Home

- Scientific Software Technologies Center

1. Model script rich editor Allows to edit model script The script is written with Script programming language Description page The Description page contains the description of the selected model An example view of the opened Description page is given below Le Custom model configuration Alias FCS Analysis atab ase o o X Fit parameters Formula Description Subtype a Conformational This i Conformational model written on the Script Programming Language Model 1 Model 2 Cancel Help Test Add model by Wizard lel ES ud ELLE ELM CM Eri f Filter Search New model wizard This wizard provides simple adding new custom model For more information see Custom model configuration dialog box topic An example view of the New model wizard is given below 36 FFSDP User Manual Step 1 5 New model wizard Models Parameter E Parameterz m Gl 37 FFSDP User Manual Step 3 Fa New model wizard OF ES Program model script Input variables Fit parameters Parameters result 1 Parameterd Parameters External parameters t Temperature Repeat umber NumChannels Duration Timestep SamplePos SamplePozsr LIrderMumber Back Net Cancel Step 4 Fa New model wizard SE ES Input model description optional oy can write the model description here Custom model integrity restore dialog box Custom model integr
2. Noise properties 1 Initial Rando specifies the initial value of the random variable generator This value should be odd 2 Constant level defines the amplitude of the constant noise component 3 Variable level defines the amplitude of the time dependent noise component 4 Time Power defines the noise distribution along the correlation function curve References to literature 1 E L Elson D Madge 1974 Fluorescence correlation spectroscopy I Conceptual basis and theory Biopolymers 13 1 27 2 J Widengren R Rigler 1998 Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solutions and on cell surfaces Cell Moll Biol 44 857 879 3 L Edman U Mets R Rigler 1996 Conformational transitions monitored for single molecules in solution Proc Natl Acad Sci USA 93 6710 6715 4 S Wennmalm L Edman and R Rigler 1997 Conformational fluctuations in single DNA molecules Proc Natl Acad Sci USA 94 10641 10646 5 Ulrich Haupts Sudipta Maiti Petra Schwille and Watt W Webb 1998 Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy Proc Natl Acad Sci USA 95 13573 13578 6 S R Aragon and R Pecora 1976 Fluorescence correlation spectroscopy as a probe of molecular dynamics J Chem Phys 64 1791 1803 7 R Brock M A Hink and T M Jovin 1998 Fluorescence Correlation Microscopy of Cells in the Prese
3. To unlink all parameters related to the given parameter group gt Select the current group in the Linked parameters treeview or Parameters settings table gt Press right mouse button on the selection to display the local menu and select the Empty local menu command If you want to delete the given parameter group while freeing all it s parameters then you should use the 55 FFSDP User Manual Delete local menu command instead of Empty local menu command The same operation for the parameter group currently selected in the Linked parameters treeview can be done using Delete selected parameter group button of the Parameters toolbar Linking parameters In this application the special object parameter group is used for linking parameters The parameters that belong to one parameter group are considered as linked to each other To link two or more parameters together you should perform the following steps 1 Create the new parameter group There are some ways to create parameter group Press the button Create new parameter group on the Toolbar of the Parameters page Right click on the Linked parameters treeview to open it s local menu Choose New group menu command from this local menu 2 Select the parameters you want to link in Free parameters list drag them and drop on the newly created group If you have previously created parameter groups you can add any free parameter s to any of these groups Also you can use local men
4. 8g P a gt Ng T Ay Y nr Ng I ULT 7 where g is the mean number of photons expressed in counts per second per molecule detected in a time interval T N is the mean number of molecules of the i th component in the open observation volume Bg is the mean background count rate of the detector and DG S ki Om T7 8 t nonu is the diffusion correction term los is the normalized correlation function of kth order 20 For the limit of short bin times it takes the form DT Aem We use normalization to the effective volume Pur ai ta in order to relate N obtained by both TIFCA and FCS Factor that appears in Eq 1 is due to this type of normalization factors are defined as P AA 67 PSF ya where PSF r is the normalized to unity brightness profile function To correct for deviations of the actual observation profile from its ideal approximation three different approaches can be used in FFC see Profile type property of the model 1 factors y can be fitted during the analysis applicable only in the global analysis with linking all fitted factors 10 FFSDP User Manual 2 FIDA like polynomial profile 21 22 can be used for the approximation of the actual PSF 3 out of focus correction of 2D 3D Gaussian or squared Gauss Lorenzian profiles 23 24 can be used In the case of out of focus correction Eq 1 becomes 144 A AP Y Ma 1 Fa F Zo idi 1 RIT E A Ay YN Tey 2
5. H1 M_OS0620 Alexa 488 SUng FCD Experiments Models M_030620 Alexa 488 50nq_PCD MM OS0620 Alexa 188 5Ung PCD MM OS0620 Alexa 188 5Una PCD MM 030620 Alexa 188 5Ung PCD M_OS0620 Alexa 188 5Una PCD MM OS0620 Alexa 188 5Ung PCD M_030620 Alexa 488 50ng_PCD M_OS0620 Alexa 400 S0ng PUD 460 BED SEO 161 1251 1451 1651 15851 261 M_O30620 Alexa 488 5Ong_PCD M_O30620 Alexa 488 5 ng PCD Add data set To be analyzed Analysis model name Analysis model type Osi vsTimeStept mM MivsTimeStepl Concentration vs time Be qi vsTimeStep2 hM qivsTimeStepz Brightness vs time B d R d ER d RE ud jenes e eee e er eem et mI SISSE SEDE SISSE d Hide Source Data Database Cancel Help Bellow the brief description of main components Fit parameters dependence page is given Experiments list This list displays the names of experiments currently opened in FFS DP The experiments that contain required data for creating dependencies of fit parameter on external parameter can be selected by checking corresponding checkboxes Model list This list displays the names of models that belong to experiments selected in the experiments list and have type currently choosen in the Type combobox list The models that contain fit parameters required for creating dependencies of fit parameter on external parameter can be selected by checking corresponding checkboxes Type combobox list This com
6. e A o 25 Os SS A 28 UNC IIA O STER TT T o eaten 31 Numero Expression topico iio brotan too loteria testa 22 la SUIS MS NITET ET PE E ECO O IL EET 22 GE NUR SI q ERI PTT TOT 32 Custom model configuration dialog DOX eve ee ae pastus Hs 60 nae es ra EE SN NERIS pev eO N ESSE CES SaS VN UNT R Ie E rREE Ies IPS NER TRAN NEN Sg IT VARPE NI NEUSS ES ERAS R CES R Rau 32 o 0 NN CRNERROE DORENRU n 33 A E A E A AE A A G 33 _ SS 33 A 33 A Aa 33 Modei SE notoria ia oido 33 o A 34 IO LENT CR HH PNWuT EE A 34 A uu 34 A q A EAA E EE E EOE S 34 M ODO a E A II RA E EE 34 ASS UE CO a gece cigs E m 34 Add model AAA elo E e A mi e 34 LIBE ES e OB 6 aM rr OPRIOARCeN ERe e EO TOLL 34 P TOTO EIUS TENTE 35 DEl DIRE LOU casada ODER 35 Pormuli Ste case acetates tesa apesess tonne 0 PUE E UE GAMME PE MEIN ISI NUMEN UU OE E EECMNPUS MPO ROMS OE 35 FFSDP User Manual UOC YASS RTT 35 PS a A Isleen E a Secun EE A ENEA 35 Punc 11 MS CO IDO AAA a e o E e E EE e O e Oe En e 33 zi D a e O FCO CO tacendo duds ie lnc EPIS RUE 36 o PP ES io 36 Model soip deuierk ilio EET T T OO DE Rm 36 Ps o EE T OD EO Re UR 36 New model VIZ ni M 36 Custom model integrity restore dialog DOX
7. external parameter view window is given on the following figure Fa Fit parameter vs external parameter view Options Fit parameter External parameter Ftrip hi Order umber Bellow the brief description of main components Fit parameter vs external parameter view window is given Fit parameter combobox list This combobox list is used to chose fit parameter to be displayed on the graph External parameter combobox list This combobox list is used to chose external parameter Note The External parameter is parameter concerned with external conditions of the measurements 76 FFSDP User Manual Button Help This button opens the help window that describes how to work with Fit parameter vs external parameter view window Button Close This button closes the Fit parameter vs external parameter view window 2D Chart This 2D Chart displays the Fit parameter vs external parameter curve The values of the fit parameter are displayed along the vertical axis and the values of the external parameter are displayed along the horizontal axis With 2D Chart you can move curve in the box by keeping the right mouse button down and moving the mouse pointer through the box zoom in the image by dragging the cursor diagonally across the necessary area from left top corner to right bottom corner restore whole view of the image by pressing left mouse button and dragging mouse pointer on some positions to
