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The Fluorescence Solutions Company

 

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LaserStrobe™
Fluorescence Lifetime Spectrofluorometer

 
  Software
 
Fluorescence Lifeteimes
 
Fluorescence Lifetime quote
 
Fluorescence Lifetime Brochure
 
Imaging System One Page Flyer
 
Fluorescence Lifetimes
 

Software

PTI offers a comprehensive set of the most advanced luminescence decay data analysis programs available. All of these programs are available as part of PTI's one comprehensive software package, FeliX32™. The analysis software can be purchased for use with systems from other manufacturers, as long as you format the data according to instructions available from PTI. All of the programs can perform decay curve analysis with or without deconvolution. The data spacing (time axis) can be arbitrary. The package includes the following programs:

  • Exponential decay analysis (1 to 4 exponentials)
  • Global exponential analysis (1 to 4 exponentials)
  • Energy transfer kinetics (general exponents)
  • Micelle kinetics (Tachiya-Infelta-Graetzel model)
  • Anisotropy decay kinetics
  • Exponential Series Method (ESM) lifetime distribution analysis
  • Maximum Entropy Method (MEM) lifetime distribution analysis
  • Time-Resolved and Decay-Associated spectra

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  

 

Fluorescence Decay: Laser

Int Time
This is the time window in microseconds within which the signal is integrated for each laser pulse. The window should be long enough so that the emission signal is fully contained within it. Set this parameter to 50 µs.

Shots
Enter the number of laser shots to be collected and averaged at each delay for each scan. Extra shots will improve the signal to noise ratio at the expense of additional acquisition time. For statistical reasons, it is generally preferable to average over several scans than over more shots on a single scan. Thus averaging three scans with five shots each scan is better than one scan with fifteen shots.

Frequency
This determines the frequency of laser firing and may be set at up to 20 Hz. Higher frequencies shorten the time required to acquire decay data. However, the consumption of nitrogen gas increases substantially at higher frequencies and the energy per pulse drops. Ten pulses per second is a reasonable choice for most experiments.

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Phosphorescence Decay: Laser

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Int Time
This is the width of the integration window for each laser pulse. Since, in this case, the observation window is defined by the integration time it is normal to choose the integration time to be comparable to the channel spacing. Choosing an integration time of 100 µs when the channel spacing is only 1 µs loses time resolution while choosing an integration time of 1 µs when the channel spacing is only 100 µs loses sensitivity.

Shots
Enter the number of laser shots to be collected and averaged at each delay for each scan. Extra shots will improve the signal to noise ratio at the expense of additional acquisition time. For statistical reasons, it is generally preferable to average over several scans than over more shots on a single scan. Thus averaging three scans with five shots each scan is better than one scan with fifteen shots.

Frequency
This determines the frequency of laser firing and may be set at up to 20 Hz. Higher frequencies shorten the time required to acquire decay data. However, the consumption of nitrogen gas increases substantially at higher frequencies and the energy per pulse drops. Ten pulses per second is a reasonable choice for most experiments.

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Time Resolved Spectra/Gated Scans

In time resolved spectra, the delay and the position of one monochromator are held fixed while the other monochromator is scanned between two wavelengths. Selecting different decay times along the decay curve can be useful for exciting individual species within a sample mixture. For laser systems (fluorescent or phosphorescent) and for nanosecond flash lamp systems, only emission spectra are supported. For xenon flash lamp systems, gated emission and gated excitation scans are supported. The selections common to all systems are listed below.

Background
Check the Acq. box to acquire the background when the acquisition is started. On subsequent scans the background acquisition checkbox is automatically cleared and the just-acquired background values will be subtracted from the current measured values in real time. The background correction values will remain in effect for subsequent measurements until cleared in the Display Setup dialog box, or by removing the check mark in the Use Background checkbox located beside the Acq. Background check control, or by checking the Acq. Background checkbox, which will clear the previous value and force a new background to be acquired. Toggling Use Background keeps the background value in memory for future use. It is important to measure the background during the first scan, otherwise the signal may be distorted. This function only measures the electrical background on the signal integrator, i.e. it measures the pre-acquisition signal before the light source is fired. It does not account for an optical background due to stray light, solvent, etc. It is important to re-measure the background every time the integration time is changed.

Excitation
Enter the excitation wavelength (nm) in the text box. If your instrument has an excitation monochromator, this will be the wavelength used for the spectrum.
If your instrument has a dye laser but no excitation monochromator, enter the reading on the dye laser counter (half this value with a frequency doubler). This will have no effect on the hardware but allows the excitation wavelength to be recorded with the spectrum.
If your instrument uses filters to select the excitation wavelength, enter the filter's center wavelength. This will have no effect on the hardware but allows the excitation wavelength to be recorded with the spectrum.

