY, and S0-->S1transition.
To do such a calculation, first select the
chromophore which to calculate the lifetime for from the drop-down
menu. This can either be the currently selected chromophore on the list,
the donor, or the acceptor. Verify or input the values that are in
the textfields with the yellow background (values in these textfields
are only used for calculating the lifetime). Now press the "Tau"
button and the lifetime value will now be displayed in the "Tau"
textfield. Note, please remember to select the correct units for
the lifetime using the drop-down menu. Selection of the wrong
units will inherently lead to huge errors in the energy transfer rate and lifetime
The textfield labeled D/A is used to set the donor to acceptor ratio.
This is normally 1 : 1, but can be set to any value just as long
as the X : X format is used. An error will result if the wrong
format is used.
The wavelength (where excitation occurs) and distance (distance between
donor and acceptor) range are the next parameters that need to be set.
These parameters are entered into their respective
textfields and can be positive numbers that have some basis
in reality. For example, setting the distance range from 0
to 1000 makes as much sense as setting the wavelength range from 0 to
1000 nm. As far as FRET calculations are concerned, use of such
excitation and distance ranges will result in meaningless values
Once all the input parameters have been verified, pressing the
button will evaluate the FRET parameters with the results being
in the results table. Because both the excitation wavelength
and distance are
varied during the calculation, the user must select which of these
variables should be held constant and which one should be varied.
is accomplished by using the respective drop-down menus, then pressing
the "Update" button. The "Update" button must be pressed whenever
any of the values in the drop-down menus are changed in order
for the new data to be loaded into the results table. The data in
the results table can be copied to the system clipboard by pressing
the "Copy" button. Unfortunately, no printing is possible at
this time, but that is a feature that is scheduled for the next release
of the program.
In addition to viewing the results in a table format, they can also
be plotted. When the "Plot" button next to the "Calculate" button
is pressed, the "Plot Option Dialog" will be displayed. From this
dialog, plots which compare the variation in efficiency (E),
intensity of the donor (Fd), intensity of acceptor (Fa), total
intensity (Ft), transfer rate (Kt), and lifetime of acceptor (Tau A) as
a result of variation in either the D-A distance or excitation
wavelength can be displayed.
Plots showing the simulated spectra or decay profiles of the
selected D-A pair can also be viewed by pressing either the
"Lifetime" buttons. For spectral simulations, the mathematical
model employed only
considers energy transfer between the donor and acceptor. For example,
if there are any other type of interaction occuring between the donor
and acceptor (i.e. electron transfer, or excimer
formation), the simulated spectra will not match the experimental.
Lifetime simulation make use of the equations found in reference
which assumess only a monoexponential decay for the donor. The
percent of acceptors in the excited state at time zero is initially set
to 5% of the excited donors
in order to properly model the decay profiles. Modification of
this value can be done to better simulate the decay profiles of the
donor and acceptor.
Editing the Spectral Database As the name implies, the tab
labeled "Manage Spectral Database" allows just that. Entries that
are already in the database can be edited
or new entries can be added in one of three ways. Spectral
data can be imported from Invitrogen
data files (text file), a pair of absorption and emission tab
delimited text files (both files are required), or taken directly from
program using the "Grab" button. When grabbing data from the
InstruView program, both
the absorption and emission data must be selected as pairs, otherwise
will occur. Importing data from an Invitrogen data file that
contain any emission data (for example, the data for a quencher) will
cause an error. In order to prevent this error, open the
file in any text editor and add some dummy X,Y data to the first two
rows of data. For example, if the data file is initially
The exact values of the numbers added does not matter.
Once any modification has
been done to the spectral database, it can be
saved to the disk by using the "Save" or "Save As" button. This
file will automatically be reopened the next time the program is run.
The "Open" button is used to load a new spectral database
while the "Download" button is used to load a spectral database
from a webserver. This is useful for having a centralized
database within a group, or for sharing a database with the public at
large. Pressing the "DBinfo" button will open up a dialog
that displays editable information about the database.
The properties of the currently selected chromophore from the list will be
displayed in the "Current Dye Spectra" panel. The values for
these properties can be modified here, but the "Update" button must be
pressed in order to save the new values. However, these values
are not saved to the disk file until the "Save" or "Save As" button is
used. Pressing the "Abs" or "Ems" will open a dialog that
displays the absorption and emission data which can be copied to the
system clipboard for importing into another program. The "Remove"
button is used to remove the currently selected chromophore from the
The "Console" is currently used to view program output such as errors,
but in future releases will have the ability to execute a
predefined list of commands when the "Run Command" button is pressed.
Pressing the "Plot" button when this tab is selected will display
a dialog that allows the absorption and emission spectra of up to
six chromophores to be plotted simultaneously.
 Lakowicz, J. R. "Principles of Fluorescence Spectroscopy 2nd. edition"; Kluwer Academic: New York, NY, 1999.
 Lamouche, G.; Lavallard, P. Physical Review, 1999, 59, 4668-4674.
 Maliwal, B. P.; Gryczynski, Z.; Lakowicz, J. R. Anal. Chem., 2001, 73, 4277-4285.
Permission to use, copy and modify this software and its documentation
purposes is granted, without fee, provided that an acknowledgment to
the author, Nathan Stevens at
appears in all copies. Nathan Stevens makes no
representations about the suitability or fitness of the software for
any or for a particular purpose. Nathan Stevens shall not
be liable for any damages suffered as a result of using, modifying or
distributing this software or its derivatives.