An example view of the Raw data chart is given below:
Local menu of the Raw Data chart
This local menu contains following items:
Customize markers: Opens Markers editor for changing style of marker lines and setting marker positions.
Standard local menu commands of 2D Chart
Statistical characteristics panel
Allows to calculate the statistical characteristics from the selected range (with help of data markers) of raw data and manage them.
It is possible to calculate:
- Autocorrelation function (linear, quasi-logarithmic and quasi-logarithmic without rebinning, i.e. calculated with the same bin time for all points)
- Crosscorrelation function (linear, quasi-logarithmic, quasi-logarithmic without rebinning, i.e. calculated with the same bin time for all points, and quasi-logarithmic with leaking correction)
- Inter-Event Time distribution (linear or quasi-logarithmic)
- Photon Counting distribution
- First Event distribution
- Single Event distribution
- Fluorescence Factorial Cumulants
- Intensity
- Coincidence Values histogram
An example view of the Statistical characteristics panel is given below:
Click the corresponding item on the picture displayed above to get more information about it.
Available data
Allows to select the active channel (for further calculation of characteristics).
Number of points
Allows to specify number of points of statistical characteristic to be calculated. For autocorrelation and crosscorrelation Number of points equals the number of time lags. For fluorescence factorial cumulants Number of points is the number of calculated cumulants. For the other types, Number of points equals the number of bins of the correspondent histogram.
Time step
The meaning of Time step depends on type of the calculated characteristic. For autocorrelation and crosscorrelation functions and coincidence histogram Time step has the meaning of time lag (for logarithmic scale - minimal one). For PCD and FFC Time step has the meaning of Time Window (bin time). For the other types Time step has the meaning of bin time of the correspondent histogram.
Note: for coincidence histogram Time step has the meaning of time lag at which the coincidence values are calculated but not the bin time of the histogram.
Weight
Set on/off the calculation of weight factors.
For autocorrelation and crosscorrelation weight is calculated by the algorithm proposed by T.Wohland. The standard deviation in Fluorescence Correlation Spectroscopy// Biophysical Journal, Volume 80, June 2001, Pages 2987-2999 (third method).
For FFC the weight is calculated as standard deviations of factorial cumulants.
For the other types of statistical characteristics the weight is calculated assumed the binomial statistics of data in a channel of the correspondent histogram.
Count
The meaning of Count depends on type of calculated characteristic. For autocorrelation and crosscorrelation this up/down edit allows to specify the number of parts on which the raw data time trace is subdivided for weight calculation (for better statistic it should be not less then 6). For Coincidence histogram Count specifies the number of parts from which the coincidence values are calculated (actually it specifies the total number of coincidence values used for building the histogram).
Filter combobox
Allows to filter the Files table by the type.
Button "AC"
Calculates the autocorrelation function (AC). It is possible to choose linear time scale or quasi-logarithmic time scale with time lag increasing with time.
Quasi-logarithmic without averaging autocorrelation function is calculated at increased time lags (quasi-logarithmic time scale) but without rebinning, i.e. with initial bin width. It allows to minimize triangular averaging effect.
Typical TimeStep (time lag) value for FCS measurements is 2E-7 sec.
"AC Linear"
Calculates the autocorrelation function with constant time lag.
Typical TimeStep (time lag) value for FCS measurements is 2E-7 sec.
"AC Logarithmic"
Calculates the autocorrelation function based on quasi-logarithmic time scale with time lag increasing with time.
Typical TimeStep (time lag) value for FCS measurements is 2E-7 sec.
"AC Logarithmic (without averaging)"
Calculates the autocorrelation function based on quasi-logarithmic time scale. It is calculated at increased time lags but without rebinning, i.e. with initial bin width that allows to minimize triangular averaging effect.
Typical TimeStep (time lag) value for FCS measurements is 2E-7 sec.