8. if Diffusion correction property is true 5 a is the structural parameter exists only if Diffusion correction property is true and Profile type property is 3D Gaussian 6 Fit parameters of selected profile 11 FFSDP User Manual To find out more information about selecting the model and specifying related properties and parameters see Experiment configuration box Properties table and Parameters table respectively 2D Gaussian profile 2x44 zl 2 PSP y z sali y factors are calculated by pei where d is the dimensionality d 2 3D Gaussian profile S 2 Peet y Z a n a e p y factors are calculated by y 1a where d is the dimensionality d 3 First order correction 2D Gaussian 1 st corr 3D Gaussian 1 st corr Squared Gaussian Lorenzian 1 st corr all Fx 0 except F1 Second order correction 2D Gaussian 2 nd corr 3D Gaussian 2 nd corr Squared Gaussian Lorenzian 2 nd corr all Fx 0 except F7 and F2 Specific FFC model parameters 1 Fcl is first order correction factor 2 Fc2 is second order correction factor 3D Gaussian fitted profile E 2 E Pere y Z expl A 5 S 0 7 factors for two first comulants are calculated as Mes 1 242 Par 1 8 respectively All other factors actually products 7275 2 are fit parameters Squared Gaussian Lorenzian profile E 2 z E Polley zZ exp eR uni zm cz wp cz 7 factors are calculat
9. 002 Tripl p T1 M_PINHOL30 003 Tripl l s A T1 M PINHOL30 004 Tripl vee P T1 M_PINHOL30 008 Tripl ven P T1 M_PINHOL30 00 Tripl m T1 fM PINHAL Mn Trini Configuration Configuration Analusis type Global Cl Probability 0 67 Cl Analysis type Exhaustive 5 PP a s Group 054 0 500 20 000 falze EF T trip s all 6 175E 3 1 000E 6 1 000 falze PERRERA 5 215E 2 1 000E 3 10 000 false 1 043 1 000E 3 100 000 false M 1 035 1 000E 3 100 000 false M M M 1 031 1 000 5 100 000 false 1030 100 2100 000 false Main window The view of the FFS Data Processor Main Window without any experiments being opened is given on the following figure 2 FFS Data Processor File View Tools Window Help Beh we a a Df Main Window contains Main menu and Main toolbar 40 FFSDP User Manual Main menu Main menu consists of the following popup items Items Description File Contains menu commands for managing the experiments View Contains menu commands for opening some application windows Tools Contains menu commands for configuring active experiment and opening Databases Window Contains menu commands for managing the view of opened experiment windows and provides the ability to switch between them Help Contains menu commands for accessing the online Help system and information about the copyright File menu Use commands of the File menu for ma
10. ES kinetics T C motion T where C inf is the level of autocorrelation function when T t by default C inf 1 Af denotes the average number of fluorescent particles Apc 1 bg denotes the background correction multiplier hg bg My dhg denotes background ratio where Z5 is the sample signal and Z amp is the uncorrelated background signal Correction to background is performed if property Background correction is set on kinetics t denotes some kinetic process emotion T describes the motion type of the particles The models are classified by kinetic term Akmetics t For each model with particular A amp kmeticsl t several types of motion are available trough the Motion type property The following motion types of the particles are implemented Free 2D Diffusion Free 3D Diffusion Anomalous 2D Diffusion Anomalous 3D Diffusion Confined 2D Diffusion Confined3D Diffusion TIRR FCS The fitting models designed in the FFSDP for the analysis of correlation functions are following Pure diffusion model Triplet State model Conformational model Protonation model Flow model Custom model Free 2D Diffusion term E l C motion 7 p DES CHE i 1 gt ue x 1 where and 7i id are respectively fraction and translational diffusion relaxation time of molecules of the i th fluorescent component Fit parameters added to the model by Free 2D Diffusion term 1 F is the contribution of mol
11. FFS Data Processor the Experiment is associated with a special object Experiment Object contains all other objects that are used for preparation of data and analysis execution Experiment icon is following a Experiment properties 1 Configuration provides access to Configuration dialog box Press the button on the right side of the property value box to open this dialog box 2 Analysis type allows to select a method for analysis The following Analysis types are currently available Global and Sequential 3 CI Probability specifies the value of confidential probability Minimum possible value is 0 001 Maximum possible value is 0 999 Default value 1s 0 67 4 CI Analysis type allows to select a method for confidential intervals analysis The following CI Analysis types are currently available Exhaustive search and Standard errors Data Set Data Set Object contains all necessary information that describes the data related to the one measured simulated characteristic autocorrelation function crosscorrelation function etc that corresponds to the sample under study Also this information defines the conditions under which the data of the given characteristic will be analyzed Data Set icon 1s following Ds 81 FFSDP User Manual Data Set properties 1 2 Local fit criterion displays a value of the Chi square calculated separately for current data set External parameters provides access to External parameters dialo
12. be created The names assigned to the parameters can then be used in the model script Custom model properties 1 IG type determines how the initial guesses of all model parameters are generated See list of IG type values for details 2 Configuration provides access to Custom model configuration dialog box Press the button on the right side of the property value box to open this dialog box 3 Subtype provides quick selection of the needed custom model from the database Custom model parameters The parameters are defined by user See Custom model configuration dialog box for more information To learn more about custom models see Using Custom model secton bellow PCH model The PCH model is used to analyse Photon Counting Distribution PCD The total PCD from a number of molecules is calculated by successive convolutions of a single molecule PCD 23 24 25 pin O g E Poila aT PSF Mar Ee 1 where Poi n A denotes the Poisson distribution with the mean value 2 Veris the effective volume and O is taken so that the product OVef is large enough to completely include the illuminated volume The total 1 distribution P n will be the weighted average of pl 2 9 convolved M times 25 P n Ng Y p n Qq Poi M QN MN AI p 0 0 q 13 5 p k Q q 2 ln 0 4 pm aie p n p amp p amp amp p n Y M 1 times PCH model properties 1 IG type determines how the ini
13. created To create and add a new Simulation Data Set to the current experiment click the Add simulation data set button Noise combobox list This combobox list is used to choose noise type The noise of this type will be used as default source noise for new Simulation Data Sets that will be created To create and add a new Simulation Data Set to the current experiment click the Add simulation data set button Button Add simulation data set This button creates and adds a new simulation Data Set to the current experiment Fit parameter dependence page An example view of the Fit parameters dependence page of Experiment configuration dialog box is given below F Experiment configuration Experiment 2 Measuremets database Simulator Fit parameter dependence Options E periments Models V Experiment 1 Experiment mmm External parameter Fit parameter FiepeatN umber H1 R d R d ER d kE ud ay Let ee rx ee A ey SER ds ree a cuaoopaeudoopoeg Add data set Ta be analyzed Analysis model name Analysis model type Os NivsTimeStept hj MivsTimeStepl Concentration vs time Os qlvsTimeStep hM qivgTimeStepz Brightness vs time Hide Source Data DataBase Cancel Help 66 FFSDP User Manual Fa Experiment configuration Experiment 2 Measuremets database Simulator Fit parameter dependence Options External parameter Fit parameter Type PLA RepeatNumber
