Software

PTI's FeliX32™ is the most comprehensive software package on the market. It's easy to use Windows™ based interface offers one software solution for all your fluorescence measurements. FeliX32™ uses full 32-bit implementation graphic capabilities, including sophisticated 3-dimensional plotting and full motion rotation. All major data handling packages are included: multi-exponential fits, global analysis, non-exponential analysis, anisotropy decay as well as maximum entropy methods. FeliX32™ also uses script controlled data acquisition so that specialized experimental routines can be easily created by the end user via FeliX32™ macro commands. This allows for unsurpassed flexibility in acquisition, calculation, and illustration of data.

• Excitation Ratio
• Emission Ratio
• Excitation Scan
• Emission Scan
• Synchronous Scan
• Multiple Dyes
• Timebased
• Timebased Polarization
• Calculate G-Factor
• Four Position Sample Turret
• Temperature Control
• Common Math Controls
• Fluorescence Resonance Energy Transfer (FRET)
• Calculate FRET Parameters (steady-state)
• Determine R_o
• Transform Commands
• Concentration Map
• Polarization
• Concentration Equation
• Lookup Table
• Data Analysis
• 1 To 4 Exponential Lifetime
• Multi 1 To 4 Exponential
• Global 1 To 4 Exponential
• Anisotropy Decays
• Micelle Kinetics
• Non-Exponential Decay
• ESM - Exponential Series Method
• MEM - Maximum Entropy Method
• DAS/TRES

Common Math Controls

Display Commands

Excitation Ratio

Excitation Ratio is used to set up and run experiments for intracellular ion determinations using excitation-shifted probes such as Fura-2 for calcium and BCECF for pH. In this experiment, the excitation source must alternate between two different excitation wavelengths that are characteristic of the probe. The emission intensity at both excitation wavelengths is measured at a longer emission wavelength and the ratio of these intensities is calculated. The ratio is proportional to the concentration of the ion under investigation. Excitation 1, 2 Enter the excitation wavelengths in the text boxes. Your instrument will automatically alternate between excitation wavelength 1 and excitation wavelength 2. The rate of alternation is dependent upon the illuminator type. The patented PTI DeltaRam X™ can provide up to 250 ratios/sec. A model 101 monochromator must move from one excitation wavelength to the other at the slewing speed set in the hardware configuration. Emission Enter the emission wavelength in the text box. If your instrument has two emission monochromators, FeliX32™ will ask for two emission wavelengths. The wavelengths you enter will be the wavelengths to which the monochromators will automatically move prior to data acquisition. If your system uses filters for wavelength selection, simply enter the peak wavelengths of the filters in these boxes. Enable Single Point Screening This is used to collect single data points into a spreadsheet display.

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Emission Ratio

Emission Ratio is used to set up and run experiments for intracellular ion determinations using emission-shifted probes such as Indo-1 for calcium and SNAFL for pH. In this experiment, a constant excitation wavelength is used and two emission wavelengths must be selected. This is normally done with two monochromators in a cuvette system, but one monochromator can be utilized. In a microscope-based system, the two emission wavelengths are selected using a dichroic assembly in the photometer. The emission intensity at both emission wavelengths is measured and the ratio of these intensities is calculated. The ratio is proportional to the concentration of the ion being determined. Excitation Enter the excitation wavelength in the text box. The wavelengths you enter will be the wavelengths to which the monochromators will automatically move prior to data acquisition. If your system uses filters for wavelength selection, simply enter the peak wavelengths of the filters in these boxes. Emission 1, 2 Enter the emission wavelengths in the text boxes. Your instrument will automatically alternate between wavelength 1 and wavelength 2. The rate of alternation is dependent upon the configuration. Dual emission systems provide up to 1000 ratios per second. Single monochromator emission systems slew between the wavelengths to provide up to 1 ratio per second. A model 101 monochromator must move from one excitation wavelength to the other at the slewing speed set in the hardware configuration. Enable Single Point Screening This is used to collect single data points into a spreadsheet display.