Button "CC"
Calculates the crosscorrelation function (CC). It is possible to choose linear time scale or quasi-logarithmic time scale with time step increasing with time.
Quasi-logarithmic without averaging crosscorrelation function is calculated at increased time lags (quasi-logarithmic time scale) but without rebinning, i.e. with initial bin width. It allows to minimize triangular averaging effect.
Typical TimeStep value for FCS measurements is 2E-7 sec.
"CC Linear"
Calculates the crosscorrelation function with constant time lag.
Typical TimeStep (time lag) value for FCS measurements is 2E-7 sec.
"CC Logarithmic"
Calculates the crosscorrelation function based on quasi-logarithmic time scale with time step increasing with lag.
Typical TimeStep (time lag) value for FCS measurements is 2E-7 sec.
"CC Logarithmic (without averaging)"
Calculates the crosscorrelation function based on quasi-logarithmic time scale. It is calculated at increased time lags but without rebinning, i.e. with initial bin width that allows to minimize triangular averaging effect.
Typical TimeStep (time lag) value for FCS measurements is 2E-7 sec.
Button "IED"
Calculates the Inter-event Time Distribution (IED): histogram of the time intervals between the concequtive photocounts. It is possible to choose linear time scale or quasi-logarithmic time scale with time step increasing with time.
Typical TimeStep value for FCS measurements is 5E-8 sec.
"IED Linear"
Calculates the Inter-event Time Distribution: histogram of the time intervals between the concequtive photocounts based on the constant time step.
Typical TimeStep value for FCS measurements is 5E-8 sec.
"IED Logarithmic"
Calculates the Inter-event Time Distribution (IED): histogram of the time intervals between the concequtive photocounts based on the quasi-logarithmic time scale with time step increasing with time.
Typical TimeStep value for FCS measurements is 5E-8 sec.
Button "PCD"
Calculates the Photon Counting Distribution (PCD): histogram of the number of photocounts arriving within the Time window (Time window = Time step).
Button "FED"
Calculates the First Event Distribution (FED): histogram of the arrival times of the first photon within the Time window (Time window = Number of points * Time step).
Button "SED"
Calculates the Single Event Distribution (SED): histogram of the arrival times of only single photon within the Time Window (Time Window = Number of points * Time step).
Button "FFC"
Factorial cumulants Kk are calculated from experimental data by formulas:
where P*(n) is the measured photon counting distribution i.e. probability to detect n photons within a counting time interval T (Time Step), the angular brackets indicate averaging with the set of probabilities P*(n).
Button "INT"
Intensity is a histogram of photon arrival times (Bin time=Time Step). One can inspect any part of the raw data in details with help of this characteristic.
Button "Coin"
Coincidence Values Histogram is a histogram of coincidence values calculated at given sampling time (Time Step).
Coincidence value K(n) are calculated from experimental data by formula:
where Ni and Nj are the absolute photon count numbers of the emission signals in consecutive time channels m, each with sampling time Time Step.
Button "Append"
Appends the calculated statistical characteristic to the Files table.
Button "Update"
Updates the selected record in the Files table with the calculated statistical characteristic (i.e. replace the stored characteristic with calculated one). Related parameters are updated when necessary.
Button "Single"
If this button is down the single statistical characteristic is calculated.
Button "Multiple"
If this button is down a set of statistical characteristics with varying bin time are calculated. Number of characteristics and bin time range are specified in the Calculation options dialog box that is displayed each time before starting calculation.
Autocorrelation function
Autocorrelation function can be either liner or quasi-logarithmic (time lag either increased with time or not).
Linear autocorrelation function is calculated by the formula:
Quasi-logarithmic autocorrelation function is calculated by the formula:
Here M denotes the total number of channels, n(t) - the intensity (or number of photons in a bin of time t),
- the bin width (Time Step).Quasi-logarithmic without averaging autocorrelation function is calculated at increased time lags (quasi-logarithmic time scale) but without rebinning, i.e. with initial bin width. It allows to minimize triangular averaging effect.
