The Burst Analyzer Tutorial
Contents
1. xw Gm 1 9 de us Figure 34 Left Experimentally obtained auto correlation function of free Rhodamine 6G in solution blue curve overlayed with the fit result of a model for freely diffusing particles red curve The model has three parameters the number of particles N the diffusion time tauDU and the relation of the radii of the laser focus elipsoid omega In the curve 1 SFelekyan R K hnemutz V Kudryavtsev C Sandhagen W Becker C A M Seidel Full correlation from picoseconds to seconds by time resolved and time correlated single photon detection Rev Sci Instr 76 8 p 083104 2005 The Burst Analyzer Tutorial fining window initial values as well as lower and upper bounds can be set for each parameter in adlton parameters tan also be fied to user defined values In the menu band of the main window global parameters regarding FCS can be configured The correlation function is always computed using a logarithmic x axis The longer the underlying time trace the longer the highest possible xvalue will be The resolution of the correlation function can be set by determining the number of points per cascade in FC5 General Parameter Points per Casc see Figure 35 In the same control group the range of the correlation time r can be set Initially the lower bound Start Carr Func is set to 0 and the upper bound Stop Corr Func is set to Infinity in order to disable the effe2. e Fred Name 3 over Bound jet TT enr n 3 Tins E ones E a s Figure 37 Fit function definition window This user interface provides the opportunity ta define your own functions with arbitrary names and function strings The parameters involved can be extracted from the function string automatically The Burst Analyzer Tutorial Auto Each porameter hax a set of properties Mis rame ital value and bounds otherwise free parameter i set to the Initial value and ignored by the fk algorithm fixed is checked the As soon as the function string is error free the background color of textbox turns from red to green The function is checked at every key stroke there is no need to explicitly compile or check the function string New parameters can be introduced just by typing them into the function string A click on the Auto button afterwards extracts all parameters to the list in the lower part of the window and gives the possibilty to edit their properties For every parameter an initial value lower and upper bounds to restrict the value range during the fit and the setting Fixed is available Whenever Fixed is checked the corresponding parameter will be turned into a constant and will not be optimized by the fitting procedure This may be advantageous if a model with many parameters does not converge All these parameter settings can also be access
3. Figure 43 1D fluorescence lifetime histogram with automatic scaling The range is set to match the range of etimes in the data set and Auto Y have been clicked E Soa a MB lt j e 7 1 BIB TH A H ERES SG ABGAxissdAsanusi Figure amp Lifetime histogram with customized scale Range limits and maximum have been entered manually 31 The Burst Analyzer Tutorial L Bose eeu ve ema Figure 45 Lifetime Channel 1 vs Lifetime Channel 2 Two dimensional histogram The color scale is set automatically The bin which contains most data points is black Zero counts in a are shown in white psssssuemesaa x zosusssiarive 208 E d o d amp 4 X The Burst Analyzer Tutorial Figure 46 Two dimensional histogram By changing the number of bins per axis you can create a histogram which resembles a two dimensional scatter plot 5 6 Savingand Exporting Histograms When you save a project histograms and data tables will be saved in the project file When you load 2 project file the data sets and histograms are loaded from the fie To create a bitmap file from a histogram you have to 1 Right click the histogr
4. Prem Figure 27 The user control group Corrections provides user dialog for parameter settings and a toggle button On to enable resp disable the correction process 46 Intensity Weighted Auxiliary Function Using the Configurtion Aux Func Intensity Weighted button see Figure 22 on page 15 the time bins involved in the calculation of mean and standard deviation of the auxiliary function for a burst or step can be weighted by their intensity sum the button is pressed each involved time bin will have a weight whichis the total intensity ofall intensity traces which are used in the auxiliary function string This way time bins with higher count rates have more influence than time bins with low signal intensity If this option is not checked each time bin is treated equally For more information on calculation of custom functions see section 4 8 2 Auxiliary Function Property on page 23 4 7 Creation of Bursts und Steps Burst and steps can be created by marking a time area in the data chart with the mouse A newly marked area presents a burst a newly marked area inside a burst presents a step Bursts and steps will automatically be added to the project and appear in the project panel with their start time and duration and their additional attached properties see section 4 1 5 Project In addition to the time consuming manual marking bursts can also be generated automatically in a very efficient way The
5. The Burst Analyzer Tutorial 3
6. Click the button in the histograms bar Select the data set you want to use Select a property or properties for histogramming Click Ok to apply your selection 29 The Burst Analyzer Tutorial Figure 41 To create histogram lick the button You can create a standard 10 histogram from each column ina data table Or you can create 20 histogram from each par of columns 54 Histogram Options Figure 42 Left Settings for dimensional histograms Right Settings for 2 dimensional histograms For each histogram you can set the number of bins and the minimum and maximum of the data range What you specify here is the center position of the first and last bin in each dimension Clicking Auto or Auto Y will determine these limits from the data For 1 dimensional histograms Auto will automatically scale the y axis based on the histogram data Most of the histogram settings can be changed after histogram creation For this simply right click the histogram area and make the changes Click Apply when you are done 5 5 Examples In this section we show a few example histograms and the corresponding histogram settings The Burst Analyzer Tutorial toate tu MJ eei Xe eem HHUH eire
7. The project file stores the properties of the marked areas only and not the individual photon arrival times The software keeps track of the burst position within the raw data stream This way a correction of parameters such as the background level which deals directly with the raw data can be done with a simple click as the burst properties are automatically recalculated from the raw data The project file is stored in an xml format and is thus readable by any text editor This facilitates further data analysis with external tools Programs like Matlab Mathworks IGOR Pro WaveMetrics or Origin Origintab can easily import the project files Identification of single molecule bursts in the time traces of the data is the main functionality of Burst Analyzer Bursts can be marked manually or automatically by defining parameters such as minium and maximum burst intensity Furthermore sub areas of the bursts can be marked as steps This can be Useful as visible steps within a burst may correspond e g to certain conformational states of proteins Under appropriate circumstances the changes inconformational states correspond to steps in the FRET trace and can be identified in this way 4 1 User Interface A screenshot of the user interface is shown in Figure 15 The user interface shows four measurement panels 1 to 4 and a tree view with a listing of burst and step properties panel 5 All control elements are located in the head row pane
8. attached channel increases the calculation time for the subsequent data processing algorithms like the correlation function calculation The Burst Analyzer Tutorial ieee s Aum o ui eee 5 S Rori Ei E iB aW ves Router D set 77 B sori E1 E Router Fi We Figure 23 Twa measurement channels were mapped to three time traces Trace 1 Blue shows photons from router trace 2 green ls assigned to router 1 race 3 orange shows the photons of both traces and describes therefore the sum Intensity of both traces 44 Assigning Traces to rotime Windows Using microtime information it is possible to split one measurement channel to different traces In this way the FRET acceptor fluorophore in a scenario of a pulsed FRET experiment can be excited by a second synchronized laser but recorded with the same detector as the donor fluorescence As donor and acceptor fluorescence show up in different parts of the microtime window they can easily be assigned to different time traces Figure 24 shows an example configuration Timm aa ars sl Reuter D E Meratine Start nst Stap mt 81 2M es Router 1 Wi Mone seme 53 E Bm E 1 EI XD S Wes 8 m Figure 24 Measurement channel Router 0 is assigned t T