14. example 13 01 2003 100 Analysis of single curve example 13 01 2003 101 Global analysis example 13 01 2003 Filter Database Er RD Experiment Name Is used for entering the name of the experiment you want to load from the Analysis Database You can quickly find the experiment you want to load by typing the first few characters of its name In this case the application will automatically select for you the first experiment that has the name with this few characters at the beginning Previously saved experiments Displays the experiments previously saved in the Analysis Database Button Help This button opens the help window that describes how to work with Open an existing experiment dialog box Button Open This button finishes work with Open an existing experiment dialog box and loads the selected experiment Button Cancel This button finishes work with Open an existing experiment dialog box without loading any experiments Button Filter This button opens Filter dialog window that allows to create the filter that can be applied to the Previously saved experiments list If this filter is applied the only experiment names that correspond to the filter criteria will be displayed 70 FFSDP User Manual Button DataBase This button opens Analysis Database and sets the record selected in the Open an existing experiment dialog box as the current record in the Database Save experiment as dialog box Save
15. experiment as dialog box is used to save the active experiment with a different name An example view of the Save experiment as dialog box is given on the following figure Save experiment as Experiment name Experiment 1 Dave Previously saved experiments Cancel 98 Analysis of simulated curve example 13 01 2003 Help 99 Custom model example 13 01 2003 100 Analysis of single curve example 13 01 2003 101 Global analysis example 13 01 2003 Filter Er RE Database Experiment Name Is used for entering the name for the experiment you are saving Previously saved experiments Displays the previously saved experiments in the Analysis Database Button Help This button opens the help window that describes how to work with Save experiment as dialog box Button Save This button finishes work with Save experiment as dialog box and saves current experiment using selected name Button Cancel This button finishes work with Save experiment as dialog box without saving the current experiment Button Filter This button opens Filter dialog window that allows to create the filter that can be applied to the 71 FFSDP User Manual Previously saved experiments list If this filter is applied the only experiment names that correspond to the filter criteria will be displayed Button DataBase This button opens Analysis Database and sets the record selected in the Save experiment as dialog box as
16. must be a letter Case sensitivity Script programming language identifiers are case sensitive so that Sum sum and suM are distinct identifiers Uniqueness Although identifier names are arbitrary within the rules stated errors are generated if the same name is used for more than one identifier within the script Variables Variable is a named storage location that can contain data that can be modified during script execution Each variable has a name that uniquely identifies it within script The variable names are sometimes referred to as 1dentifiers Variables can be of three types external variables fit parameters time and external parameters internal variables can be defined by user in the script result variable contains script execution result Internal variables declaration Internal variables declaration 1s a list of variable identifiers The declaration begins with keyword var The identifiers are separated by commas and the list is terminated by a semicolon Example var varl var2 var3 where varl var2 var3 are any sequence of distinct identifiers Operators Operators are tokens that trigger some computation when applied to variables and other objects 1n an expression 25 The following operators are available in the Script programming language Operator Operator Operator Operator Operator Operator This operator assigns the value of an expression to an internal v
17. passes over a toolbar button Button Export parameters This button executes the export procedure that exports all values from Parameters setting table to the standard MS Excel file After pressing this button Save dialog box will be opened in which the path and the name of the target MS Excel file can be specified Button New Group m This button creates new parameter group within the current experiment 49 FFSDP User Manual Button Delete selected parameter group es This button deletes parameter group selected in the Linked parameters treeview All parameters of this group will be moved in the Free parameters list This action 1s equivalent to the unlinkage of all parameters of this group Button Link mode This button sets the Link mode for the drag drop operation within the Linked parameters treeview This mode is used to relink the parameters related to the current experiment If you drag any parameter from one parameter group to another then this parameter will be moved from the source group to the target group If you drag any parameter group to another parameter group then all parameters from this eroup will be moved to the target group and the dragged group will be deleted iP Button Order mode E This Button sets the Order mode for the drag drop operation within the Linked parameters treeview This mode is used to order the parameters related to the current experiment If you want to change the position of any p
18. scripts Lexical elements e Whitespace e Tokens Variables Expressions Built in functions Whitespace Whitespace 1s the collective name given to spaces blanks horizontal and vertical tabs and newline characters Whitespace can serve to indicate where tokens start and end For example the following two sequences are lexically equivalent var a X y a x y and var a X y a XTy Tokens Tokens are word like units recognized by a language Script programming language recognizes five classes of tokens 24 FFSDP User Manual Here is the formal definition of a token e keyword e identifier e operator e built in function e punctuator also known as separator As the source code is scanned tokens are extracted 1n such a way that the longest possible token from the character sequence is selected For example external would be parsed as a single identifier rather than as the keyword extern followed by the identifier al Keywords Keywords are words reserved for special purposes and must not be used as normal identifier names Keyword var is used to declare new variables Example var x y Z In this example three variables x y and z are declared Variable identifiers Identifiers are arbitrary names of any length given to variables Naming and length restrictions Identifiers can contain the letters a to z and A to Z and the digits 0 to 9 There is only one restriction The first character
19. term motion t in the model equation see general formula Conformational model parameters 1 Ginf is the level of autocorrelation function when T ca 2 N is the average number of fluorescent molecules in the detection volume exists only if brightness correction is not performed 3 A 1s the pre exponential factor 4 beta is the stretch parameter S lau is the characteristic time of conformational relaxation 6 Fit parameters of selected motion term C motion UT To find out more information about selecting the model and specifying related properties and parameters see Experiment configuration dialog box Properties table and Parameters table respectively 20 FFSDP User Manual Protonation model For Protonation model 5 kinetic term kietics t in general formula is defined by the following equation A A IS e Fe Be a gt where P and P are the pre exponential factors and 2are the decay constants associated respectively with the external and internal protonation processes Protonation model properties 1 IG type determines how the initial guesses of all model parameters are generated See list of IG type values for details 2 ComponentsCount defines number of fluorescent components 3 Background correction determines if background correction is taken into account If this property 1s true the Data Set associated with the model should contain the external parameter BG ratio 4 Bright
20. the current record in the Database Export data dialog box This dialog box is used to export the target criterion value parameter groups free parameters and arays of characteristics and residuals to the text file An example view of the Export data dialog box is given below File ric Browse Data to be sawed x Parameters groups Target criterion value Gi Meme ml a Arrays to be saved X asis values Check all _ PINHOL30 004 Experimental data Check all PINHOL30 005 v PINHOL30 006 W Theoretical data PIMHOL30 007 Clear all PINHOL30 008 Residuals PINHOL30 009 Iv PINHOL30 010 e Related free parameters Check all Clear all Bellow the brief description of main components Export data dialog box is given File edit This box contains the name of the file where experiment data will be exported Button Browse This button displays the standard Open dialog box in which the path and the name of the file can be chosen Target criterion value check box If this check box is checked the target criterion value will be exported 12 FFSDP User Manual Related free parameters check box If this check box 1s checked the information about unlinked parameters related to each data set selected in the Data Sets list will be exported Data Sets list This list is used to choose the Data Sets the data will be exported from Button Check All This button selects all data s