Typical TimeStep value for FCS measurements is 2E-7 sec.
Crosscorrelation function
Crosscorrelation function can be either liner or quasi-logarithmic (time lag increased with time or not).
Linear crosscorrelation function calculated by the formula:
Quasi-logarithmic crosscorrelation function calculated by the formula:
Here M denotes the total number of channels, n(t) - the intensity (or number of photons in a bin of time t),
- the bin width (Time Step).Quasi-logarithmic without averaging crosscorrelation function is calculated at increased time lags (quasi-logarithmic time scale) but without rebinning, i.e. with initial bin width. It allows to minimize triangular averaging effect.
Typical TimeStep value for FCS measurements is 2E-7 sec.
Photon Counting distribution
Photon Counting distribution (photon counting histogram) is a histogram of the number of photocounts arriving within the Time window (Time window = Time step). Number of histogram points is automatically increased when number of photocounts exceeds the value specified in the Number of Points edit.
Inter-event Time distribution
Inter-event Time distribution is a histogram of the time intervals between the concequtive photocounts. It is possible to choose linear time scale or quasi-logarithmic time scale with time step increasing with time.
Typical TimeStep value for FCS measurements is 5E-8 sec.
First Event distribution
First Event distribution is a histogram of the arrival times of the first photon within the Time window (Time window = Number of points * Time step).
Single Event distribution
Single Event distribution is a histogram of the arrival times of only single photon within the Time Window (Time Window = Number of points * Time step).
Fluorescence Factorial Cumulants
Factorial cumulants Kk are calculated from experimental data by formulas:
where P*(n) is the measured photon counting distribution i.e. probability to detect n photons within a counting time interval T (Time Step), the angular brackets indicate averaging with the set of probabilities P*(n).
Note: Weight factors are calculated for the five first cumulants (if Weight calc. check box is checked). Weight factors for all other cumulants are zero (if Weight calc. check box is checked). If Weight calc. check box is not checked, wheight factors for all cumulants equal unity. Therefore they do not participate in the analysis (i.e. do not participate in the calculation of the criterion value), though residuals are displayed.
Coincidence Values Histogram
Coincidence Values Histogram is a histogram of coincidence values calculated at given sampling time (Time Step).
Coincidence value K(n) are calculated from experimental data by formula:
where Ni and Nj are the absolute photon count numbers of the emission signals in consecutive time channels m, each with sampling time Time Step.
Intensity
Intensity is a histogram of photon arrival times (Bin time=Time Step). One can inspect any part of the raw data in details with help of this characteristic.
Local menu of the characteristics chart
This local menu contains following items:
Standard local menu commands of 2D Chart
View Characteristics: Set the visibility of the characteristic itself.
View StDev: View standard deviations (weighting factors) of each point of the characteristic. This local menu item is unavailable if standard deviations have not been calculated. To calculate them check Weight cacl. check box (it allows to calculate standard deviations of each point of the characteristic from the raw data) or use Calc.Weight local menu command of the DataSet panel of the main window (it allows to calculate standard deviations of each point of the characteristic from the set of characteristics measured (or calculated from the raw data) under the same conditions).
View Rel. StDev : View relative standard deviations of each point of the characteristic (i.e. standard deviations of the point divided by the point value).
Files table
Displays the calculated statistical characteristics. It displays the Observation files (Measurements Database) related to the linked with the selected raw rata Files Group as well if such files were imported together with raw data.
Notes
- This table can be sorted by any field. To perform the sorting by the given field, select this field and choose Ascending/Descending menu items in the local menu.
- Choose
2D Chart
This 2D Chart displays the calculated statistical characteristics and imported Observation files .