9. 6 Panel 1 shows an overview diagram panel 2 holds the measurement data as intensity time traces panel 3 contains a diagram showing auxiliary functions 11 The Burst Analyzer Tutorial panel 4 contains a the microtime histogram panel 5 the project window and panel 6 the active tab of control elements Mesa ELM t LIMIT a GEGCSGOR GER 444 Overview Diagram The small diagram in the center contains an overview trace of the currently selected data set The data set can be selected by clicking a filename in the project panel 5 The overview diagram displays the total intensity of all measurement channels as a function of measurement time Additionally this diagram shows the marked areas of bursts and steps in yellow Their absolute position in time can clearly be seen 412 Measurement Data Diagram FEEL Bin iat inst os Figure 16 This group of control elements provides user controls to set on and off the visibility of the intensity traces and to determine the width of their time binning This diagram shows the photon arrival times in terms of up to four intensity traces The visibility of the individual time traces can be set by the four user control elements in Star Traces Visibility see Figure 16 The width of the time bins used for this diagram can be set through the Bin W
10. Burst Finder group of the menu band Min Width Max Width and Dark Width see Figure 29 Min Width and Max Width defines the borders of The Burst Analyzer Tutorial the tolerated burst length in Milliseconds Dark Width allows a drop of the parameter Flank Level for the configured duration In this way two adjacent bursts can be combined by increasing this parameter A By Me wen 0 lax Width 2000 Find Settings NAS With Bursts Dark Width 20 Figure 29 The user control group Auto Burst Finder provides all parameters for the automatic burst finder algorithm Figure 30 demonstrates the burst finding process The burst finder algorithm moves a marker from time bin to time bin until the intensities of the current time bin exceed one of the defined Min Level thresholds In Figure 30 this happened for the first time in this data stream at time 86979 ms From this position a potential burst is initiated with start 86979 and width 0 Next the left and the right border will be extended until the intensities of the current time bin falls below the thresholds Flank Level For subsequent burst detection these two borders define a potentially new burst E s Figure 30 An automaticaly created burst with the parameters Min value 50 for trace 1 blue and Min value 40for trace 2 green Hank level was set to 10 for both traces The burst detection started at position 86279 ms ed li
11. Using a focused laser beam for excitation a small focal volume of the solution is excited Through confocal detection the signal from a femtoliter volume is collected The average number of fluorescent molecules in the detection volume is small and the molecules diffusing in and out of focus give rise to a fluorescence correlation The Burst Analyzer Tutorial function which is governed by the diffusion coefficient of the molecules the shape of the detection volume the type of diffusion e g two dimensional or three dimensionall etc With Burst Analyzer you can compute and analyze FCS FCCS curves fram single molecule TCSPC data Several predefined fitting models and user defined functions are supported FreeDIBDIDT Name Vane T ge pcs pense BEH HESI pne D xw am dM 1 d uw Figure 5 Left Experimentally obtained uto correlation funcion of free Rhodamine 6G in solution blue curve overlayed with the fit result of 2 model for freely diffusing particles red curve The model has three parameters the number of parties N the diffusion time taDf and the relation of the radii of the laser focus ellipsoid omega In the curve Fluorescence Lifetime Distributions 244 User defined fit functions for Fluorescence Correlation Spectroscopy A software based correlation algorithm is implemented to generate auto and cross correlation functions out of the TCSPC data
12. a multi module TCSPC system such as the SPC 154 to the different inputs of a DPC 230 photon correlator board or to a router attached to a single SPC module In most cases different measurement channels have to be visualized by differently colored time traces With Burst Analyzer time traces can be defined in an extremely flexible way You can map the photons from a measurement channel or part of the photons based on the microtime to one or more time traces and you can define time traces which show the sum of different measurement channels The default setting in Burst Analyzer is to map the first four measurement channels to the four available time traces To display other measurement channels or do more a more sophisticated selection based on the microtimes you have to change the trace definitions manually This can be done inthe me Trace Definitions dialog located at Configuration Time Traces Setting A mouse click on the plus button opens a list of available measurement channels With successive clicks the button you can add the photons from multiple measurement channels to one trace A button removes a measurement channel from a trace definition See Figure 23 for an example Here a third time trace is generated from a two channel setup just by mapping both measurement channels to the third trace as well mouse click on the Any of the four available traces can hold any number of measurement channels However every
13. correlation function reveals correlations between individual fluorescence bursts resulting from single molecules or complexes of molecules diffusing through the focal volume of the measurement system From the resulting graph one can see directly how the measured fluorescence signal is composed of correlated fluctuations of different lengths Large particles lead to a slow decrease of the FCS curve whereas small particles result in a fast decrease of the FCS curve The reason is that large molecules are diffusing much slower than smaller ones The correlation function for each burst or step is calculated numerically from the intensity signal without any explicit assumptions about the measured molecules For this a multiple tau algorithm is used which uses logarithmically spaced groups of time bins ref The correlation function is visualized by Burst Analyzer as a blue curve in panel 3 see fig 29 The curve can be automatically fitted to an appropriate model i e freely diffusion particles of one type of equally sized and equally bright particles This is the first step where assumptions about the particles enter the analysis process The model parameters i e mean number of particles or diffusion time are determined through the fitting process The resulting fit function is shown as a red curve which is shown in the same diagram as the correlation function In this way one can judge the goodness of fit at a glance see Figure 34 0
14. criteria Each data list has its own set of filter conditions and itis possible to create multiple lists from the same raw data set All time related values in the lists are usually saved in milliseconds except for the fluorescence lifetimes which are given in nanoseconds Bursts and steps always contain Start and Width entries All further entries appear only when the corresponding property was selected during the creation of the burst or step respectively The following entry identifiers exist Intensity group containing average intensity values PhotonCount mean intensity counts per ms o Peakintensity maximum amplitude per bin counts per ms AuxFunc group containing means and deviations of the auxiliary function AuxiliaryFunctionName Mean mean value of the auxiliary function AuxiliaryFunctionName Std standard deviation of the auxiliary function LifeTime group containing lifetime information based on the lifetime histogram taul tau2 the lifetime value for every available time trace ns group containing fit results of the autocorrelation function first entry name of the choosen fitting function following entries fitting parameter names with their fit results CCF group containing fit results of the crosscorrelation function first entry name of the choosen fitting function o following entries fitting parameter names with their fit results 5 2 Filte E Ea