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Excitation Scan

In an Excitation Scan, the excitation monochromator is scanned between two wavelengths while the emission monochromator is fixed. The emission intensity is measured as a function of excitation wavelength.Due to the nature of fluorescence, the emission wavelength is set at a wavelength that is longer than the excitation wavelength range (red-shifted). Start and Stop Enter the initial excitation wavelength and the final excitation wavelength for the scan in these text boxes. Emission Enter the emission wavelength in the text box. If your instrument has two emission monochromators, FeliX32™ will ask for two emission wavelengths. Length This shows the length of the scan that will be run. If the starting wavelength and the length are entered, FeliX32™ will calculate the ending wavelength corresponding to these parameters.

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Emission Scan

In an Emission Scan, the emission wavelength is scanned between two wavelengths while the excitation monochromator is fixed. The emission intensity is measured as a function of excitation wavelength. Due to the nature of fluorescence, the excitation wavelength is set at a shorter wavelength than the emission wavelength range. Excitation Enter the excitation wavelength in the text box. Start and End wavelength Enter the emission wavelength scanning range in the Emission 1 text boxes. If the system is equipped with two emission monochromators, FeliX32™ will request two wavelength ranges. Length This shows the length of the scan that will be run. If the starting wavelength and the length are entered, FeliX32™ will calculate the ending wavelength corresponding to these parameters. For dual emission systems, the length of the scan will be the identical for both emission channels. FeliX32™ will adjust the emission ranges to ensure they both have the same length.

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Synchronous Scan

In a Synchronous Scan, the excitation and emission monochromators are scanned simultaneously at identical scan rates with a constant wavelength difference between them. A synchronous scan often results in the simplification of complex excitation or emission scans. Excitation Range Enter the excitation wavelength range in the text boxes. Emission Range(s) Enter the emission wavelength range in the text boxes. If the system is equipped with two emission monochromators, FeliX32™ will request two wavelength ranges. Length Enter the scan range in nanometers. This value will be calculated for you if a range is entered. If starting wavelengths and length is entered, FeliX32™ will calculate the ending wavelengths based upon these parameters. The length for all monochromators, excitation and emission, will be identical. FeliX32™ will adjust the range values to ensure that the length is the same.

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Multiple Dyes

The Multiple Dyes function is used to set up and run experiments for intracellular ion determinations using several indicators in combination, such as Fura-2 for calcium and BCECF for pH. In this experiment, the excitation light source must alternate between four different excitation wavelengths that are characteristic of the two probes (e.g. 340, 380, 440, 490 nm). In addition the isosbestic wavelength for Fura-2 is frequently monitored at 361 nm to obtain a calcium-independent signal. The emission intensity resulting from excitation at the above five wavelengths is measured at longer emission wavelengths (510 and 525 nm, respectively) and the ratio of these intensities is calculated. The ratio is proportional to the concentration of the ion under investigation. Any combination of up to 10 excitation and 10 emission wavelengths may be defined to accommodate the simultaneous measurement of both excitation- and emission-shifted dyes. Use Use the checkboxes to select the number of wavelength pairs for the experiment. Ex Enter the excitation wavelengths in these text boxes. Emi. 1 and Emi. 2 Enter the emission wavelengths in these text boxes. If your system only has a single emission channel, the system will only display a single column for entering emission wavelengths. Enable Single Point Screening This is used to collect single data points into a spreadsheet display.

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Timebased

In a Timebased experiment, the excitation and emission wavelengths remain fixed throughout the experiment. The emission intensity is measured as a function of time. Timebased experiments typically involve kinetic measurements. Excitation Enter the excitation wavelength in the text box. Emission Enter the emission wavelength in the text box. If your instrument has two emission channels, FeliX32™ will ask for two emission wavelengths. The wavelengths you enter will be the wavelengths to which the monochromators will automatically move prior to data acquisition. If your system uses filters for wavelength selection, simply enter the peak wavelengths of the filters in these boxes. Enable Single Point Screening This is used to collect single data points into a spreadsheet display.

