### FRETView 1.0 Help Manual

Introduction 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 [1] with a database of commercially available and user defined chromophores. The core 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 intuitive graphical user interface (GUI) which allows the inputting of experimental parameters and 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 [2] 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 note 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 and 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 [2] 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 simulation results.
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 being returned.
Once all the input parameters have been verified, pressing the "Calculate" button will evaluate the FRET parameters with the results being displayed 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. This 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 "Spectra" or "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 3 and 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 the InstruView program using the "Grab" button. When grabbing data from the InstruView program, both the absorption and emission data must be selected as pairs, otherwise an error will occur. Importing data from an Invitrogen data file that doesn't 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
wl,abs
250,221.6772
251,209.3986
change to.
wl,abs,wl,em
250,221.6772,100, 0
251,209.3986,101,1
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 database.
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.
References
[1] Lakowicz, J. R. "Principles of Fluorescence Spectroscopy 2nd. edition"; Kluwer Academic: New York, NY, 1999.
[2] Lamouche, G.; Lavallard, P. Physical Review, 1999, 59, 4668-4674.
[3] Maliwal, B. P.; Gryczynski, Z.; Lakowicz, J. R. Anal. Chem., 2001, 73, 4277-4285.
Please note:
Permission to use, copy and modify this software and its documentation for NON-COMMERCIAL purposes is granted, without fee, provided that an acknowledgment to the author, Nathan Stevens at "http://casilab10.sci.ccny.cuny.edu/~nathan", 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.