15. entries of the same type the feature Bursis General Parameter Refresh all Bursts Steps can be used to update all project entries which includes the alignment of their parameter sets Afterwards the project file can be exported and processed further 6 1 Importing Data into Matlab ASCII data exported from BurstAnalyzer can easily be read by Matlab We recommend to use the following Matlab function function bursts steps baread filename id fopencfilename bursts struct Hf steps struct ursts 0 moofsteps 0 while feof Cid Str fgetl fid C textscan str Xs Delimiter b CDS switch case Wwe Pareto Mure m ie EP width str2nun C 1 355 bursts noofursts par ie or 124 arval nean bursts noofsursts char barval 1 parval 2 en case Step noof steps noofsteps 1 Steps noofscens tine strinunccn un Steps noofsteps width str2num C 1 3 3 par size c 1 The Burst Analyzer Tutorial BEES FY end end end fclosecfid end The function bapxread returns two structure arrays corresponding to burst and step data The fields of the structures correspond to the various properties determined by Burst Analyzer and can be used in any of the matlab plotting and analysis functions e g gt gt burst step bapxread myproject txt gt
16. files The number and size of SPC files is not important when working with Burst Analyzer In Burst Analyzer the photons are always loaded into the PC s memory dynamically even over the file borders of several SPC files a system with multiple measurement modules ie SPC 154 itis sufficient to select only one of the created SET files The Burst Analyzer recognizes the number of modules automatically and adds the relevant SET files the set of measurement files The file open dialog is located at Start Data File Add SET SPC File The first time that a data set is opened an index file is automatically created this enables the concerted loading of the photons corresponding to the currently selected zoom area without the need to scan all photon data For every SET file an own index file will created so every module gets its own index file An index file has the file extension idx Its file size is about 1 1000 of the sum of all attached SPC files The IDX file is stored in the same directory as the source SET and SPC data files This means it is mandatory to have write access to this directory There is no further data processing possible without an existing index file Whenever it is missing the Burst Analyzer automatically tries to create it 43 Assigning Traces to Measurement Channels In a typical measurement photons from multiple detector channels are recorded The detectors can be connected to different recording channels of
17. of photons a 0 5 10 15 E 5 10 15 Microtime ns Microtime ns Figure 12 Convolution of the mono exponentil photon distribution with instrument response of the detection system Left instrument reponse green dotted and exponential decay red Right Convolution of exponential and instrument response The finite width of the system response is clearly visible in the rising part of the resulting curve blue The red curve lea pure mono exponental decay JENU xd t turns out that for a mono exponential decay F x exp x tau wD oc rf This means you can obtain the true lifetime of the fluorophore by subtracting the first moment of the normalized instrument response function from the first moment of the measurement data The system response function IRF can be recorded by feeding an attenuated back scattered laser beam to the detector and recording a data set The microtime histogram derived from this data set shows the system response the first moment of which must be subtracted from the lifetimes of the measurement data to obtain the true lifetime values for fluorescence measurements Therefore the software allows to define a constant offset value for each time trace to account for the influence of the IRF Let 00 rer 2 me 7450 feti 82 Figure 13 A click on the round bution at the right side of the lifetime information opens a menu where you can enter a co
18. stream of a user defined section of the traces Levenberg Marquardt fit algorithm adjusts the free parameters of a selected model The results of the parameter values are shown together with an overlaid function curve to show the quality of the fit see Figure 6 While fitting the data the model parameters can be chosen to run free or to be hold fixed at their initial value Name Vane I g fas ossa 2 ime 1 0 Xm ous Figure 6 Single Rhodamine6 in water Upper autocorrelation function blue curve with model red curve The model parameters are listed in the right window and show the result of the curve ft The model itself is stored into a library of fit models which can be extended by user defined models All of these models can have an arbitrary number of parameters where their name initial value and value bounds can be individually defined 242 Filtered FCS In Burst Analyzer you can calculate a fluorescence correlation curve for a complete data set as well as for particular regions within a data set addition to this you can also filter a data set and exclude selected regions bursts from analysis This can be used to remove artifacts from FCS FCCS measurements The Burst Analyzer Tutorial 2 5 Burst Step Property Histograms 254 Lifetime Information Fluorescence Decay Histogram For TCSPC data histograms of the measured
19. 1 Intensity Traces 32 Microtime Histogram Software Description 41 User Interface 4 1 1 Overview Diagram 41 2 Measurement Data Diagram 4 13 Diagram of Auxiliary Functions 414 Microtime Histogram 4 15 Project Panel 4 16 Menu Band 42 Datafiles Assigning Traces to Measurement Channels 44 Assigning Traces to Microtime Windows 45 Correction of Traces 4 6 Intensity Weighted Auxiliary Function 4 7 Creation of Bursts und Steps a a 2 12 13 14 14 as as 16 18 ES ES The Burst Analyzer Tutorial 481 482 483 484 52 53 54 55 56 61 Attaching Properties to Bursts and Steps Intensity Property Auxiliary Function Property Lifetime Property and CCF Properties Burst and Step Data Analysis Property Data Tables Filtering Creating Histograms Histogram Options Examples Saving and Exporting Histograms Exporting Data Importing Data into Matlab Glossary n 23 23 24 27 27 28 28 30 30 33 33 E 36 The Burst Analyzer Tutorial 1 Getting Started 14 What s New in Version 2 Multiple Table views of burst step properties Support for multiple filter sets Histogramming of filtered burst step properties Huge speedup in FCS calculation Exclusion of artefacts from FCS calculation Automatic or manual recalculation of FCS data FCS fitting with weights derived from raw single photon data 12 System Requi
20. Becker amp Hickl GmbH Burst Analyzer 2 0 For Correlation Analysis and Single Molecule Spectroscopy FCS FCCS smFRET PIE ALEX FILDA etc 2nd Edition March 2014 The Burst Analyzer Tutorial Becker amp Hick GmbH Nahmitzer Damm 30 12277 Berlin Germany Te 49 30 2128002 0 FAX 49 30 2128002 13 http www becker hick com mall info becker hickl com 2nd Edition February 2014 This handbook is subject to copyright However reproduction of small portions of the material in scientific papers or other non commercial publications is considered fair use under the copyright law tis requested that a complete citation be included in the publication If you require confirmation please feel free to contact Becker amp Hickl GmbH The Burst Analyzer Tutorial Contents 1 Getting Started 11 What s New in Version 2 12 System Requirements 13 Installation 14 Getting Help 2 Overview 21 Visualizing Intensity Traces 22 Marking Bursts and Steps 23 Generation of Time Curves Based On User Defined Functions 24 Fluorescence Cross Correlation Spectroscopy FCCS FCS 2 4 1 User defined fit functions for Fluorescence Correlation Spectroscopy 242 Filtered FCS 25 Burst Step Property Histograms 2 5 1 Lifetime Information Fluorescence Decay Histogram 25 2 Lifetime Distributions 25 3 FRET Histograms 3 Structure of Measurement Data Macrotime and 3
21. This wil bring up an info window from where you can edit your license key The Burst Analyzer Tutorial 14 Getting Help By clicking you get access to the Burst Analyzer User s Manual tis a PDF document To view it you most have Adobe Acrobat Reader installed or a similar PDF A click on Help wil open the manual in your PDF viewer dura Arayer 2013 p in ted ingle shot cura dite ey valid tor this product tem s Figure 1 Burst Analyzer information Window t shows contact information software version number license key and ives you access to the user s manual Burst Analyzer 2 comes with a number of tutorials which are accessible through the start menu You also check the B amp H website www becker hicklcom for application notes and other literature For an introduction into Fluorescence Correlation Spectroscopy have a look at following resource by Petra Schwille and Elke Haustein www biophysics org Portals 1 PDFs Education schwille pdf 2 Overview BurstAnalyzer is a tool for analysis of time trace data It can identify and analyze peaks in time traces and also analyze user defined time intervals In addition to this BurstAnalyzer can also perform calculations on the full traces e g compute auto correlation or crass correlation of time traces BurstAnalyzer has been specifically designed for analysis of data obtained from time resolved single molecule fluorescenc