Emission
This option is only used for gated excitation scans (Xenon flash lamp). Enter the emission wavelength (nm) in the text box. If your instrument has an emission monochromator, this will be the wavelength used for the spectrum. If your instrument uses filters to select the emission wavelength, enter the filter's center wavelength. This will have no effect on the hardware but allows the emission wavelength to be recorded with the spectrum.

Start/Stop
Enter the wavelengths (nm) between which the spectrum will be run. These will be excitation wavelengths for gated excitation scans (Xenon flash lamp) and emission wavelengths for all other time resolved scans.

Step Size
Enter the step size (nm) to be used in recording the spectra. The minimum value is 0.25 nm with a standard grating monochromator.

Delay
Enter the delay at which the detection window will be opened. For fluorescence systems this is in nanoseconds. For phosphorescence systems this is in microseconds. Phosphorescence systems may be used to acquire fluorescence and phosphorescence spectra by choosing the appropriate delay and integration time. See the discussion under Int Time for details.

Averages
Enter the number of complete scans to be averaged to give the final spectrum.

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Fluorescence Time Resolved Spectra: Laser

Int Time
This is the time window in microseconds within which the signal is integrated for each pulse. The window should be long enough so that the emission signal is fully contained within. Set this parameter to 50 µs.

Shots
Enter the number of laser shots to be collected and averaged at each delay for each scan. Extra shots will improve the signal to noise ration at the expense of additional acquisition time. For statistical reasons, it is generally preferable to average over several scans than over more shots on a single scan. Thus averaging three scans with five shots each scan is better than one scan with fifteen shots.

Frequency
This determines the frequency of laser firing and may be set at up to 20 Hz. Higher frequencies shorten the time required to acquire decay data. However, the consumption of nitrogen gas increases substantially at higher frequencies and the energy per pulse drops. Ten pulses per second is a reasonable choice for most experiments.

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Phosphorescence Steady State Emission Scan: Laser (Gated Emission Scan)

Int Time
This is the width of the integration window for each laser pulse. Since, in this case, the observation window is defined by the integration time, increasing the integration time will increase the signal at the expense of lifetime resolution while decreasing the integration time will increase the lifetime resolution at the expense of signal strength. In particular, when the instrument is being used to separate fluorescence spectra from phosphorescence spectra, care must be used in selecting the integration time. Since fluorescence is essentially over in the first 5 to 10 µs after the excitation pulse, the delay should be set to the excitation peak and the integration time to 5 to 10 µs. Longer integration times will contaminate the fluorescence with phosphorescence. When collecting phosphorescence, the delay should be set 5 to 10 µs after the excitation pulse and the integration time chosen to be larger to maximize sensitivity.

Shots
Enter the number of laser shots to be collected and averaged at each delay for each scan. Extra shots will improve the signal to noise ration at the expense of additional acquisition time. For statistical reasons, it is generally preferable to average over several scans than over more shots on a single scan. Thus averaging three scans with five shots each scan is better than one scan with fifteen shots.

Frequency
This determines the frequency of laser firing and may be set at up to 20 Hz. Higher frequencies shorten the time required to acquire decay data. However, the consumption of nitrogen gas increases substantially at higher frequencies and the energy per pulse drops. Ten pulses per second is a reasonable choice for most experiments.

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Fluorescence Timebased: Laser

PTI

Points/sec
The value in this box is completely defined by the choice of shots and frequency and cannot be chosen directly.

Int Time
This is the time window in microseconds within which the signal is integrated for each laser pulse. The window should be long enough so that the emission signal is fully contained within. Set this parameter to 50 µs.

Shots
Enter the number of laser shots to be collected and averaged for each point for each scan. Extra shots will improve the signal to noise ratio at the expense of time resolution. When using a timebased experiment to adjust the instrument hardware this value is set rather low so that the effects of adjustments can be seen quickly.

Frequency
This determines the frequency of laser firing and may be set at up to 20 Hz. Higher frequencies shorten the time required to acquire data and can improve time resolution. However, the consumption of nitrogen gas increases substantially at higher frequencies and the energy per pulse drops. Ten pulses per second is a reasonable choice for most experiments.

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Phosphorescence Timebased: Laser

Points/sec
The value in this box is completely defined by the choice of shots and frequency and cannot be chosen directly.