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Timebased Polarization

Polarization measurements can be done in several ways in FeliX32™. Some methods include using a timebased experiment or excitation/emission scans. One of the easier ways to perform polarization is to use the Timebased Polarization acquisition, which is useful for performing the polarization measurements under complete FeliX32™ automation, including measuring the G-factor. Anisotropy and polarization will be calculated in real time and displayed in the workspace. This experiment is mainly intended for systems with a single emission channel using motorized polarizers, although manual polarizers may be used as well. The various parameters have much in common with the other experiments. Excitation Enter the excitation wavelength in the text box. Emission Set the emission wavelength for the experiment in the text box. Enable Single Point Screening This is used to collect single data points into a spreadsheet display. It is useful for measuring the polarization of a number of samples. The two intensities, polarization, and anisotropy for the sample will be determined. Background/G-Factor The system can perform background subtractions and G-Factor calculations automatically. The parameters for these measurements may be set to different values than the above section, which contains the information to run the polarization experiment on the sample. The measurement of G-Factor and background may be controlled using either the Calculate G-Factor or Acquire Background checkbox.

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Calculate G-Factor

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Four Position Sample Turret

The Sample dialog box controls the mode of automatic data acquisition of multiple samples (1–4) for instruments having a four position sample turret accessory. Data is acquired with the parameters established in the acquisition setup. Using the Display Setup dialog box, specify how the curves will be displayed. Samples are identified as S1, S2, S3, and S4. The curves produced by each sample may be displayed in separate groups or they may all be displayed together in one group.
Click on images to enlarge.

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Temperature Control

There are three modes of temperature control, Set Temp., Ramp Temp., and Increment Temp. with the latter only available during macros and ramping only during timebased acquisitions. Once a mode is selected you need to configure the experimental parameters. Additional temperature based controls can be found in Acquisition Preferences. Here you may select the temperature delta (how close the sample temperature must approach the set temperature before the set temperature is reached) and the units of temperature.

Set Temperature
Use this set of commands to bring the sample to a specific temperature.

Ramp Temperature
The following parameters allow you to ramp the temperature over a user defined range and speed.

Temperature after acquisition
After the scan is finished, this controls whether the temperature should be 1) uncontrolled, in which case it will tend towards the ambient temperature; 2) return to the temperature at the start of the forward ramp; or 3) hold at the final temperature.

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Common Math Controls

Create New Data
If checked, a new curve will be created. The original (source) data will be preserved.

Replace Old Data
If checked, the original curve will be permanently lost, as it will be replaced by the new data.

Label
Type the name of the new curve in the text box. If no label is specified, the new curve will be listed in the legend with a name comprised of a generic math function descriptor (e.g., Smooth, or Logarithm) added to the source curve's original name.

Execute
Carries out the operation. If you type in new values to select an X-axis region, Execute is required to perform the new calculation.

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Antilog

Calculates the antilogarithm of the selected curve.

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Average

Calculates the average value of the Y-axis parameter on a selected region of a curve. The average value is the sum of the values divided by the number of points.
The standard deviation is also determined using the equation: Where x _i is a data point and n is the total number of data points in the portion of the data trace being averaged.

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Combine

The combine command allows you to add one curve to another, subtract a curve from another, multiply a curve by another, or divide a curve by another. The math is performed in a point-by-point fashion. Only the portions of the curves that overlap are combined.

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Combine Constant

This command allows you to apply an arithmetic operation between a curve and a constant.

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XY Combine

This feature allows the user to construct a new data trace, using the X values of one trace, and the Y values of another trace. In this way, complex data, such as time-dependent temperature ramps and correlated data, can be converted into new traces that have compatible X axes to simplify the display and treatment of the data.

Source trace with X data
Use the drop-down menu to choose the trace from which to create the X data. Alternatively, select a curve from the legend and click on the Pick icon beneath the Source trace with X data header.

Source trace with Y data
Use the drop-down menu to choose the trace from which to create the Y data. Alternatively, select a curve from the legend and click on the Pick icon beneath the Source trace with Y data header.