22. a dichroic filter a fraction of an intensity signal may appear in the wrong channel due to an imperfect match of the emission spectra of the fluorophore with the corresponding optical filters With the crosstalk parameter provided by the user dialog shown in Figure 26 the available time traces can be corrected by the fraction originating from signal crosstalk Like the scale parameter in Figure 25 the crosstalk parameters are dimensionless relative factors of the source trace The figure shows how to compensate a crosstalk of 2 from channel 2 which falsely appears as a signal in channel 4 Time Trace Crosstalk Correction Parameter Crosstalk IntoTrace 1 into Trace 2 into Trace 3 into Trace 4 from Trace L o o fem Trace2 0 o 002 fomWae3 0 E o oo o o Figure 26 The Time Trace Crosstalk Correction Parameter dialog gives access to options for signal cross talk correction The values entered represent a fraction of the source trace Intensity showing up In the destination trace The crosstalk correction will be done fst afterwards the scale parameter willbe processed and at last the offe correction will be applied The Burst Analyzer Tutorial After setting the correction parameters they must be activated by clicking the On element in the Corrections control group see Figure 27 and setting it to the On state Clicking On once more will deactivate the corrections we he Nose
23. am area 2 Click Save bitmap This will bring up the following dialog Fesoutor Width em Height emi Here you can set the resolution and size of the bitmap When you click OK you will be asked for a filename 6 Exporting Data Starting from a burst analyzer project file with entries of bursts and steps the data can be imported external software Furthermore the export function see Figure 47 The project panel exhibits an export button which saves the currently opened project file as an ascii file generates a project file in a spreadsheet like shape with tabs and equal signs as separator therefore direct import into Matlab Mathworks Origin Origintab or Excel Microsoft and similar software is possible Averaging of time slices over multiple experiments can be performed with one of these programs psc fin 4 O05 GOO ms 10182 N 7 i 30020 0000 1 50020 20000 me 36 55 30000 Gono ms 96 52 120000 GOO m 75 43 N tauDIFL O3 uD 130000 ANON mst 2522 A12 6 tS A iru D4O S26 bcc tisch Figure 47 The project panel exhibits an export Button which saves the currently opened project fle as an asl md 3 The Burst Analyzer Tutorial To import an ASCII project file in i e Excel the column separator has to be set to the tab sign and additionally to the equal sign Hereupon the cells of the spreadsheet wi
24. arrival times are recorded The time measurement is started when a photon arrives at the detector and itis stopped when a laser pulse is detected Thus all photon arrival times are measured with respect to an excitation pulse This time difference can be recorded very accurately but with this method there is no common reference point for all arrival times which means the absolute arrival time of a photon measured from the start of the experiment is lost Modern TCSPC systems go one step further In addition to the time difference between photon and excitation pulse they also measure and register the absolute time from the start of the experiment Basically they count the number of laser pulses from the start of the experiments and convert this number into a time value which is possible because the lasers run at a constant repetition rate This arrival time is measured in units of the laser repetition rate It is called macrotime while the time between pulse and photon is called microtime 3 1 Intensity Traces Burst Analyzer the macrotime is used to generate fluorescence intensity traces You can define a time unit e g 1 ms and the number of photons within each successive time bin is counted and displayed as a time trace This shows how the fluorescence intensity changes over time Burst Analyzer the time trace analysis is based on the idea that whenever a fluoresceing molecule crosses the laser focus a burst of photons is e
25. ation of the burst 2 3 Generation of Time Curves Based On User Defined Functions Arbitrary functions of the signal intensity can be visualized For example a FRET trace is generated by just entering the string 12 11 12 as an auxiliary function The chart which shows this user defined function is always synchronized with the main data chart containing the underlying intensity traces see Figure 4 tes Figure 4 On the basis of the user defined function string an additional graph shows the outcome of the function which uses the intensity values of the original trace by the parameter 11 12 13 and on In this manner any aualllary function Le FRET traces can be generated and visualized together with the underlying intensity traces A green background of the textbox containing the auxilary function string indicates an error free function string 24 Fluorescence Cross Correlation Spectroscopy FCCS FCS The cross correlation CCF function of two time dependent signals f t and g t is defined as lt f t g tetau gt lt t gt lt glt gt where lt gt denotes an average over the complete time range considered for calculation If function f t is correlated with itself i e tet the result is called auto correlation function ACF I ft and g t are fluorescence signals the functions are named FCS and FCCS respectively a typical FCS experiments the sample is a dilute solution of molecules
26. automatic and manual selection of bursts from intensity traces and calculates several burst properties automatically such as mean fluorescence lifetime intensity auto correlation and cross correlation The various properties can then be further analyzed to determine molecular parameters such as molecule size and structure 2 2 Marking Bursts and Steps Arbitrary areas of intensity traces can be marked as bursts Usually a burst is a high intensity region of the trace indicating the diffusion of a molecule through the laser focus Marking a burst is done by just by clicking and drawing the mouse over the desired region see Figure 3 As soon as the mouse button is released a burst is created and added to the project list together with its attached properties Each burst can be sub divided into steps Steps can be defined in the same way as the bursts but must always start within a burst Burst Analyzer provides numerous ways to analyze burst and step data but it also possible to store burst properties within a project file and analyze them with external software The Burst Analyzer Tutorial JA tens imei iens Figure 3 Marking a burst in three steps First press the left mouse button at the desired start position of the burst Second move the mouse while pressing the button to the desired end position of the burst Third release the button to create the burst The blue area turns into yellow and hence shows the localiz
27. ch data list is associated with a set of filter conditions which can be used to select a subset of items far further analysis add a filter condition Click the button in the top right corner of the table view Select the property you want to use for filtering Specify minimum and or maximum values Click checkbox to enable filter Click Execute Filter to apply all active filter conditions to the table The Burst Analyzer Tutorial Add filter Figure 39 Each data table can be fitered Fiter conditions can be added by clicking Filters can be activated and deactivated by clicking a checkbox Active filers are applied when Execute Filter is clicked Lm Ea Tn Ere TA i TE mee H 13 e ee rd Figure 40 Table entries which do not match the fiter conditions are greyed out They will not show up in data set histograms remove a filter condition 1 lick Del 2 Click Execute Filter Alternatively you can deactivate a filter condition by unchecking the appropriate checkbox see Figure 40 5 3 Creating Histograms The filtered data tables described in section 5 1 are the basis for further analysis You can create 1 and 2 dimensional histograms from these data sets as well as create new data sets with different filter conditions To create a histogram you need to