Int Time
This is the width of the integration window for each laser pulse. Since, in this case, the observation window is defined by the integration time, increasing the integration time will increase the signal at the expense of lifetime resolution while decreasing the integration time will increase the lifetime resolution at the expense of signal strength. In particular, when the instrument is being used to separate fluorescence spectra from phosphorescence spectra, care must be used in selecting the integration time. Since fluorescence is essentially over in the first 5 to 10 µs after the excitation pulse, the delay should be set to the excitation peak and the integration time to 5 to 10 µs. Longer integration times will contaminate the fluorescence with phosphorescence. When collecting phosphorescence, the delay should be set 5 to 10 µs after the excitation pulse and the integration time chosen to be larger to maximize sensitivity.

Shots
Enter the number of laser pulses to be collected and averaged at each point for each scan. Extra shots will improve the signal to noise ration at the expense of time resolution. When using a timebased experiment to adjust the instrument hardware this value is set rather low so that the effects of adjustments can be seen quickly.

Frequency
This determines the frequency of laser firing and may be set at up to 20 Hz. Higher frequencies can improve time resolution. However, the consumption of nitrogen gas increases substantially at higher frequencies and the energy per pulse drops. Smaller frequencies may be useful when very long timebases are run, otherwise extremely large amounts of data will be collected.

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Data Collection Options

This dialog box controls the several aspects of how decay data are collected. These controls are available only for fluorescence and phosphorescence decay acquisitions.

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Scan Type
Radio buttons allow the selection of Automated or Manual when a four position turret is installed.

Automated: Collects data for the selected positions of the four position turret without pausing between samples. This option is useful when all the samples can be run with the same instrument settings.

Manual: Pauses data collection after each change in the four position turret's position. This allows the user to change neutral density filters etc between samples. Acquisition is continued by pressing the continue button.

Collect Mode
Radio buttons allow the selection of Sequential or Random, which controls the order in which data points are collected.

Sequential: Causes the data to be collected in "conventional" order, i.e. from the shortest delay to the longest delay.

Random: Causes the data to be collected in random order. This can be useful in situations where photochemical reactions are suspected of producing systematic effects on sample lifetimes.

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Collect Step

Radio buttons allow the selection of Linear, Arithmetic or Logarithmic, which controls the spacing between consecutive time delays.

Linear: The conventional choice and divides the time between the start delay and end delay into equal time increments.

Arithmetic: Adds a constant time increment on to the previous time step to obtain the next time step. Thus the time between data points increases as the delay increases.

Logarithmic: Multiplies the previous time step by a constant factor to obtain the next time step. With this option, time between data points increases even faster than it does with the Arithmetic option.

The Arithmetic and Logarithmic options are particularly useful when the sample decays with several very different lifetimes. In such cases, it may be necessary to have good data at both short and long time delays. Good data at short time delays could be obtained by choosing Linear and a small time increment. However, this would require many channels for this small time increment to be extended to long delays. Choosing Arithmetic or Logarithmic concentrates the points in the short delay region but still gives coverage in the long delay region.

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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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Acquisition Preferences

Several aspects of the way FeliX32™ looks and behaves can be adjusted to suit the user. All changes made in this menu are automatically applied to all acquisitions. Selecting View then Preferences in an Acquisition dialog will access the dialog box.

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Acquisition

Reset clock for Time-Based acquisitions: Selecting this option will reset the time counter in the status window to zero at the beginning of each acquisition cycle.

Manual Pause pauses acquisition clock: If pause is selected from the acquisition dialog window during an experiment the clock will also be paused. The counter will resume upon selecting continue.

Display manual polarizer cues: If the hardware configuration used for the acquisition contains manual polarizers this option will create pop up windows to inform the user when and by what degree to rotate the polarizers during the experiment.

Polarizer calibration before starting acquisition: This forces the motorized polarizers to confirm their angle against the optical encoder prior to starting an experiment. This feature adds to the time before the experiment is actually run.

Temperature

Delta: This function is utilized during temperature controlled experiments. Once the temperature controller is within this range, FeliX32™ will allow the user to start the acquisition. The controller temperature will continue towards its set temperature. If an external temperature probe is used such as the DP41, the acquisition will be allowed to start once the temperature of the probe is within the delta range of the controller temperature. The controller temperature must first reach its set temperature before FeliX32™ looks for agreement with the temperature of the probe. In both cases, a smaller delta will produce greater precision of temperature. However, due to heat transfer, especially at the extremes, the sample temperature may never reach the set temperature. If the delta is set too small, it may take an excessive amount of time for the range to be breached. Testing with a dummy sample is encouraged to determine the best tradeoff between waiting on the experiment and temperature accuracy.

Units: Sets the default units for temperature control. The user can select Kelvin (K), Celsius (°C), or Fahrenheit (oF).