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Differentiate

Differentiate takes the derivative of the selected curve. Subsequent application of the differentiate command results in the second derivative, etc. Differentiation is done using the 5 point Savitzky-Golay algorithm, which provides a smoothed derivative.

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Integrate

This function integrates within the range of the selected region of a curve. The Total Area is the integral of the data above the absolute X-axis. The Peak Area is used to integrate a peak within a curve.

Total Area
Displays the total integrated area within the selected range. If there is negative data, then the total integrated area may also be negative.

Peak Area
Displays the integral of the peak above the background. FeliX32™ projects a line between the points where the boundaries of the range intersect the curve. Peak Area is the integrated area above that line. If most of the curve data lies below this line, then the Peak Area will be a negative number.

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Linear Fit

Calculates and overlays a linear fit to the selected region of a curve. The slope, intercept, and correlation coefficient are displayed.

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Linear Scale

The Linear Scale is used to shift a curve or a selected region of a curve on either the X or the Y-axis. The curve can be shifted on the Y-axis by a multiplier, divisor, or an addend. The curve can be shifted on the X-axis by an addend only.

Y and X Value

Multiplier: Multiplies all Y values in the curve by the specified multiplier.

Divisor: Divides all Y values in the curve by the specified divisor.

Offset: Adds the specified value to each X or Y point in the curve.

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Logarithm

Calculates the logarithm of the selected curve.

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Normalize

Normalizes a curve to a set value. The normalization function reference may be either a peak or a specified point.

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Reciprocal

Calculates the reciprocal (1/Y) of the Y-axis data in the selected curve.

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Smooth

This function performs a Savitzky-Golay smoothing of the selected curve.

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Truncate

Truncate is used to reduce the X-axis range on the selected curve. The selected region of the curve is preserved and all X values above and below this region are permanently deleted. The region may also be selected using the Mark Region icon in the toolbar and clicking and dragging the mouse over the desired range in the workspace.

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Baseline

This function is useful when noise in the baseline of the scatterer affects the lifetime of the sample. This happens because the IRF (scatterer) is convoluted with the sample lifetimes to give the observed decay. Thus noise in the scatterer is also convoluted and becomes a major problem for long-lived samples when observations are recorded out to many sample lifetimes.

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Peak Finder

This function finds the global peak as the highest Y-value and local peaks as being higher than immediate left and right neighboring points.

Global peak
The peak within the selected range with the highest Y-axis value.

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Analysis

The various analysis programs are accessed through a drop-down menu. Only users with the correct Customer Access Code can access them.

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Conversion: Energy to Quantum

Converts the selected spectrum from energy units to quantum units proportional to the number of photons per second.

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Conversion: Quantum to Energy

Converts the selected spectrum from quantum units, expressed as the number of photons detected at a given wavelength (or wavenumber), to energy units proportional to the number of photons detected at a given wavelength (or wavenumber) multiplied by the photon energy.

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Conversion: Wavelength to Wavenumber

Converts the selected trace from units of wavelength (nm) to wavenumber (1/cm). This command will also convert the trace to wavelength from wavenumber. Selection of which direction to convert is performed automatically.

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Fluorescence Resonance Energy Transfer (FRET)

The Fluorescence Resonance Energy Transfer (FRET) takes place between an excited donor molecule (D) and the ground-state acceptor molecule (A) over a range of distances, typically 10-100 Å. FRET is a non-radiative process (i.e. there is no photon emitted or absorbed during the energy exchange). The efficiency of FRET is strongly dependent on the D-A distance and is characterized by the Förster critical radius R_o, a unique parameter for each D-A pair. When the D-A distance is R_o, the efficiency of energy transfer is 50%. Once R_o is known, the D-A pair can be used as a molecular ruler to determine the distance between sites labeled by D and A.

FRET can access the FRET Calculator. The FRET drop down menu gives three choices: Determine Ro, Calculate FRET Parameters (steady- state) and Calculate FRET Parameters (lifetimes).

