PTI--RatioMaster™

Software

FeliX32™ software represents the ultimate workhorse platform for professional live cell experiments, setting the standard for reliability, efficiency, and integration with a multi-wavelength illuminator.

Felix Software Tutorials

From fixed cell preparations to dynamic events, FeliX32™ fluorescence software represents the ultimate, expandable platform for professional live cell single region of interest applications. There is no other system that delivers the sensitivity, flexibility, and capability of options as the FeliX32™ software platform.

Excitation Ratio
Emission Ratio
Excitation Scan
Emission Scan
Multiple Dyes
Timebased
Fluorescence Resonance Energy Transfer (FRET)
Calculate FRET Parameters (steady-state)
Calculate FRET Parameters (lifetimes)
Determine R_o
Transform Commands
Concentration Map
Polarization
Concentration Equation
Lookup Table
Data Analysis
Micelle Kinetics
Non-Exponential Decay
ESM - Exponential Series Method
MEM - Maximum Entropy Method
DAS/TRES

Common Math Controls

Antilog	Average	Combine
Combine Constant	XY Combine	Integrate
Linear Fit	Differentiate	Linear Scale
Logarithm	Normalize	Reciprocal
Smooth	Truncate	Baseline
Peak Finder	Analysis	Logarithm
Conversion: Energy to Quantum	Conversion: Quantum to Energy	Conversion: Wavelength to Wavenumber

Display Commands

Normal View	3D View	Annotations
Toggle Visibility	Display Options

About the Software
From fixed cell preparations to dynamic events, FeliX32™ software represents the ultimate, expandable platform for professional live cell imaging applications. There is no other system that delivers the sensitivity, flexibility, and breadth of options of FeliX32™ software.

Key Features

Peace of Mind

Day to Day Routines

Non-Destructive
Modular Design

RatioMaster
Security Administrator Tools
All multi users to be set up with levels of authorization ability.

Protocol Template
Common experiments can be saved as a template and run with one button operation.

Data Integrity
All data is saved to ODBC database structure.Can be exported to common formats

Expandable
All PTI optical building blocks can be controlled by FeliX32™.Configure your system and go!

Key Feature:
PTI's BryteBox data acquisition computer streams data to host computer for acquisition. This gives the user the benefit of running experiments without worrying about host computer interruptions or the limitation of the computer. You can high speed stream and control TTL inputs and outputs with minimal latency. Competitive systems offer add in cards or USB modules that become outdated fast, due the ever changing computers. Not with PTI BryteBox

Key Feature:
PTI knows how valuable your data is, which is why we use a database to archive and store experiment data. The raw data integrity is kept, allowing the researcher to experiment with peace of mind that their data remains in its original form on the hard drive.

Key Feature:
FeliX32™ software also allows for the maximum flexibility for your future needs with our modular software architecture. Components can be added to the system to increase functionality. This feature allows researchers to get started inexpensively and then add functionality later as their research grows.

Peace of Mind
A database file keeps track of all experiment data on the hard disk or server. Allowing you to never worry about where you saved your data. The database keeps track of all hardware configurations, protocols, wavelength, channels, integration times, and all vital controls.

Day to day routine
Session templates for common experiments are provided and you can create your own templates. Ready to run common experiments “out of the box” with minimal input from the user. Just push acquire and go!

Other Features of the software:

Square, area of interest photometry in real-time or in post acquisition.
Trace math analysis functions such as anti-log, average, combine, XY combine, differentiate, integrate, and linear fit, peak finder.
Real-time generation of user defined event markers and event journaling.
Real-time non-destructive ratio-metric calculation, and background subtraction.
Export of data to popular formats such as ana, ang, spc or text files.
Control of up to 10 excitation, emission, or unlimited derived channels.

You need an RatioMaster if:
Collection and analysis of ratio-metric data for Calcium, pH and intracellular ion imaging are important to you. PTI has poured all of our 20 years of ratio-metric imaging expertise into this new package. The package is modular and expandable offering flexibility for different applications. It allows researchers get "control back" into their research by reducing the learning curve associated with complex general purpose imaging platforms. This allows researchers to focus on their research application specifically, rather than a complex system with difficult learning curve.

Modular Design
If the out of the box acquisition protocols do not quite fit your applications, the PTI Macro editor allows the user to create their own specialized experiments by drag and dropping the command library into a loop logic. You do not have to be a programmer or understand a scripting language to implement these macros. You simply select the function you want, then apply it to the loop. This allows the researcher to increment the start excitation wavelength, emission wavelength, number of points per second, increase temperature, add solutions with a titrator and a host of other acquisition modes. The macro editor is simply one of the easiest to use ever designed by PTI!

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 V can provide up to 250 ratios/sec while the DeltaScan X can produce ultra-fast switching allowing for 650 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.

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.

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.

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.

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.

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.

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.

Back to Common Math Controls.

Antilog

Calculates the antilogarithm of the selected curve.

Back to Common Math Controls.

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.
PTI The standard deviation is also determined using the equation: Where xi is a data point and n is the total number of data points in the portion of the data trace being averaged.

Back to Common Math Controls.

Combine

PTI
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.

Back to Common Math Controls.

Combine Constant

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

Back to Common Math Controls.

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.

Back to Common Math Controls.

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.

Back to Common Math Controls.

Linear Fit

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

Back to Common Math Controls.

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.

Back to Common Math Controls.

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.

Back to Common Math Controls.

Reciprocal

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

Back to Common Math Controls.

Smooth

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

Back to Common Math Controls.

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.

Back to Common Math Controls.

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.

Back to Common Math Controls.

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.

Back to Common Math Controls.

Analysis

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

Back to Common Math Controls.

Conversion: Energy to Quantum

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

Back to Common Math Controls.

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.

Back to Common Math Controls.

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.

Back to Common Math Controls.

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 R_o, Calculate FRET Parameters (steady- state) and Calculate FRET Parameters (lifetimes).

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.

PTI

Calculate FRET Parameters (steady-state)

PTI

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.

Calculate FRET Parameters (lifetimes)

PTI

In the Input box, enter the lifetime value of donor in the presence of acceptor (tDA) and the donor alone (tD). At the bottom of the Input box, either enter the value of R_o or retain the value calculated in the Determine R_o option.

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.

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.

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.

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.

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.

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.

Back to Display Commands.

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: wire frame, 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.

Back to Display Commands.

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.

Back to Display Commands.

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.

Back to Display Commands.

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.).

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.

Back to Display Commands.

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.

Micelle Kinetics

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

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.

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
Fluorescent probes in biomembranes and other biological systems
Ffluorophores 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 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.

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
Fluorescent probes in micelles and liposomes
Ffluorescent 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.

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..