21. the mouse should be moved in vertical direction to zoom the 3D image Button Move Mode If this button is down 3D image moving will be performed if the left mouse button is pressed and mouse is moved over the 3D Chart 3D Characteristics and Residuals charts 3D Characteristics chart displays measured green and theoretical red characteristics in three dimensional space 3D Residuals chart displays weighted residuals in three dimensional space 3D charts display all curves within the current experiment Here the curves sequence corresponds to the Data Sets sequence in the Configuration treeview An example view of the 3D charts is given below 61 FFSDP User Manual Charactenstics Residuals F P M ee M en pid E o E E En Y le ui L Ln r srl REA i pu I P 1 CIA I Smin 1 ETE 1 044H 2 rec v ET i 1 nl 1 LI E E 1 1 1 oT g T e r cord 5 51 3E 4 A Lin ay alae f 1 174E 2 oy s For 3D chart you can rotate zoom or move the 3D image by keeping the left mouse button down and moving the mouse pointer through the 3D chart The active transformation of 3D image depends on the mode that 1s set through the Toolbar or the 3D chart configuration dialog box Additional actions with 3D Chart available through the local menu Local menu of 3D chart This local menu contains following items 1 Default vie
22. the template you want to load from the Analysis Database You can quickly find the template you want to load by typing the first few characters of its name In this case the application will automatically select for you the first template that has the name with this few characters at the beginning Button Filter This button opens Filter dialog box that allows to create the filter that can be applied to the Previously saved templates list If this filter is applied the only template names that correspond to the filter criteria will be displayed Button Apply This loads and applies the selected template After pressing this button the progress bar window appears This progress bar window contains a number progress bars for displaying the progress of the template loading procedure Button Cancel This button finishes work with the dialog box without loading and applying any templates 75 FFSDP User Manual Button DataBase This button opens Analysis Database runs the template viewer and sets the record selected in the Load and apply an existing template dialog box as the current record in the template viewer Button Help This button opens the help window that describes how to work with Load and apply an existing template dialog box Fit parameter vs external parameter view window This window displays the dependence of the value of the given fit parameter on the external parameter An example of the Fit parameter vs
23. to prepare the source data in the Simulation Data Sets 44 FFSDP User Manual Button Execute This button executes the fit Button Stop e This button interrupts the fit d Button Chi square This button calculates Chi square for the current values of fit parameters Also theoretical curves and residuals curves will be built and displayed in the corresponding graphical windows Button View Properties If this button is down properties and parameters tables are shown otherwise they are hidden Button CI Analysis a If this button 1s down confidential intervals for the estimated parameters will be calculated after execution of the fit Button Configuration 4l This button opens the Configuration dialog box for configuring the current experiment Configuration treeview Configuration treeview displays the structure of the experiment and provides the access to any object included in it An example view of the Configuration treeview is given below El a De DataSet M ame seen uf tal Source measured data a Mi Model Model Name Conformational EI a Ds DataSet M ame E B tal Source simulation EMT M Model Conformational Lo HN Nose Normal Lo MI Model Model Mame Conformational Se Bg PH4GFP10 002 vee y tal Source measured data o MI Model Model Mame Conformational The objects that can be displayed in this treewiev are divided into the following classes E Experiment BS Dat
24. upper left Additional actions with 2D Chart available through the local menu Local menu of 2D chart This local menu contains following items 1 Default view 2 Save graph 3 Copy graph to Clipboard 4 Export data 5 Graph configuration 6 Save settings as default 7 Load default settings 8 Reset default settings Local menu command Default view Resets a view of the current 2D Chart to the initial state Local menu command Save graph This menu command saves the current 2D Chart image to BMP file Local menu command Copy graph to Clipboard This menu command stores the current 2D Chart image to Clipboard Local menu command Export data Exports data for the dependences currently displayed in the 2D Chart TI FFSDP User Manual Local menu command Graph configuration This menu command opens the 2D chart configuration dialog box for configuring the current 2D Chart Local menu command Save settings as default Saves current settings of the current 2D Chart as the default settings Local menu command Load default settings Loads and Applies the default settings to the current 2D Chart Local menu command Reset default settings Resets and Applies the default settings to the current 2D Chart separation of coincidence values histograms window This window is used to calculate the separation quality for two coincidence values histograms that can be choosen from experiments currently opened in FFS Data Proce
25. values that are used for configuring the corresponding object within the current experiment Parameters Parameters are the values that can be estimated during the analysis Structural parameter The structural parameter is given by Pz f Fry 80 FFSDP User Manual where rxy and rz are the distances from the center of the laser beam focus in the lateral and axial directions respectively at which the collected fluorescence intensity has dropped by a factor of e compared to its peak value for Gaussian beam profile Parameter group In this application the special object parameter group is used for linking parameters The parameters that belong to one parameter group are considered as linked to each other Parameter groups are displayed in the Linked parameters treeview Templates Template contains the all information the types of the models and their properties settings and links of the parameters being estimated that is related to the analysis of the Data Sets within the current experiment Template provides the quick way for analyzing the large amount of data with the same analysis scheme Local menus FFS Data Processor offers local menus context menus for several interface elements All these menus can be opened through a right mouse click when mouse pointer is over the element that contains local menu Experiment In the FFS Data Processor the notion Experiment means the numerical experiment Within the
26. was calculated while the fit 1s running while the analysis of the confidential intervals 1s running Parameters page This page is used to manage the settings of the fit parameters and parameters linkage An example view of the Parameters page is given below 48 FFSDP User Manual Parameters 3D Graphics 2D Graphics Linked parameters Free parameters FHER T1 s Group A 71 M_PINHOL30 001 Triplet State En Ttrip s Group P Ttip M_PINHOL30 001 Triplet S tate P Ftrip 1 008 1 000E 3 100 000 false M_PIMHOL30 003 Triplet State 1 013 1 000E 3 100 000 false M_PINHOL30 002 Triplet State 1 020 1 000E 3 100 000 false M_PINHOL30 001 Triplet State PABA Ne 5 093E 2 1 000E 3 10 000 false BBI Ttip s Group 4 833E 3 1 000E 6 1 000 false C E a 7 215 0500 20000 false M_PINHOL3O 001 TripletState q The following components are related to the Parameters page Parameters toolbar Linked parameters treeview Free parameters list Parameters settings table Click the corresponding item to get more information about it Toolbar of the parameters page Toolbar contains buttons for exporting the parameters settings managing the parameters and parameter groups within the Linked parameters treeview and setting the initial guesses of the parameters 3 ejm mm 1 The Parameters toolbar has short Help Hints Help Hint is the pop up text that appears when the mouse pointer