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Determine R_o

The Donor Emission button selects the curve to be used as donor emission spectrum. Select a curve by clicking on its name at the left side of the FeliX32™ screen and then click on the Donor Emission button. The name of the selected curve will appear on the box beside the button.

The Acceptor Absorption button selects the curve to be used as acceptor absorption (excitation) spectrum. Select a curve by clicking on its name at the left side of the FeliX32™ screen and then click on the Acceptor Absorption button. The name of the selected curve will appear on the box beside the button.

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Calculate FRET Parameters (steady-state)

The D only emission button selects the donor emission curve. Select a curve by clicking on its name at the left side of the FeliX32™ screen, then click on the D only emission button.

The D/A emission button selects the donor emission curve measured in the presence of acceptor. Select a curve by clicking on its name at the left side of the FeliX32™ screen, then click on the D/A emission button.

Range for D: To select the averaging range for the donor alone, click on the D radio button in the Range box, position the mouse pointer at the desired start of the integration, click and hold down the left mouse button, drag the mouse to the desired end of the range and release the button. The average intensity value for D will be displayed in the Intensity Values box. Alternatively, type in the start and end values for the range and click on the UPDATE button. The average D intensity will be displayed in the Intensity values box. If the averaging is to be carried out over the entire range, just click on the FULL button and the intensity will be captured and displayed.

Range for D/A: To select the averaging range for the donor in the presence of acceptor, click on the A radio button in the Range box, position the mouse pointer at the desired start of the integration, click and hold down the left mouse button, drag the mouse to the desired end of the range and release the button. The average intensity value for D/A will be displayed in the Intensity Values box. Alternatively, type in the start and end values for the range and click on the UPDATE button. The average D/A intensity will be displayed in the Intensity values box. If the averaging is to be carried out over the entire range, just click on the FULL button and the intensity will be captured and displayed.

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Transform Commands

Settings and controls that are common to all dialog boxes are presented together at the end of the chapter under the heading Common Transform Controls. The descriptions for the configuration dialog boxes that follow provide details on the specific math functions as well as settings and controls that are unique to them. For further information on commands in the Transform menu please see the online Help utility.

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Concentration Map

Concentration mapping is used to convert saved experimental data to concentration. The experimental data may be intensity or the ratio of two intensities.

Lookup Tables
Lookup tables can be constructed to calculate the concentration in several different ways.

Intensity to Concentration: For most steady state experiments, the intensity is related directly to concentration.

Ratio to Concentration: For most ratio fluorescence experiments, the ratio of two intensities is related to concentration.

Ratio to pH: Converts ratio values to pH.

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Polarization

This function is used for post-acquisition calculation of polarization or anisotropy from saved experimental data. Use the radio buttons to select the operation to perform anisotropy or polarization.

Config. G-Factor
The G Factor is used in calculating polarization or anisotropy. It is the ratio of the relative transmission efficiencies of the emission channel for horizontally and vertically polarized light. The G Factor can be measured with any sample. The excitation polarizer is rotated to the horizontal position. Emission is measured with the emission polarizer in the horizontal and vertical positions.

G = I(HV)/I(HH)

G-Factor: Enter a pre-determined G-Factor manually or highlight a region of a G-Factor curve (or select a curve) using Mark Region and select Capture. The average Y value over the selected range will be entered into the G-Factor text box. Prior to clicking Capture you can see the value that will be captured in the Capture Value text box. If you enter HV and HH values into their text boxes, the G-Factor will be calculated automatically.

HV: Select the curve from the legend having polarizers with horizontal excitation and vertical emission orientation. Click on Capture to enter the average value of the selected curve. Alternatively, enter an HV value manually or select a region of a curve using Mark Region and click Capture to acquire the region's average value into the text box. Prior to clicking Capture you can see the value that will be captured in the Capture Value text box.

HH: Select the curve from the legend having polarizers with horizontal excitation and horizontal emission orientation. Click on Capture to enter the average value of the selected curve. Alternatively, enter an HH value manually or select a region of a curve using Mark Region and click Capture to acquire the region's average value into the text box. Prior to clicking Capture you can see the value that will be captured in the Capture Value text box.