Clock Display

Select hours:minutes:seconds or absolute seconds to be displayed in the acquisition status window.

Default Settings

Store Acquired Data: Select yes, no, or ask.

Name of Dataset: Enter the default name of new acquisition datasets. This name will be used if Auto Generated Name is not selected. If it is selected, the database will be titled with the type of acquisition and the date and time. Add to Open Acquisition will force acquisitions with the same dataset name (entered in the Display Setup menu) to have their trace groups inserted into the open dataset. If not selected, a new dataset with the same name will be created. This option is irrelevant if Auto Generated Name is selected because the name constantly changes with the time.

Background

Select the number of data points to be acquired that will be averaged to form the background value.

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TimeMaster Output

When doing any kind of Data Analysis (except for DAS/TRES) a notepad window named TimeMaster Outputs pops up containing identification information, fitted parameters and various statistics associated with the fit. Since this is a Notepad window, the text may be edited, saved or printed as the user desires. The results are not deleted from this window when another analysis is run. This feature allows the results of several analyses to be combined. However, this feature may also lead to very long files if many trial analyses are run without clearing the window.

Identification Information

  • Analysis Function: Type of analysis.
  • Curves: Curve names the analysis is based on.
  • Time Range: Characterized by Start Time and End Time.
  • Start Parameters: Fixed or floating start values of the used parameters.

Statistic Results

  • Fitted Curve: Curve generated by the fitting procedure.
  • Residuals: Curve displaying the difference between the calculated fit and the real data.
  • Autocorrelation: Autocorrelation curve.
  • Deconvoluted: Deconvoluted curve.
  • Chi2: Chi Square Statistic for testing correlation.
  • Durbin Watson: Durbin-Watson parameter for testing correlation.
  • Z: Parameter expressing the result of a Runs Test.
  • Pre-exponential: Defined as ai in the equation I(t) = å[(ai)exp(-t/ti)], where t is time and ti is the lifetime.
  • Lifetime: Defined as ti in the equation I(t) = å [(ai)exp(-t/ti)], where t is time and ai is the pre-exponential factor.
  • F1: Relative integrated intensities defined as Fi= [(ai)(ti)]/[å(ai)(ti)], where ai and ti are the pre-exponential factors and lifetimes, respectively.
  • Tau-av1: Steady state average lifetime defined as Tau-av1= å[(ai)(ti)²]/å[(ai)(ti)], where ai and ti are the pre-exponential factors and lifetimes, respectively.
  • Tau-av2: Amplitude average lifetime defined as Tau-av2= å[(ai)(ti)]/å(ai), where ai and ti are the pre-exponential factors and lifetimes, respectively.
  • Fitted Parameters: Values and deviations of the curve parameters resulting from the fit.

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TimeMaster Results

TimeMaster decay fits that are saved using the Save Results button can be opened and viewed from this menu. The output results are listed in alphabetical order in the display window along with the fitting procedure, user and last date modified. Once a file has been selected, the user can view and export the data.

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Import: TimeMaster results from other FeliX programs or workstations can be imported by selecting this button. A typical Windows open file window will appear allowing the appropriate file to be selected. Files of extension .res, .exp, .mex, .glo, .ast, .mkn, .esm, .mem, and .nex can be imported into Felix32™.

Export: Opens a typical Windows file save window. Results will be saved as TimeMaster data with the extension res.

Refresh: Updates the window to show the influence of any changes to the listed files.

Show Results: Opens a TimeMaster Output window where the data can be viewed and exported as a text file.

Delete: Erases the file from the database.

Filter: Can be used to sort the TimeMaster result files based on fitting procedure, date, and/or the user.

Close: Closes the TimeMaster Results window.

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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.
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.

 

 

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Combine

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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 . These programs are discussed in Data Analysis.

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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 Ro, a unique parameter for each D-A pair. When the D-A distance is Ro, the efficiency of energy transfer is 50%. Once Ro 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 Ro

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)

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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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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 Ro or retain the value calculated in the Determine Ro option.

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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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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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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

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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. 1 To 4 Exp. Lifetime
  2. Multi 1 To 4 Exp.
  3. Global 1 To 4 Exp.
  4. Anisotropy Decays
  5. Micelle Kinetics
  6. ESM, MEM
  7. Non Exponential
  8. 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:

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Eq. 1 where D(t) is the delta function generated decay at time t. This fitting function allows for negative ai's so that risetimes can also be determined with this program.

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Multi 1 To 4 Exponential

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The multiple file 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

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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
  • Fluorophores attached to polymers
  • Fluorescent 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 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
  • 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.

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DAS/TRES

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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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