Local menu of the Files table
This local menu contains following items:
| Items | Description |
| Delete | Deletes current record. |
| Edit | Switches state of the table in edit mode. |
| Post | Saves current record in the Database. |
| Cancel | Cancels changes. |
| Refresh | Refreshes the information in the table. Valuable in multi-user application when several users work with the Database simultaneously. |
| Sort | Sorts the table. |
| Find Record | Allows to set the filter on the table and to search the information by typing the first few characters. |
| Append | Appends the calculated statistical characteristic to the Files table. |
| Update | Updates the selected record in the Files table with the calculated statistical characteristic. |
Use the Raw Data Simulator to simulate the raw data time trace with constant intensity and given autocorrelation function.
An example view of the Raw Data Simulator is given below:
Click the corresponding item on the picture displayed above to get more information about it.
Button "Start"
This button start the simulation of the raw data. At the beginnig of the simulation Export to the Database dialog box are opened.
Button "Stop"
This button stops the simulation.
Note:
Uncompleted raw data trace will be saved to file and exported to Database.
Button "Export to ASCII file"
Exports simulated raw data to the ASCII file. Opens standard Save File dialog box to select the file.
Button "Export to the Database"
Opens Export to the Database dialog box to export simulated raw data time trace to Database (RawData table).
Flow source combobox
Allows to select the source used for simulation.
Button "Help"
This button opens the help window that describes how to work with Raw Data Simulator.
Parameters
Here you can specify parameters of the raw data simulation.
Model parameters
Here you can specify parameters of the model used for the raw data simulation.
Simulation information
This window monitors the information about the simulation progress.
Export to the Database dialog box
Use Export to the Database dialog box to export simulated raw data time trace to RawData table (Measurements Database).
An example view of the Export to the Database dialog box is given below:
Click the corresponding item on the picture displayed above to get more information about it.
RawData table
Displays the stored raw data in the Measurements Database.
Notes
- Field ID is read only.
- This table can be sorted by any field. To perform the sorting by the given field, select this field and choose Ascending/Descending menu items in the local menu.
- Choose Quick Search menu items in local menu for quick searching the information in the selected field.
RawData table
Displays the stored raw data.
Raw Data Description
Describes the stored raw data.
Channel
Allows to choose red or blue channel where simulated data will be stored.
Button "OK"
This button finishes work with the dialog box and applies all changes.
Raw data file
Use this edit box to specify the raw data file.
Button "Cancel"
This button finishes work with the dialog box and discard any changes.
Button "Browse"
This button opens a standard Save File dialog box for specifying of raw data file.
Button "Help"
This button opens the help window that describes how to work with Export to the Database dialog box.
Local menu of the Raw data table
This local menu contains following items:
| Items | Description |
| Delete | Deletes current record. |
| Edit | Switches state of the table in edit mode. |
| Post | Saves current record in the Database. |
| Cancel | Cancels changes. |
| Refresh | Refreshes the information in the table. Valuable in multi-user application when several users work with the Database simultaneously. |
| Sort | Sorts the table. |
| Find Record | Allows to set the filter on the table and to search the information by typing the first few characters. |
Photon Counts Calculator dialog box
Use Photon Counts Calculator dialog box to calculate number of photons in a time range of raw data defined by data markers.
An example view of the Photon Counts Calculator dialog box is given below:
Click the corresponding item on the picture displayed above to get more information about it.
Button "Close"
This button closes the dialog box.
Button "Calculate"
This button starts the calculation.
Combobox "Markers"
This combobox is used for selection of needed pair of data markers.
Group box "Count rate"
This group box displays statistics of photon counting:
Bin time displayes the binning time;
Bins number displayes the number of bins each of bin time width;
Average displayes the average number of photons in a bin;
Std. deviations displayes the standard deviations of number of photons in a bin.
"Raw data" grid
Displays names of raw data files linked to the selected record in the raw data table (it is viewed only if a number of raw data files is more than one).
The number of raw data files is indicated below this grid.