28. ched properties should by updated This can be done by clicking the Bursts General Parameter Refresy all Bursts Steps button see Figure 32 482 Auxiliary Function Property By means of the user editable auxiliary function a time trace of a user defined function of signal intensities can be generated see section 4 1 3 Diagram of Auxiliary Functions By selecting the Auxiliary Function property the mean value and its standard deviation of this auxiliary function will be calculated and stored for each burst or step respectively In this way you can for example calculate FRET efficiencies for steps by setting the function string to 12 11 12 provided that 12 and I2 are acceptor and donor intensities In this case every step in the project file contains its average FRET efficiency and standard deviation Note All calculations are based on the background corrected count rates see section 4 5 Correction of Traces Moreover the values depend on the time bin size used for analyis see section 4 1 2 Measurement Data A larger time bin size leads to less fluctuations of the auxiliary function and thus to reduced standard deviations If the button Configuration Aux Func Intensity Weighted see Figure 22 on page 15 is checked the calculation of mean and deviation is weighted by the total intensity of the involved time traces within each bin Otherwise each time bin is treated equally see section 4 6 Intensity Weighted Auxiliary Func
29. ct of a restriction of x Im qe lg Figure 35 The FCS menu band provides global settings concerning FCS FCCS The access to the fit function s brary is also located here as well as the possibility to export the generated correlation funcion Including its fit results In the Curve Fitting control group the fit model can be selected The fit can be done for a user defined interval of correlation time which introduces the possibility to perform fits of only a part of the numerical function i e to achieve a tail fit The lower bound is set with the control element Start the upper bound is defined by the element Stop There isa library containing models for free diffusion in two and three dimensions each with up to two diffusion terms and up to one additional bunching term It can be accessed by the control element FCS Curve Fitting Library The library can be extended by user defined functions as required see Figure 36 Lory mI Fest cp restes zo reet 209 204 Figure 36 The fit functions library contains italy models for freely effusion partides in two and three dimensions with each upto three features All of these models are editable Moreover further models can be added as required 25 The Burst Analyzer Tutorial To add a new function first click the button which adds a new entry to the en
30. d of the function list Select this entry and click the Edit button to open a new window which provides settings for the function definition see Figure 37 model consists of an arbitrary function name This function name will appear in the list of available functions The list has a limited width a reasonably short name should be chosen Below the name the mathematical formula of the model can be entered Any parameter names may appear here Nested brackets can be used at any depth Moreover mathematical functions can be called by using the keyword Math followed by the function name For a complete list of supported math functions see Table 1 1 Complete tof all supported mathematical functions of the function editor Constants E 2 71828 Euler number Pi 3 14159 Circle number Trigonometric functions Acosiradiantj Asiniradiantj Atan radiant AtanZly x Cos radiant Cosh radiant Sin radiant Sinh radiant Tan radiant Tanh radiant Exponential functions Exp number Pow base exponent Loginumber Loginumber base LogiO aumber Other Absinumber Signinumber Min number1 number2 Max number1 number2 Rounding Ceiling number Round number Truncate number Floor number Polynomial Sqrt number Please note the function and variable names are case sensitive Dein So LUN KD Mant omega au
31. e spectroscopy where a small focal volume is observed and a fluorescent molecule entering the focus causes a large fluorescence burst For this reason time intervals in BurstAnalyzer are called Bursts When a molecule changes its state it may also change its fluorescence properties Such a sudden change shows up as a step in fluorescence intensity Thus sub regions of Bursts are called Steps Burst Selection of arbitrary length within a fluorescence time trace The common case is a peak in fluorescence originating from a single molecule or a complex of molecules travelling through the detection volume The Burst Analyzer Tutorial Step Selection of a time range within a burst Normally a step reflects a change of the properties of a molecule while observed by the detection system T Pai sub um 2 De Go RS 0 ZR GR G8 20 d Figure 2 Arbitrary time regions in an Intensity trace can be selected for further analysis 1 These regions are called burst Subregions within bursts are called steps 2 burst containing two steps 2 1 Visuali ing Intensity Traces The Burst Analyzer software helps you to visualize intensity traces from your single molecule TCSPC data fast and easily Data from multiple detection channels can be combined into one trace or separated by arrival time Up to four different intensity traces can be defined Burst Analyzer supports
32. ed from the main window during the fitting process see Figure 34 Note the default variable name for the correlation time t is x basic requirement for fit models is that the biochemical conditions do not change within the observation time The determination of the correlation function eventually represents a time average of the observed time span For a more detailed introduction into the method of fluorescence correlation spectroscopy see ref 5 Burst and Step Data Analysis 5 1 Property Data Tables When a burst or step has been defined it will show up in the project tree and in the corresponding data tables You can select an item in the project tree or in a data table The corresponding time trace range will automatically be highlighted and centered in the trace view i Medina M A Schwille P Fluorescence correlation spectroscopy for the detection and study of single molecules in biology 2002 BioEssays 24 758 764 x The Burst Analyzer Tutorial Figure 38 Project Tree and data lists in the single molecule view Data lists can be sorted by clicking a column header Right Many different fiter criteria can be applied to a data set The data lists which contain all range properties are the basis for further analysis and visualization They contain all the different attached properties as individual data columns You can plot various histograms from each property and you can do this based on a number of filter
33. efined function of up to four intensity traces or the correlation function with a user defined fitting model A cross correlation or auto correlation function can be shown The chart type can be chosen in the control group FCS Curve Type see Figure 19 If you select Auxiliary Function you can define your own function by typing a string into a text box In the function definition I1 to indicate the intensity values of the four available time traces Showing the Auxiliary Function is the default startup behavior TPT ZTE Assis fureien c o NN or mmm Figure 19 Choosing the type of auiliary chart auto correlation cross correlation or auxiliary function Depending on the selection you can select the traces for correlation or enter the auxiliary function definition In the Auxillary Function diagram cach time bin is computed from the intensity values of the time traces within the bin using the function definition you typed in The default is to use 2 12 This is called proximity factor ttl related to the donor acceptor distance in FRET experiment an example the funcion string 2 1 2 2 1 gives you an anisotropy trace in case you defined trace 1 as photons with parrale polarization and trace 2 as photons being perpendicularly polarized photons For assigning time traces to available routing channels see sections 4 3 Assigning Traces to Measurement Channels and section 44 Assigning Traces to Microti
34. gt hist burst width gt gt scatter burst width burst tau1 35 The Burst Analyzer Tutorial 7 Glossary Burst A burst is an aggregation of data points here fluorescence photons They indicate the presence of a molecule near the center of the laser focus The Burst Analyzer enables the user to mark any areas of intensity traces Those areas are called bursts due to the fact that usually molecules in the laser focus have to be marked Bursts itself may contain gt steps cw continuous wave Acw laser generates a continuous laser beam without interuptions in contrast to a pulsed laser where photons were emitted in periodic packages Data Set A data set means the entity of information of one measurement which is stored into a set of multiple files in different data formats For identification of a data set a SET file has to be specified here It contains internal links to binary files which store all the collected photon arrival times of the measurement FCS Fluorescence Correlation Spectroscopy The data analysis is based the correlation function of the measured time resolved fluorescence intensity trace This kind of spectroscopy is able to determine many physical parameters like i e the size or the concentration of the investigated molecule In contrast to other single molecule methods an intrinsic averaging over many molecules is performed FRET F rster Resonance Energy Transfer The energy of the excited