27. NMG DCL Tagi where Fk are correction parameters defined n relative difference between integral of the kth power of the actual observation profile y and that of its approximation PB v o AE In the most cases just first order correction 1s needed all Fx 0 except F1 and sometimes second order correction all Fx 0 except F 1 and F2 is necessary to get best fit to the experimental data FFC model properties 1 IG type determines how the initial guesses of all model parameters are generated See list of IG type values for details 2 ComponentsCount defines a number of molecular components with different brightness 3 Motion correction determines if motion correction due to diffusion is taken into account 4 Profile type specifies the type of PSF x y z The following profile types are available 2D Gaussian 2D Gaussian 1 st corr 2D Gaussian 2 nd corr 3D Gaussian 3D Gaussian 1 st corr 3D Gaussian 2 nd corr 3D Gaussian fitted Squared Gaussian Lorenzian Squared Gaussian Lorenzian 1 st corr Squared Gaussian Lorenzian 2 nd corr Polynomial FFC model parameters 1 Bg is the mean background count rate of detector 2 Nj is the mean number of molecules of j th brightness component 3 qj is the mean number of photons detected in a time interval brightness cpsm of j th brightness component 4 Tj is the translational diffusion relaxation time of molecules of the i th diffusion component exists only
28. Tab Bellow the brief description of main components of Custom model configuration dialog box is given button Makes a copy of the current record 7 button Deletes the current record button Inserts new empty record button Saves the current record button Cancels changes Models list Contains the list of custom models available from the custom models database 33 FFSDP User Manual Filter button Opens Filter dialog box that allows creating the filter that can be applied to the custom models table If this filter 1s applied the only table records that correspond to the filter criteria will be displayed Search button Displays a search box for the subtype field of the custom models database If you want to find quickly the necessary record you should type the first few characters and press Enter In this case the application will automatically select for you the first record that contains these characters at the beginning To close the search box press key Esc OK button Finishes work with the Custom model configuration dialog box and changes the configuration of the current model according to the dialog box settings Close button Finishes work with the Custom model configuration dialog box without changing the configuration of the current custom model Help button Opens the help window that describes how to work with Custom model configuration dialog box lest button Tests the script of the curr
29. a Set Data Source M Model 45 FFSDP User Manual H Noise The names of Experiment and Data Sets or additional information about Data Sources Models and Noises are displayed on the right of the corresponding object icon A red dot e on the left of any object means that this object is modified If the object 1s not modified the green tick is displayed on the left of the given object If the current object contains some child objects then it has one of the special indicators or El Press this indicator to show or hide the child objects Properties and parameters of the object selected in the Configuration treeview are displayed in the Properties and Parameters tables correspondingly Local menu of the Configuration treeview This local menu contains following items Rename Export Data Apply Template Save Template Simulate Execute Stop Calculate Chi square Show Properties CI Analysis Configuration pe ee T m o Local menu command Rename This menu command allows to change the name of the Experiment or Simulation Data Sets The Rename menu command appears only if the Experiment or Simulation Data Set object is selected in the Experiment configuration treeview After you have chosen this menu command the inplace editor will be displayed instead of the selected treeview node Local menu command Export Data This menu command opens Export data dialog box that allows exporting the data from any Data Set with
30. ae ote A e tet da 73 Arrays LOIS saved Pron DIR dan deed inter ta oret edm ete Deep A adea nati 73 Dutton PX 9191 gman RE ne Mon itat s tette TCPOM II dmt en EE ote staat eod 73 Button Help A m 73 A cc hd t race are dde tns d duae ce dental Cat ttai ote 73 Create and save new template dialog BOX eoe o rate o Ix See T eoe a vaa a Pane Viae obe Ene SOR Ue eon ea You ve ire nani inning orina iron Na ea ONU Pea qe 74 Buron A ic NL T T ovt 74 DUUOH O sca ste te eat a iM cec n gn tte aN D pa d M eM Me M ema aoa CE LS 74 Template ame RTT TEE 74 Button Database ii T 74 Buton A A nas orate ears ann ouacdouem eaten ouatesamies 74 Load and apply an existino template dialog DOX scrccessesnssesccceateossecsusetesseceusecnecssuscevevoneceaveenaceuseseesecdundeonteusssusueevesuasvaessesexoavenses 74 Previously saved templates alisar aa fotu pada td noa e eoa Leda E car T3 Template aMule Suenos 75 Dutton TEE eia Aa A N E IM E ee 75 Buton APII sah pcs T TS Dutton CAN Cele illa liada 75 Button DataBase Giese MC ica 76 Dutton Helper duoc died A uu ME M I 76 Fit parameter vs external parameter view window cceeee eee e eee eee ee e eee e ee eee tette e eee eese sees sss sas e eee ee eee ee esee eese esses sse aaae 76 Pit Pabalmelet COMUOWOK Sans aedi a diqumstuse asdita o buuspep iuo dita n itesuuepe EEEN 76 FFSDP User Manual Extemabparameter combOoDOX SL carte lili li
31. alysis If Fixed is true then the value of the current parameter 53 FFSDP User Manual will be constant during the analysis otherwise the parameter will participate in the fit CI Left CI Right Contain left and right bounds of Confidential Interval which was obtained for the current parameter or parameter group if Confidential Interval analysis was performed for it These fields are read only CI Analysis Indicates whether the Confidential Interval should be calculated for the current parameter or parameter group If button CI Analysis on the Configuration toolbar is down and CI Analysis value is true then the Confidential Interval will be calculated for the current parameter or parameter group while Confidential Interval Analysis 1s executed Owner Contains the Name of the model which generated the current parameter for parameters only It is read only Notes Fields CI Left CI Right CI Analysis are visible only if button CI Analysis is down on the Configuration toolbar The parameters that are displayed in this table can be sorted by any field To perform the sorting by the given field click the header of the appropriate column To change the sorting direction of the parameters click a second time on the same header A small triangle located on the right of the header name shows the sort direction Local menu of Parameters settings table This local menu contains following items Rename Empty Delete Link all paramete
32. ame s for selected Data Sets There are two special local menu commands in this submenu Same New applies the same newly created model names for all currently selected Data Sets Different New applies the different newly created model names for all currently selected Data Sets Submenu Analysis Model Type This submenu contains local menu commands for specifying Analysis model type for selected Data Sets Submenu Analysis Model Type is only available if analysis model names are supplied for all currently selected Data Sets Local menu command Delete This menu command deletes the Data Sets selected 1n For analysis table Local menu command Locate in source data Finds the currently selected Data Set in the Files list The File group that contains this Data Set will be found also in the File groups list Local menu command Find in Measurements Database This local menu command executes Measurements Database and forces it to find the currently selected Data Set Open an existing experiment dialog box Open an existing experiment dialog box is used to load a previously saved experiment An example view of the Open an existing experiment dialog box is given on the following figure 69 FFSDP User Manual if Open an existing experiment Experiment name Analysis of simulated curve example Open Previously saved experiments Cancel 398 Analysis of simulated curve example 13 01 2003 Help 99 Custom model