Bursts Counting windowU
Bursts Counting window is designed to perform coincidence bursts analysis.
It allows:
- Automatic selection and counting of bursts in both channels separately by setting min and max threshold;
- Counting and selection of coincidence bursts by analyzing of start/end positions of each burst in both channels. Only bursts with given time overlap and photon counts ratio (in percent, implemented for the accounting the leaking of the fluorescence from blue to red channel) are counted;
- Highlighting of the leaked bursts (in the red channel chart) by blue color;
- Calculating of percentage of coincidence bursts from a number of bursts in selected channel.
Bursts Counting window consist of the following components:
- Raw Data grid
- Red channel page
- Blue channel page
- Coincidence page
- Status Bar
An example view of the Bursts Counting window is given below:
Click the corresponding item on the picture displayed above to get more information about it.
The Coincidence page allows selection and counting of coincidence bursts by analyzing of start/end positions of each burst in both channels and calculating of percentage of coincidence bursts from a number of bursts in a selected channel. Only bursts with given time overlap and photon counts ratio (in percent, implemented for the accounting the leaking of the fluorescence from blue to red channel) are counted.
Status bar, which is a horizontal area in Bursts Counting window below the all panels, provides information about the current state of application and any other contextual information.
"Coincidence bursts" grid
Displays coincidence bursts. It display start/end positions of the burst in both channels, time overlap and min photon ratio (implemented for the accounting the leaking the fluorescence from blue to red channel).
Use local menu for exporting start/end positions and number of photons in CSV file and highlighting the leaked bursts in the red channel by blue color (redraw red channel chart for removing such highlighting).
Count button
Starts the selection and counting of coincidence bursts at given time overlap and min photon ratio.
Red/blue channel radiobutton
Is used to select the channel for calculation of percentage of coincidence bursts in it.
Delete button
Deletes the selected burst.
"Min time overlap" edit
Use this edit to set min time overlap (in percent) between bursts in both channels. The burst is marked as coincidence burst if time overlap between correspondent bursts in both channels is more or equal then given one.
"Min photon ratio" edit
Use this edit to set min photon ratio (in percent) for the accounting the leaking of the fluorescence from blue to red channel.
Blue Channel page
The Blue Channel page allows automatic selection and counting of bursts in the blue channel by setting min and max threshold. It also allows:
- Plotting the intensity in linear and logarithmic mode;
- Lee filtration of intensity graph;
- Possibility to include thresh-holding in order to define bursts;
- Manual editing of bursts (add, delete, edit) with help of data markers;
- Counting of number of photons is each bursts automatically or manually with help of Photon Counts Calculator dialog box;
- Export of start/end positions and number of photons in CSV file.
Bursts Chart
This Bursts Chart displays intensities in a channels.
One can select necessary time range of raw data for bursts selection by means of data markers.
Button "Count"
This button starts the bursts selection and counting procedure.
Button "Add"
Is used for manual selection of bursts. Adds the selected by means of yellow data markers burst to the grid.
Button "Delete"
Is used for manual selection of bursts. Deletes the bursts selected in the grid.
Button "Update"
Is used for manual selection of bursts. Updates the selected burst with a new selection.
Button "Clear"
Is used for manual selection of bursts. Clears the list of bursts.
Button "Redraw"
Calculates raw data intensity and redraws the chart.
Button "Log"
Sets logarithmic scale of Y axis and redraws the chart.
Button "Filter"
Performs the Lee filtration of intensity and redraws the chart.
"Min photons count" edit
Use this edit to set the minimal count of photons in a burst. The only bursts with number of photons more or equal to given value are selected. This limit does not work while manual selection of bursts.
"Filter half width" edit
Use this edit to set the half width of Lee filter.
Bursts chart
Local menu
Bursts chart is a standard 2D Chart with embedded vertical and horizontal markers. It displays intensities in red or blue channels.
One can select necessary time range of raw data by means data markers.
You can:
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