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Concentration Equation

The Concentration Equation establishes the conditions for converting intensity ratios directly to intracellular ion concentrations using the equation from Grynkiewicz, Poenie, and Tsien (J. Biological Chemistry, 260, 3440-3450(1985)) in the calculation of concentrations from ratio fluorescence data.

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Lookup Table

This command is used to construct a lookup table to relate intensities or ratios of intensities to concentration. A lookup table is a plot of intensity (or an intensity ratio) versus concentration or pH. The concentration of an unknown sample is calculated by measuring the intensity and interpolating between values on the lookup table to calculate the concentration. An LUT can also be used for calculating G-Factor in anisotropy and polarization experiments. The G-Factor LUT can similarly be reached under Function in the Display Setup dialog box or by selecting Configure G-Factor in Transform/Polarization or in a Timebased Polarization acquisition.

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Display Commands

The display of curves in the FeliX workspace is controlled by commands in the Display and Axes menus.

Normal View

Changes the display mode to a graphical plot of X and Y values. Curve(s) will be presented graphically. The X and Y scales can be adjusted in order to best display data.

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3D View

FeliX has been developed to provide scientists with a software package that aids the data plotting and visualization and helps present your work in its best light. And when you think of a new, better, or different way to present the data, FeliX gives you full control over 2D and 3D plot parameters. FeliX is a complete package that contains plotting software with extensive 2D and 3D capabilities for visualizing data from analyses, and experiments. Whether you're doing scientific analyses, or experiments, FeliX allows you to explore the data, produce informative 2D and 3D views and create presentation-quality plots and animations.

Viewing Style: Gives one the options of color and monochrome.

Font Size: You can select three different (small, medium, large) sizes for plot features such as title, axes titles.

Numeric Precision: Allows one to select the number of decimal places to plot the data to on all the axes.

Grid Lines: You can display grid lines on both axes, one individually or not at all.

Show Bounding Box: This option encloses the 3D plot in a cube which allows one a better appreciation of the depth being displayed. There are three choices under this selection; 1) While Rotating will display the bounding box only when the image is being rotated; 2) Always will display at all times; and 3) Never will disable this option. Rotation Animation: By selecting this option the 3D image is put in an animated environment where it rotates clockwise through a 360° angle in increments.

Rotation Increment: This option allows one to choose a particular angle rotation for the selected image. The following angles of rotation are available through selecting this option (15, 10, 5, 2, 1, -1, -2, -5, -10, -15).

Rotation Detail: This option lets you set how much detail is shown during graph rotation.

Plotting Method: This option gives the following choices for plotting the data: wireframe, surface, surface with shading, surface with contouring, and pixels.

Shading Style: There are white and various color types of shading available under this option.

2D Contour: The Contour option performs the calculations on the data allowing the representation to be projected onto either equal angle or equal area stereograms. The contour option allows the user to set contour lines on top or bottom as well as color or black and white contours.

Maximize: Maximize viewing area for plot.

Customization Dialog: This dialog box provides the user with more options in customizing the looks of the generated plot. This menu has submenus that set other plot parameters, such as font style, plot style, color, etc.

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Annotations

Use Annotation to add boxes, data pointers and text directly to the experimental output. These annotations are attached to the X-Y coordinates of the dataset and are disabled in Grid View and 3D View. If the particular X-Y coordinates of an annotation are off the display then so will be the annotation.

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Toggle Visibility

Use this toolbar command to toggle the display of the selected curve(s). When a curve is visible in the workspace, the trace name will be in bold color in the legend. When the curve is hidden, the name will appear as plain gray text. Multiple curves (hidden, visible, and mixed sets) can be toggled at one time. Selecting a group or multiple groups enables the user to Hide All curves, Show All curves, or toggle the visibility of all curves within the group(s). Hide All and Show All commands are located in a user menu that can be found by right clicking on one of the selected group names.