33. arameter within the given parameter group or the position of any parameter group within the current experiment you should drag this parameter or parameter group and drop on the necessary position Button Initial guesses IG This button sets initial guesses to all parameters and parameter groups within the current experiment Initial guess for every parameter depends on the value of the IG type property of model that contains this parameter Linked parameters treeview Linked parameters treeview is used for linking parameters and displaying the linked parameter groups To find out more about how to link and unlink parameters see Linking parameters and Unlinking parameters operations An example view of the Linked parameters treeview is given below Linked parameters i pb a s Group P a TSModel Triplet State E E a TSMadelz Triplet State Fl pel Ftrip s Group Fl ppl Ttrip a Group Epp T1 s Group Parameter groups are marked with the following icon IPF Parameters are marked with the following icon IP The names of the parameter groups and parameters are displayed on the right of the corresponding icons If the current parameter group contains some parameters then it has one of the special indicators El or E Press this indicator to show or hide the parameters related to the current parameter group 50 FFSDP User Manual Local menu of the Linked parameters treeview This local menu contains following items Rena
34. ariable Syntax varname expression The operator syntax has following parts Part Description varname Required any internal variable expression Required any numeric expression Example var Var Varl Var2 Varl 5 Var2 Varl 10 Var Varl Var2 Operator This operator is used to sum two numbers Syntax result expressionl expression2 The operator syntax has following parts Part Description result Required any internal variable expression Required any numeric expression expression2 Required any numeric expression Example var MyNumber Varl Var2 MyNumber 2 2 Returns 4 MyNumber 4257 04 98112 Returns 102369 04 Varl 34 Var2 6 Initialize variables MyNumber Varl Var2 Returns 40 Operator FFSDP User Manual This operator is used to find the difference between two numbers or to indicate the negative value of a numeric expression Syntax 1 result expressionl expression2 Syntax 2 expression 26 FFSDP User Manual The operator syntax has following parts Part Description result Required any internal variable expression Required any numeric expression expression2 Required any numeric expression expression Required any numeric expression Remarks In Syntax 1 the operator is the arithmetic subtraction operator used to find the difference between two numbers In Syntax 2 the operator is used as the unary negation oper
35. ator to indicate the negative value of an expression Example var MyResult MyVar MyResult 4 2 Returns 2 MyResult 459 35 334 90 Returns 124 45 MyVar 2 MyResult MyVar Returns 2 Operator This operator is used to multiply two numbers Syntax result expressionl expression2 The operator syntax has following parts Part Description result Required any internal variable expression Required any numeric expression expression2 Required any numeric expression Example var My Value MyValue 2 2 Returns 4 MyValue 459 35 334 90 Returns 153836 315 Operator This operator is used to divide two numbers Syntax result expression expression2 The operator syntax has following parts Part Description result Required any internal variable expression Required any numeric expression expression2 Required any numeric expression Note 21 FFSDP User Manual expression2 must be nonzero expression2 0 results in an runtime error You can t divide by zero Example var MyValue MyValue 10 4 Returns 2 5 MyValue 10 3 Returns 3 333333 Built in functions Built in functions are used to perform mathematical calculations The following functions are available in the Script programming language abs acos asin atan cos erf erfc exp fact Gamma log loglO logGamma pow sin sqrt tan abs Syntax abs x Description Returns the absolu
36. bobox list is used to chose model type The models of this type will be displayed in Models list located bellow Button Add data set This button creates new Data Set that contains the dependence of fit parameter on external parameter and adds it to the current experiment 67 FFSDP User Manual For Analysis table For Analysis table is used for displaying Data Sets within the experiment and the models that correspond to them This Table contains the following columns Data set displays the names of the Data Sets contained in the current experiment The values in this column can be changed only for Simulation Data Sets e Source model displays the type of the source model for the simulation Data Sets only Source noise displays type of the noise for the simulation Data Sets only Analysis model name displays the name of the analysis model related to the current Data Set Analysis model type displays the analysis model type Button Hide Show Source Data This button hides shows In Database box and Simulator box Button DataBase This button opens Measurements Database You can use this DataBase as more advanced way to navigate through your data and add some of them to the current experiment for analysis Button Ok This button finishes work with the Experiment configuration dialog box and applies all configuration changes to the current experiment Button Cancel This button finishes work with the Experim
37. ce 2D Residuals chart displays weighted residuals in two dimensional space 2D charts display curves according to the selection in the Configuration treeview If the Experiment object is selected in the Configuration treeview then the curves related to all Data Sets within the current Experiment are displayed in 2D charts If any Data Set or child objects Data Source Model Noise are selected then the only curves that belong to this Data Set are displayed in 2D charts On these 2D charts the values of the characteristic in the 2D Characteristics chart and residuals in the 2D Residuals chart are displayed along the vertical axes and arguments are displayed along the horizontal axes In the case if all curves are displayed in 2D chart particular curve can be selected by double click on it On these 2D charts the values of the characteristic in the 2D Characteristics chart and residuals in the 2D Residuals chart are displayed along the vertical axes and arguments are displayed along the horizontal axes An example view of the 2D charts is given below Charactenstics For 2D chart you can move curves in the box by keeping the right mouse button down and moving the mouse pointer through the chart zoom in the image by dragging the cursor diagonally across the necessary area from left top corner to right bottom corner restore whole view of the image by pressing left mouse button and dragging mouse pointer on some 58 FFSDP Us
38. cent component 3 Y is dy Fxy 4 Z 1s dzr 5 a is the structural parameter 17 FFSDP User Manual TIR FCS term ai F gt t Cal D to 75 ie e zx a a T where and 77 id are respectively fraction and axial diffusion relaxation time 1 of molecules of the i th fluorescent component a is the structural parameter a zy 0 erfcx x exp x erfc x is the scaled comlementary error function Fit parameters added to the model by TIR FCS term 1 Fj is the contribution of molecules of the i th fluorescent component In the case if brightness of the particles should be taken into account F 1s replaced by Nj the number of particles for i th component For more information see brightness correction 2 Tj 1s the axial diffusion relaxation time of molecules of the i th fluorescent component 3 a is the structural parameter Brightness correction Brightness correction for correlation function model determines if different quantum efficiency of each component is taken into account In the case of brightness correction the following replacement is made in the motion term of a model equation 2 EE Nj pe where qi and Ni are respectively brightness and number of particles for i th component 2 Mi is the average number of fluorescent particles Ai is the contribution of molecules of the i th fluorescent component AM N Fi in the case if brightne
39. cons of all minimized experiment windows Minimize All Minimizes all opened experiment windows Window List Displays the list of currently opened experiments One can switch to required experiment by all items below clicking it s name the separator Help menu Use commands of the Help menu to access the help system and get information about the copyright The Help menu contains the following commands Commands Description Contents Opens Help topic contents About Displays the copyright and version number for FFS Data Processor Main toolbar The Main toolbar provides shortcuts for menu commands DEA e t 5d The correspondence between buttons and menu commands is the following Button Menu commands FilelNew FilelOpen FilelSave ToolsIMeasurements DataBase ToolslExperiments DataBase ViewlCVH separation WindowlCascade 9 0 e eje ou jo WindowlTile The Main toolbar has short Help Hints Help Hint is the pop up text that appears when the mouse pointer passes over a toolbar button Experiment window Components of this window are used for creating and configuring an experiment executing the analysis procedure and displaying the results Experiment window has two parts Configuration part left and Analysis part right An example view of the Experiment window is given on the following figure 42 FFSDP User Manual X Global analysis example E ST e gt ee Y g amp U CIA i yf El Global analysis exampl