35. idth ms control which is also located in the Start Traces Visibility group Values below 1 ms can be defined by decimal values using a decimal point independent of the language settings see Figure 16 The Burst Analyzer Tutorial The zoom area of the measurement data diagram can be moved using the elements in Start Scroll and resized with elements in Start Zoom see Figure 17 MM U 4 FP Te Fast Left Right Fat To Zoom Zoom iE Bege det Rigt 1 Px Figure 17 Control elements for moving and scaling the visible area of the measurement d The om area visuaied asa Hue rectangle inthe overview diagram see Figure 18 To move the oom area without rescaling the view just click on the desired poston inthe overview diagram or drag and drop the blue rectangle Moving the mouse cursor on the edges of the blue rectangle will resize the mom area Enlarging the width ofthe rectangle zooms out leading tn increased memory consumption due to higher number of visualized photons Shrinking the width coms in yielding to fewer datapoints and les memory demand H LI TII I E LEN ANNE EN NN Figure 18 Overview Diagram The zooming area for panels 2 3 and s shown as a blue rectangle ts size and position can be changed directly by moving and squeezing i with the mouse The measurement diagram wil be updated accordingly 413 Diagram of Auxiliary Functions Panel 3 either shows a user d
36. ions within a sample Figure 8 Flurescence distribution calculated form a large number of bursts coming from dextran molecules labelled with Alexa 488 Left Two dimensional histogram showing correlations between burst intensity and lifetime The Burst Analyzer Tutorial 2 53 FRET Histograms With Burst Analyzer you can analyze single molecule FRET experiments You can identify single molecule fluorescence bursts by specifying intensity thresholds for donor and acceptor channels Thus you can separate bursts from interacting donor molecules from those which do not interact Figure 9 Left Donor lifetime distribution ofa complex molecule labelled with CFP and YFP simulated data The histogram shows 4 diferent FRET states Right Two dimensional histogram plot auf FRET efficiency obtained from donor and acceptor intensities proximity factor 12 1912 vs donar lifetime Based on your selection you can then get FRET efficiencies by analyzing the donor lifetime or donor acceptor intensity ratios In principle both approaches should yield the same results This is demonstrated in Figure 9 where the FRET efficiency obtained from donor and acceptor intensities is plotted versus the donor lifetime in a FRET experiment which shows multiple FRET states The Burst Analyzer Tutorial 3 Structure of Measurement Data Macrotime and Microtime In a classic TCSPC measurement photon
37. ll be filled with the data of the project file The first column is named with the type of the entry file burst or step all other columns are pairwise filled with the parameter name and the parameter value Make sure to have the decimal separator set to a point before importing the project file Otherwise Excel will not well recognize the number format The data setin the spreadsheet can naw be used as a starting point for further analysis A very common task is to generate histograms of a subset of all data points Let s assume that we want to create histogram of the proximity factors of all steps where the steps should have a minimum average intensity of the second trace To achieve that sort the worksheet by the first column to accumulate all steps to a contiguous range Then copy this range to a new worksheet and sort that new worksheet by the desired property In our example this is the column containing the PhotonCount2 entry The desired data points are now again in a contiguous range from which you can create a histogram with Excel Project entries of different types e bursts and steps can have different parameter sets Their values will occupy different columns Furthermore it may happen that even project entries of the same type may exhibit different parameter sets This situation can arise when the user changes the selection of attached properties when some bursts or steps have already been created To align all project
38. me Windows Within the function definition string you can also use a few mathematical functions by entering e g Math Logli1 12 The available mathematical functions are listed in Table 1 on p 26 The zoom area of the Auxiliary Function diagram is synchronized with the intensity traces of panel 2 Bursts and steps are visualized in yellow in both diagrams 13 The Burst Analyzer Tutorial If you choose Auto Correlation or Cross Correlation the x axis in the diagram indicates the correlation time and the graph displays a correlation function computed for the measurement data shown in panel 2 414 Microtime Histogram This chartshowsa histogram of photon arrival times for the photons within the currently selected time range In the border above the diagram the lifetime information derived from the first moments of the distributions is located see Figure 20 Here you can also define an offset to correct for an instrument response which is not centered at t 0 Additional information about microtimes can be found in section 3 2 Microtime Histogram seme nek 26 179 00 00 bert 1600 Maemum 14 ns 1638 omes teu rs Figure 20 Microtime Histogram The first moment af the photon distribution gives an estimate of 4 ns for the lifetime This Valve must be corrected by subtracting the first moment of the instrument response This offset can be defined for each t
39. menu band Bursts provides a set of user controls specifically for this purpose see Figure 22 Bursts Auto Burst Finder Settings presents a user dialog for configuring the thresholds for the burst finder algorithm see Figure 28 The automatic burst finder algorithm can be started by pressing the Find Bursts button in the Sursts Auto Burst Finder control group 19 The Burst Analyzer Tutorial Parameter ink Tel Burat Pan Ege eel oan lul I 40 Bo Bo Flank level me M Ho Eo oo 1 mo Ao Ho sorot ur El o Ab Ho oo mers Figure 28 User dialog for configuring thresholds for the automatic burst finder algorithm The first two parameters Min Level and Flank Level determine the size of the bursts Min Level is the minimum count rates a burst must have During the scan of the measurement data set every time bin will checked against this value As soon as this value is reached a burst is created with its start value at this time bin and zero length Next the left and right border of this new burst will be extended until the current count rate drops below the Flank Level value of the corresponding trace Now the burst created in this way will be checked against further parameters Min AVG and Max AVG are the minimum resp maximum mean count rates of the entire burst Peak Level is the maximum value which mus
40. mitted which gives rise to a peak in an intensity trace With Burst Analyzer you can find and analyzer such bursts in many different ways You can use the Burst Finder to identify bursts automatically or you can mark manually by simply using the mouse to select an area in the intensity chart This is done in the same manner as editing audio files with a wave editor 5 Figure 10 Measurement fle with two spectral channels blue and green and one marked burst yellow area Each photon arrival time is stored with routing information attached The routing information usually identifies a distinct detection channel from the measurement system In Burst Analyzer routing information is used to generate separate intensity traces for the detectors In addition to this you can even split routing channels into multiple intensity traces by using the microtime information or combine portions of different routing channels based on microtime 3 2 Microtime Histogram The microtime obtained from TCSPC experiments is the time difference between a photon and a reference signal In most configurations a pulsed laser is used to excite fluorescence and the microtime is the time difference between the fluroescence signal and the excitation pulse The measurement is done in reversed start stop configuration This means that each recognized photon starts an internal The Burst Analyzer Tutorial timer which is stopped by the next recording of a laser p