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Display Options

Viewing Style: Gives one the options of color and monochrome.

Font Size: You can select three different (small, medium, large) sizes for plot features such as title and axes values and titles.

Numeric Precision: Allows one to select the number of decimal places to plot the data to on all the axes.

Plotting Method: Select the method for which FeliX32™ will plot the data. Options include point, line, area, stick, points + best fit line, points + best fit curve, points and line, points and spline.

Data Shadows: Shadows can be selected as normal shadows, 3D, or toggled off.

Grid Lines: You can display grid lines on both axes, one individually or not at all. Grid in Front: Toggle to overlay or underlay the grid lines on the graph.

Mark Data Points: When toggled on, this command will display the data points in the plots marking them clearly visible with all plotting methods.

Show Annotations: Toggles the visibility of the annotations.

Undo Zoom: Selecting this command when zoomed in on an area of the graph will re-expand the plot to the set Y and X-axes values (depends on the axes settings for example, Full Autoscale, Autoscale from 0, Fixed Y- Min & Max, etc.).

Customization Dialog: This dialog box provides the user with more options in customizing the looks of the generated plot. This menu has submenus that set other plot parameters, such as font style, plot style, color, axis range, etc.

Export Dialog: Allows the 3D plot and key areas to be exported as Windows ordinary and enhanced metafiles (example: Bitmap and JPEG). These can be imported into many applications including CorelDraw, Word, etc. Select the export destination (clipboard, folder, or printer) and the image size. Click Export to complete the operation if the destination is either the clipboard or the printer. Click Save in the Windows save dialog box to export the image to a folder on the hard drive or disk drive.

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Data Analysis

Perhaps the most important aspect of the TimeMaster portion of the FeliX32™ software after data collection is data analysis. This chapter is devoted to this very important topic.

The various methods of data analysis are found under Math Analysis. They are: 1 To 4 Exp. Lifetime Multi 1 To 4 Exp. Global 1 To 4 Exp. Anisotropy Decays Micelle Kinetics ESM, MEM Non Exponential DAS/TRES These methods are covered in separate sections that are independent of each other. Thus, only the section of interest needs to be read. However, it is recommended that the General Introduction is read first as most of the concepts and topics used in the other sections are introduced there.

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1 To 4 Exponential Lifetime

This is the simplest and arguably the most generally useful of the fitting procedures. It is suitable for the analysis of fluorescence decays consisting of up to 4 exponentials and associated pre-exponentials. Fitting Function This analysis program can fit up to a 4 exponential decay that follows the fitting law: Eq. 1 where D(t) is the delta function generated decay at time t. This fitting function allows for negative ai's so that rise times can also be determined with this program.

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Multi 1 To 4 Exponential

The multiple one to four exponential lifetime method, as its name implies, allows the analysis of multiple scatterer/sample pairs at the same time. Each pair will be separately analyzed over the same range with the same number of exponentials and the same options. The analysis results in a set of parameters (lifetimes and pre-exponential factors) for each data pair. The theory for this method is exactly the same as that for the 1 To 4 Exp. Lifetime method. This type of analysis is useful when a series of otherwise identical decay curves has been collected as a function of some parameter (temperature, composition or wavelength for example). Trends in the values of the lifetime parameters may then be recognized rather easily.

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Global 1 To 4 Exponential

This analysis program provides for the analysis of up to 4 exponential decays for a number of data files simultaneously. The global analysis assumes that the lifetimes are linked among the data files but that the associated pre-exponentials are free to vary.

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Anisotropy Decays

This program allows for the calculation of up to four rotational correlation times plus a residual anisotropy term. The program first allows the user to calculate the fluorescence lifetime(s) from the parallel and perpendicularly polarized emission intensities. The user can then calculate the rotational correlation time(s). The Configure G-Factor dialog box is shown at left. The G-factor may be entered directly into the G-factor text box or captured from HV and HH decays. To capture the G-factor select the HV curve in the left legend and click on the Curve HV Pick button. Select the HH curve in the left legend and click on the Curve HH Pick button. Select the region of the curves to be used in calculating the G-factor in the normal manner (usually this is the whole decay curve). The ratio of the integrals under the HV and HH curves is displayed in the Capture text box. Click on Capture to accept this value for the G-factor. It will be displayed in the G-Factor text box. Click OK to return to the previous dialog box.