40. cribes how to work with Custom model integrity restore dialog box Using FFS Data Processor Starting FFS Data Processor Fluorescence Fluctuation Spectroscopy Data Processor FFS Data Processor can be started via Start button on the Windows Task Bar gt Goto the Programs FFS Data Processor and click the FFS Data Processor item Choose Run and specify the path to FFSDP EXE Windows Explorer Locate and double click the FFSDP EXE file 1f you have performed a default installation this file is located in Program Files SSTC FFS Data Processor Bin Take a look on a successfully loaded FFS Data Processor 39 FFSDP User Manual SF FFS Data Processor Global analysis example Ea File View Tools Window Help laj xl D c uw mw i PIT gt SE g IG gt 7 ee d El wf E Global analysis exampl G De PINHOL30 001 0 of RE Source measu 0 wf M Model M PINI S De PINHOL30 002 0 f RE Source measu 0 wf M Model M_PINI Ej Del PINHOL30 003 oe gf tal Source measu 0 wf M Model M PINI Ej Del PINHOL30 004 ba ta Source measu 0 wf M Model M PINI E f Del PINHOL30 005 0 of RE Source measu wf M Model M PINI E vi PINHOL30 006 m w fis Source measu k Analysis information Chi criterion value 0 892 Properties Parameters Parameters 3D Graphics 20 Graphics Linked parameters El pP a s Group z pn T1 s Group be P T1 M_PINHOL30 001 Tripl s A T1 M_PINHOL30
41. dad 76 BORO Te ION TI ISUttonm CIOS Aaa EA EE E MES TE P BICI ELE C Tcr Pc E Locale ob 2D CD EE oo as LA Loto DL EM LA LL dE T11 Separation of coincidence values histograms windOW sssssseseccececccccsosssecccccccccccoccosossssceseeecccccococccccssseecccccccocossssssssseccceeoo 78 Exper mene liS Gace aa CEE H 79 OPUS DORK a cesis cte decias 79 PBXG CIPUE c MN 79 beparation quabiby O A A veu Sian A SA ciue Moe ue uA amin eden Uu Us cM EN 79 External parameters dialog POX id eee aee EVEN RS Ue vea ee EYvER S eeu ER Ee ERN Edu ue ee Ee See Neve pne ME eR ES UE E DP eeu EE NE el a PE e eov ERES UNION 79 Tabples BxteraL patate e EST cerea te tents a acne e oda de eoe o ach dtes dete i eas dat la Cantera ftoi rat 79 A MLR en t Cn Ra dtp dte ttd dt deter t dd i dme d dba e bue a ey te O 80 Button Dele Goat irte ti n 80 BUON OS A crm 80 BUON MEO aec EET 80 Buton C ANICE M ER EH TET 80 Button Hess nas 80 TERMO 3 80 WW CS 80 FES ot E E M M ee 80 PROPERTIES mec P n 80 Paramete Saana ea ostesuesusiivauaseosoncuatescussseddessivceascelosccaluscusddedsbussieueiseduuucesesddebuccnedusdueedecidsetsecedsesadauvsive
42. der A small triangle located on the right of the header name shows the sort direction The free parameters that are going to be linked will be moved from this list to the Linked parameters treeview The linked parameters that are going to be unlinked will be moved from the Linked parameters treeview to Free parameters list To find out more about how to link and unlink parameters see Linking parameters and Unlinking parameters operations Local menu of the Free parameters list This local menu contains following items 1 Link all parameter name J s 2 Link To 3 Find Model 4 View on graph 52 FFSDP User Manual Local menu command Link All This menu command creates new parameter group with name parameter name s Group and moves all parameters with the name parameter name to this newly created parameter group Note parameter name is the name of the first parameter in the selection Local submenu Link To This submenu contains local menu commands that provide the ability to link the selected parameter s to any existing parameter group The name of the submenu command is equivalent to the name of the target parameter group Local menu command Find Model This menu command finds and selects model in the Configuration treeview which contains the parameter currently selected in the Free parameters list This local menu command is visible if only one parameter 1s selected in the Free parameters list Local menu co
43. e Properties Parameters Parameters 3D Graphics 20 Graphics sg Del PINHOL30 001 Y uf tal Source measur EN Confiquratior Cort w M Model M_PINH re ses E T1 M_PINHOL30 001 Triplet State a e F 71 M_PINHOL30 002 Triplet State E e Dig PINHOL30 002 7 V Ej Source measun C Analysis type Esha 8 T1 M_PINHOL30 002 Triplet State T Ttrip s Group me M Model M PINH Ei Be PINHOL30 003 E Tip M_PINHOL30 001 Triplet State _ ni T Frin hd IRA 20 OA T rindat Cb aka 3 I y tal Source measun Loo wf M Model M PINH Linked parameters El el 11 Group Parameters settings Mame Value Minimum M aximum F Owner a E F1 1 000 0 000 1 000 fale M PINHD F1 1 000 0 000 1 000 fale M PINHD P F1 1 000 0 000 1 000 fale M PINHD P Ftrip 3775E 2 0 000 1 000 false M PINHD Final Chi 2 criterion value P Ftiip 0108 0 000 1 000 fale M PINHD 1 207E 5 F Ftrip 0113 0 000 1 000 false M PINHD E Gin 1 000 0500 1500 tue M_PINHO T The Analysis part of the Experiment window consists of three pages Parameters page is used for working with parameters e 3D Graphics page is used for displaying correlation functions and weighted residuals in three dimensional space e 2D Graphics page is used for displaying correlation functions and weighted residuals in two dimensional space Note To choose the necessary page while working with FFS Data Proce
44. e average flow time of the fluorescent particles through the detection volume Flow model properties 1 IG type determines how the initial guesses of all model parameters are generated See list of IG type values for details 2 ComponentsCount defines number of fluorescent components 3 Background correction determines if background correction is taken into account If this property is true the Data Set associated with the model should contain the external parameter BG ratio 4 Motion type specifies the motion term motion in the model equation see general formula Flow model parameters 1 Ginf is the level of autocorrelation function when T ca 2 N is the average number of fluorescent molecules in the detection volume 3 Tf is the average flow time of the fluorescent particles through the detection volume 4 Fit parameters of selected motion term motion t To find out more information about selecting the model and specifying related properties and parameters see Experiment configuration dialog box Properties table and Parameters table respectively Methods Global fit The parameters of the models attached to the characteristics can be estimated by a global fitting procedure based on the Marquardt Levenberg non linear method of least squares 9 10 In the global analysis several measured and or simulated characteristics are combined and simultaneously fitted Certain parameters can be linked The values o
45. e confined volume diameter Fit parameters added to the model by Confined 2D Diffusion term 1 F is the contribution of molecules of the i th fluorescent component In the case if brightness of the particles should be taken into account F 1s replaced by Nj the number of particles for i th component For more information see brightness correction 16 FFSDP User Manual 2 Tj is the translational diffusion relaxation time of molecules of the i th fluorescent component 3 Y is dy Fxy Confined 3D Diffusion term on DD Ph g A ET 28 where l gy iT el x E e erf Y 1 f Jum errem st ns x efe 4 siet 24 we TRUM Ipa Elz 0 689 0 345 0 37 FHS dy pL r 4 Cea r Mand 7i id are respectively fraction and translational diffusion relaxation time of molecules of the i th fluorescent component a is the structural parameter and z are distances in lateral and axial direction at which the intensity of the exciting laser beam is dropped by a 4y and dz are the confined volume diameters Fit parameters added to the model by Confined 3D Diffusion term 1 F is the contribution of molecules of the i th fluorescent component In the case if brightness of the particles should be taken into account F 1s replaced by Nj the number of particles for i th component For more information see brightness correction 2 Tj 1s the translational diffusion relaxation time of molecules of the i th fluores
46. e ees Pene e eee P e eee ee eee eee sene eee ee 69 Experiment Name vir A ieee D tuse dea est Ue DeC a Us Seni iaa 70 Previously saved Experiments ueque sisi esee EDU Udo Ede USUS RU A S cas wou een Ueno veu USUS C OU reddent UU ups nian aaah 70 Button Help ct p A A A I 70 BUON Open a T 70 Button Poles ni A A AA ene 70 BUDA o UE Cer R 70 Buron DAA cde De PEEL C D porc en pM remeare re dir e E M 71 Saveexpermient AS dialog DOX 5 dee ede hae edi eee es 71 Experiment IN AINE utn diei De bait Doch tad duet tetto dedu feat dct fu udo fotu Gat ab tette tuos added fedt bed MN east nd rnt 71 PENOSA Saved XDOrmie fits aces d idu a 71 Button Help atte tti a M oic m Ned be ro uA cedo Abtei Nm di idu eas eee t etus 71 Buton SAVE usd ln dota toad cut Su oli 71 eU EO aC Gy PROC RN N ater ken dat aatcd E E ui yeaatst daanien ua E she Cine sa eh onetaee Gestaats 71 Dutton PIET Sg ates born each date ten E yt desit pte tasto cos iE dose a a e qoxrate Ica token Cos M ove ska cte 71 Dron IAL ALAS E aiii ili E E T2 ExporEdata CAO DON os ensem dero ardore Enid sv e ed niv Mese D eie i tupieriotu e 72 polaco m Y p P TET 72 Paton Browse assi EA E a EA 12 Iateetontenom yalllc Chec KNOX sessin sed 12 RElated res parameters Checo DO ss ntsc 73 WEIN EMI UE uu IU Tem 73 Buton a qim e nO Po O aA 73 Batom Clear Ally os I Uu ITO 73 Parameters PIOUDS E FOU DOX tea coe s