41. ne After that the borders are extended further until one intensity Link Or resp all intensites when Link And are above the parameters Flank Level in the area of the length Dark Width around the burst borders see Figure 31 In this example the Link property was set to Or therefore only one of the time traces was necessary to be continuously above its assigned Flank Level In this example it was the green time trace 2 If Link was set to And this condition should have hold true for each trace If Link was set to Sum the sum of the corresponding traces should have been above the threshold After the burst is defined in this manner its properties will be determined and checked against all the other parameters as described above If one check fails the burst will discarded Otherwise it will be added to the project Then the burst finder will proceed until the end of the file or the maximum mumber of bursts is reached The maximum number of bursts can be set by the parameter Bursts General Parameter Max Bursts see Figure 32 n The Burst Analyzer Tutorial Dark Width Flank Level Figure 31 With the help of the parameter Dark Width the burst was extended to its correct width The red areas mark the scanning regions where the Intensities are continuously above lank lever Another way to automatically create bursts is to add bursts with a fixed burst width In
42. nstant offset value which is subtracted automatically from the calculated first moment of the histogram to compensate for the IRF The Burst Analyzer Tutorial 4 Software Description All features of the software can be accessed from a set of toolbars The active toolbar is selected by selecting different tabs in the top row of this menu band Each toolbar contains subgroups of elements with similar functionality For example the dialog box for reading measurement files is located in the first tab Start and then in the first group Data File A click on the element Add SET SPC File opens the file open dialog BRE un Boswad se om Mjaa Figure 14 The tool bar stare In this manual the position of the operational controls is specified as tab group element name The example from above reads Start Data File Add SET SPC File The data analysis approach of Burst Analyzer is based on marking certain areas of the data stream attaching property values to these areas and finally extracting information from a statistical analysis of these properties The transition of a single molecule through the laser focus generates a photon burst which can be Identified and marked manually by the Burst Analyzer software This can be done for many bursts Yielding burst length statistics which can be used to derive the distribution of sizes of the observed molecules This information can be saved in a project file
43. ow proximity factor values The addition to the 4 16 Menu Band The whole functionality of the software can be accessed by the elements in the menu band They are arranged in tabs Each tab provides its own menu band containing groups of user controls Figure 22 shows all user control elements of the current software version ae E B masse s SH BE 0m oUm SE us Lela AA cer BAe Rz E Se ccm Eque So is S qus sie EE tey reine 2 Fi ee FECE E eue Co bere sean 2 Wu Wm m m ram o tx E Figure 2 The menu band consists of four tabs Start Bursts FS and Configuration Each of them provides user controls arranged in groups e Eel pd Te es ee GR ey Sey ae AE c 42 DataFiles A set of measurement data consists of one SET file and one to several SPC files If you have multiple SPC modules in your measurement system such set of files is created for each module The SET file saves the hardware settings of the measurement card and has a file size of a few kilobytes The photon 25 The Burst Analyzer Tutorial arrival times are stored into the SPC files The file size is proportional to the number of collected photons The typical file size is in the range of 10 100 megabytes Depending on the measurement parameters all photons are either stored in one large SPC file or distributed over several smaller SPC
44. photon arrival times are shown and automatically updated based on the currently selected time interval within the data stream The mean lifetimes for all intensity traces are derived from the first moments of these histograms and are shown above the decay chart The first moment is defined as m YN where N is the number of counts in time channel i and t is the time for the ith time channel which results in the following lifetime estimate Din XN a typical experimental setup the decay does not start at time t 0 but at a later time which is determined by first moment of the system s instrument response function To get accurate lifetime values this offset value must be provided It is recommended to set this to the maximum of the fluorescence decay function When you enter an offset the maximum position is indicated as a suggestion 00 00 O uu umet a9 00 00 5 o Maxam 8 am E Fo g i 45 He E E E 024 6 8 dwt D Figure 7 Histogram containing the lifetime information of the TESCP data An export function provides the histogram as ASCI fle to any further processing steps The etim i calculated from the frst moment of the decay data An offset correction can be provided 252 Lifetime Distributions The fluorescence lifetime can be calculated for each burst or step in an experiment This data can be used ta show lifetime distribut
45. race 1 loe Measurement channel Router 1 ls divided into tuo traces based on its mlcrotime information Trace 2 green and Trace 3 orange a The Burst Analyzer Tutorial 4 5 Correction of Traces Ina typical experiment the sensitivity of detectors can vary and the background level can also depend the wavelength and measurement channel In Burst Analyzer you can correct for some of these effects by assigning correction factors to the traces For every trace there are two correction parameters a constant offset and a scaling factor The offset value captures background noise the scaling factor reflects unequal detection efficiencies of the detectors During calculation the scaling factor is processed first afterwards the offset is subtracted The background value must be given in counts ms The parameters can be configured in the user dialog shown in Figure 25 The dialog is located at Configuration Corrections Settings see Figure 27 Beckaround 0 Sae 1 Figure 25 The user dialog Time Trace Noise Correction Parameter provides two correction factors for every trace background subtraction and a scaling factor for detection efficiency compensation The measured count rates wil first be multiplied with the scaling factor and afterwards corrected by subtracting the background offset value In more advanced setups with multiple detection channels e g separated by
46. race Individually 4 15 Project Panel The Project Panel shows information on the active measurement project It reflects the data stored in the project file The Project Panel has a tree structure with three hierarchical levels files bursts and steps Each file may contain bursts and each burst itself may contain steps For each burst or step the start time and length are shown In addition to this tree structure the project panel contains data tables which show burst and step data separately This lists are the basis for data analysis They are described in detail section 5 The selection of burst and step properties shown in the project tab is controlled by the settings in the Configuration tab see section 3 1 6 Attaching Properties to Bursts and Steps is described from page 22 onward The Burst Analyzer Tutorial cu with standard deviation burst steps na 55 Start Bof ma 9552 ZA burehat burch tauBuneh rg aso length burchz0 tauBunching 0 180000 OTT re Bunche taufuncing D Figure 21 Project panel Fles bursts and steps compose the project tree For each entr the ttr and length values shown Additional parameters aiso be attached to burst and steps In thls example bursts were generated with additional mean intensities and FCS curve fitting results while steps where configured to sh
47. rements Burst Analyzer can process projects of arbritary size asit creates an index file and only loads small parts of the data on demand It will run on any modern computer system including laptop and netbook systems However when large parts of a measurement are selected for viewing memory size is important Computer System recommended Intel Dual Core 2 GHz or better 2 GB RAM or better Harddisk space depends on amount of measurement data Operating System Windows XP SP3 Windows Vista Windows 7 Windows with Framework version 4 0 or higher installed 13 Installation Before install make sure you have your license key ready Start Setup exe from your installation media and follow the on screen instructions For Windows 7 or earlier versions Microsoft Framework 4 0 must be installed The setup program will check the presence of the framework and install it if needed After installing the framework you have to reboot your computer and restart setup exe 4 When running Burst Analyzer for the first time you will be asked for a license key Please enter the license key as written on your installation media If you do not see this dialog click the Burst Analyzer Icon in the system tray If you want to change your serial number g if you have added other bh products to your license or if you want to switch from a demo license to a full license click in the top right corner of the application window