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Micelle Kinetics

This program allows for the analysis of quenching processes in micelles.

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Non-Exponential Decay

This program allows for the analysis of data by a general fitting function consisting of two exponentials multiplied together each with variable exponents of time. The exponents can be either varied or fixed which provides a powerful general function for models such as Förster energy transfer and time-dependent quenching.

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ESM - Exponential Series Method

Fluorescence lifetime measurements often result in complex decays requiring a more sophisticated approach than a single or double-exponential fitting function (James and Ware, 1986, Siemiarczuk et al., 1990). This applies especially to the emission originating in such intrinsically complex systems as:

• Bichromophoric molecules exhibiting distributions of conformers in the excited state
• Fluorophores adsorbed on surfaces
• Ffluorophores attached to polymers
• Fluorescent probes in micelles and liposomes
• Ffluorescent probes in biomembranes and other biological systems
• Fluorophores in monolayers
• Iintrinsic fluorescence from proteins
• Systems undergoing Förster-type energy transfer
• And many others.

Even intuitive considerations would lead one to expect distributions of lifetimes in these systems. Quite often, however, especially for low precision data, a good fit can be obtained with a double- or triple-exponential function for a system, which in fact represents a continuous distribution of lifetimes. In general, however, the parameters recovered from such a fit have no physical meaning. The Exponential Series Method (ESM) is designed to recover lifetime distributions without any a priori assumptions about their shapes. This method uses a series of exponentials (up to 200 terms) as a probe function with fixed, logarithmically-spaced lifetimes and variable pre-exponentials. This allows covering a lifetime range of several orders of magnitude. In many situations the ESM is capable of differentiating between continuous distributions and discrete, multi-exponentials decays.

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MEM - Maximum Entropy Method

Fluorescence lifetime measurements often result in complex decays requiring a more sophisticated approach than a single or double-exponential fitting function (James and Ware, 1986, Siemiarczuk et al, 1990). This applies especially to the emission originating in such intrinsically complex systems as:

• Bichromophoric molecules exhibiting distributions of conformers in the excited state
• Fluorophores adsorbed on surfaces
• Fluorophores attached to polymers
• Ffluorescent probes in micelles and liposomes
• Fluorescent probes in biomembranes and other biological systems
• Fluorophores in monolayers
• Intrinsic fluorescence from proteins
• Systems undergoing Förster-type energy transfer
• And many others.

Even intuitive considerations would lead one to expect distributions of lifetimes in these systems. Quite often, however, especially for low precision data, a good fit can be obtained with a double- or triple-exponential function for a system, which in fact represents a continuous distribution of lifetimes. In general, however, the parameters recovered from such a fit have no physical meaning. The Maximum Entropy Method (MEM) is designed to recover lifetime distributions without any a priori assumptions about their shapes (Skilling and Bryan 1989, Smith and Grady, 1985). This method uses a series of exponentials (up to 200 terms) as a probe function with fixed, logarithmically-spaced lifetimes and variable pre-exponentials. This allows covering a lifetime range of several orders of magnitude. In many situations the MEM is capable of differentiating between continuous distributions and discrete, multi-exponentials decays.

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DAS/TRES

As discussed in the General Introduction, the analysis of time domain data acquired using a pulsed light source is complicated by convolution with the intensity profile of the light source. This is true both for decays and for time resolved spectra and is particularly serious at delay times short compared to the width of the exciting pulse. FeliX32™ allows the direct acquisition of time resolved spectra (called gated spectra for phosphorescence modes) but it must be remembered that these must suffer to some extent from convolution caused distortion. In many cases, the convenience of the direct acquisition of time resolved spectra far outweighs the effect of distortion at short time scales particularly when only qualitative comparisons are required.

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