48. state of a molecule can at appropriate conditions be transferred to an other proximate molecule This effect is very strong depandant from their mutual distance and arises noticeably only at distances below 10 nanometers The relation of the fluorescence intensities of an adequate choosen pair of fluorophores reflects their mutual distance This effect is thereby used to measure inter fluorophore distances on the nanometer scale Even more with fluorophores attached to known positions the conformational dynamic of single molecules can be investigated with a high temporal resolution Macrotime Microtime The time stamp of the photons consists of two times the absolute laboratory time Macrotime and the time duration since the preceded laser puls Microtime Out of the Macrotime correlation functions and intensity trajectories can be calculated The microtime is used to extract the lifetime Information of the excited state Step A 3 burst can hold sub areas which are called step Usually these areas are defined to identify time spans where the investigated molecule does not made a conformational change This results in steps in the FRET trace which explains the name TCSPC Time Correlated Single Photon Counting TCSPC denotes the here used measurement method single photons were not only counted but were attached and stored with a very precise time stamp The full temporal information of the detected photon stream is reflected by the data
49. t not be exceeded for a time longer than that given by the Peak Width parameter in milliseconds Min Phot and Max Phot are thresholds for the total number of photons in a burst A burst will be discarded if one or more of the property values is outside of the defined boundaries Each parameter can be activated or deactivated individually The traces on which the parameter operates can also be selected individually Furthermore each parameter can be linked between traces with the settings And Or or Sum The setting of the link mode does only play a role if more than trace is selected The link mode And means the threshold of the parameter must hold for all traces Or denotes that the threshold may exceed only for one trace to turn the parameter check to true Sum indicates that the sum of the time bin values of all selected traces will be checked against the parameter For marking bursts the parameter Min Level is to be linked with And in order to guarantee sufficient count rates for the selected traces The parameter Flank Level can be best linked with Or cause the expansion of the burst should terminate as soon as the count rate in one trace drops below the threshold value All other parameter can be linked with Or in order to ensure that a burst will not be added whenever one parameter in a trace does not fulfil the requirements There are three additional parameters in the Auto
50. this case a burst does not reflect a molecule diffusing through the focus but this mode can be useful to find out how sample properties change aver time within a measurement The control group Bursts Bursts with Fed Width contains user controls to define a burst width add adjacent bursts with this width in ms or equalize the burst width of all existing bursts automaticaly see Figure 32 Three further tools are available automatically add one step per burst delete all existing bursts and delete all existing steps of the current data set The value Max Sursts defines the maximum number of automaticaly created bursts to prevent the creation of an unwanted amount of bursts in case of a wrong parameter setting The button Refresh all Bursts Steps recaleulates all property values attached to bursts and steps see section 0 Attaching Properties to Bursts and Steps This may be necessary e g after changing the correction parameter For configuring correction parameter see section 4 5 Correction of Traces ae A 20000 ane Step Delte All Deletean 2002 arash at Per BUN Busts Steps Figure 32 Three control groups provide further automatic burst processing and step processing tals 48 Attaching Properties to Bursts and Steps There are five properties available to be attached to bursts and steps They are called Intensity Auxiliary Function Lifetime ACF and CCF They can be selected individually at Calculate B
51. tion 483 Lifetime Property If the Lifetime property is selected the mean fluorescence lifetime will be calculated from the microtime histograms of each new burst or step The calculation is based on the first moment of the microtime distribution and is carried out for all available data traces see section 4 1 4 Microtime The Burst Analyzer Tutorial Histogram for more details This can e g be used in FRET studies due to the fact that the donor lifetime reduces with increasing FRET efficiency 4 8 4 ACF and CCF Properties If one of the properties ACF and CCF is selected the auto correlation function ACF resp the cross correlation function CCF will be computed and fitted to the model selected in the FCS Cuve Fitting group The traces used for the calculation depend on the configuration the Start Ausliary Chart menu see section O Fluorescence Correlation Spectroscopy FCS FCCS for details Note the computation time increases with the number of photons in a burst and can take some time especially slower computers Fluorescence Correlation Spectroscopy FCS FCCS Fluorescence Correlation Spectroscopy is based on the analysis of temporal correlations within the fluorescence signal More specifically FCS refers to the measurement analysis of the autocorrelation function of a single intensity trace whereas FCCS is based on the cross correlation of two traces In a single molecule experiment the fluorescence
52. ulse To obtain a fluorescence decay profile from the data it is only necessary to reverse the time scale of the microtimes before the microtime histogram is built up In Burst Analyzer this is all done automatically and the histogram of microtimes directly shows the Probability of photon emission measured from the excitation pulse see Figure 11 and Figure 12 120 toa s00 400 Number of Photons 200 5 10 15 20 Microtime ns Figure 11 Theoretical distribution Function af the photon arrival times of an excited state The excited state of a quantum system Le the excited state of a single fluorophore species in a uniform environment with lifetime r will emit photons in an autonomous random process at constant average rate of In this case the microtime distribution is mono exponential The only free Parameter of such a mono exponential probability distribution is r For a mono exponential distribution this is equal to the first moment or average arrival time of the normalized photon distribution feroa freer joper However due to the finite width of the laser pulse and the limited system response time of the measurement electronics the microtime histogram does not show the distribution of photon arrival times of the excited state directly but a convolution of this distribution with the instrument response function The Burst Analyzer Tutorial g R Number
53. urst Properties and Configuration Calculate Step Properties see Figure 33 The selected properties are calculated whenever a new burst or step is created the borders of a burst or step are changed or when the Bursts General Paremeter Refresh all Bursts Steps button is clicked Figure 32 Configuratio The Burst Analyzer Tutorial Figure 33 Attached Properties can be selected for bursts and steps individually According to the settings made here every newly created burst will contain the mean Intensity vales of all available time traces and the fitting results of the auto Correlation function Every new step wil hold the mean value with standard deviation over his length of the user editable function Changing the selection of the properties does not affect existing bursts and steps Therefore it is possible that a project contains bursts or steps with different sets of properties With the user control Bursts General Parameter Refresh all Bursts Steps see Figure 32 the sets of properties for all bursts and steps within a project can be synchronized 4 8 1 Intensity Property When Intensity is selected the mean intensity of each burst is stored together with the peak intensity ofthe burst or step This is done for all available time traces All calculations are based on the corrected time traces see section 4 5 Correction of Traces As soon as the parameter for the correction changes the atta
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