47. e opened Formula page is given below Le Custom model configuration Alias FCS Analysis atab ase t Xx Fit parameters Formula Description fai Subtype Input variables Model script Conformational Fit parameters 3 ts y Cancel Model 1 F x abs al Model 2 var parti parta Help part1 14 2 exp pove titau beta TM parta 1 01 HT sartil tr a a T 111 Test result 1 partl part2 External parameters Add model by Wizard keij Repeat umber NumChannels Duration TimeStep gt S amplePaz S amplePaozY Filter Search OrderHumber Input variables list Displays model parameters that can be used as variables in the model script The name of any parameter viewed in Input variables list can be double clicked with left mouse button to be added to the current cursor position in Model script rich editor External parameters list Displays the time and external parameters that can be used as variables in the model script The name of any parameter viewed in External parameters list can be double clicked with left mouse button to be added to the current cursor position in Model script rich editor Functions combobox Contains list of all built in functions 35 FFSDP User Manual button Inserts function currently selected in the functions combobox to the current cursor position in Model script rich editor button Shows help about function currently selected in the functions combobox
48. ecules of the i th fluorescent component In the case if brightness of the particles should be taken into account F 1s replaced by Nj the number of particles for i th component 14 FFSDP User Manual For more information see brightness correction 2 Tj is the translational diffusion relaxation time of molecules of the i th fluorescent component Free 3D Diffusion term di l E l where and 7 id are respectively fraction and translational diffusion relaxation time of molecules of the i th fluorescent component a is the structural parameter Fit parameters added to the model by Free 3D Diffusion term 1 F is the contribution of molecules of the i th fluorescent component In the case if brightness of the particles should be taken into account F 1s replaced by Nj the number of particles for i th component For more information see brightness correction 2 Tj 1s the translational diffusion relaxation time of molecules of the i th fluorescent component 3 a is the structural parameter Anomalous 2D Diffusion term midi T oe where and 7i id are respectively fraction and translational diffusion relaxation time of molecules of the i th fluorescent component c denotes the anomality factor Fit parameters added to the model by Anomalous 2D Diffusion term 1 Fj is the contribution of molecules of the i th fluorescent component In the case if brightness of the particles should be taken into account F 1s re
49. ed by 12 FFSDP User Manual e ud Fk NET Ya Cam Polynomial profile For the polynomial profile Eq 1 becomes E Bg Tto Ma Ey 3 2 Na T P Tapa AB ak 6a 4 k amp where Ey En fv A vB and u Aa Poa 1 v Ad 3S a1 and a2 are the fit parameters Ao and Bo are chosen so that the normalisation conditions assumed in FIDA 21 2 are satisfied Specific FFC model parameters 1 a1 is the brightness profile parameter 2 a2 is the brightness profile parameter Gaussian model The Gaussian model is used to analyse coincidence values histogram 16 and photon counting distribution The model is based on the Gaussian distribution function am ac d a fe qct where m is the mean os the standard deviation Gaussian model properties 1 IG type determines how the initial guesses of all model parameters are generated See list of IG type values for details Gaussian model parameters 1 M is the mean of the Gaussian distribution 2 Sigma is the standard deviation of the Gaussian distribution To find out more information about selecting the model and specifying related properties and parameters see Experiment configuration box Properties table and Parameters table respectively 13 FFSDP User Manual Correlation function models The correlation function models 17 18 19 that are included in FFSDP are based on the following general formula 1 2 GIT E Cu UN A BG
50. ef description of main components Create and save new template dialog box is given Button Save This creates and saves the new template using selected name Button Cancel This button finishes work with the dialog box without creating and saving current template Template name Is used for entering the name for the template you are creating and saving Button DataBase This button opens Analysis Database runs the template viewer and sets the record selected in the Create and save new template dialog box as the current record in the template viewer Button Help This button opens the help window that describes how to work with Create and save new template dialog box Load and apply an existing template dialog box This dialog box is used to load and apply a previously saved template An example view of the Load and apply an existing template dialog box is given below 74 FFSDP User Manual LfLoad and apply an existing template Template name Template 1 Previously saved templates Filter 25 Template 1 26 Template 2 DataBase Help Apply Cancel Bellow the brief description of main components Load and apply an existing template dialog box is given Previously saved templates Displays the templates previously saved in the Analysis Database If the filter 1s applied the currently displayed template names depend on the filter criteria Template name Is used for entering the name of
51. ent configuration dialog box without changing the configuration of the current experiment Button Help This button opens the help window that describes how to work with Experiment Configuration dialog box Local menu of Configuration dialog box This local menu contains following items Rename Select AII Source Model Type Source Noise Type Analysis Model Name Analysis Model Type Delete Locate in source data Find in Measurements Database OPRIMA Local menu command Rename This menu command allows changing the name of the Simulation Data Sets The Rename menu 68 FFSDP User Manual command appears only if the name of any Simulation Data Set is selected After you have chosen this menu command the inplace editor will be displayed in the selected cell Local menu command Select All This menu command selects all Data Sets in the For analysis table Submenu Source Model Type This submenu contains local menu commands for specifying Source model type for selected Data Sets Submenu Source Model Type is only available if all currently selected Data Sets are Simulation Data Sets Submenu Source Noise Type This submenu contains local menu commands for specifying Source noise type for selected Data Sets Submenu Noise Model Type is only available if all currently selected Data Sets are Simulation Data Sets Submenu Analysis Model Name This submenu contains local menu commands for specifying Analysis model n
52. ent model for errors Add model by Wizard button Opens the New model wizard for simple adding new custom model Initial settings table Displays the initial settings of the parameters related to the current custom model Icon column contains the parameter icon Name column contains the name of the parameter It can be changed Value column contains the value that will be used as initial guess for current parameter It can be varied between the minimum value contained in the field Minimum and the maximum value contained in the field Maximum Minimum column contains the minimum possible value of the parameter Maximum column contains the maximum possible value of the parameter Fixed column indicates whether curent parameter is initially fixed when new custom model of current type is created If Fixed is true then the value of the current parameter will be constant during the analysis otherwise the parameter will participate in the fit The final state of the parameter fixed or not is defined in the Parameters settings table Notes The parameters order can be changed by dragging icon of corresponding parameter to new place within 34 FFSDP User Manual the table Add button Adds a new parameter to the given model Delete button Deletes the parameter currently selected in the Initial settings table Formula page This dialog box provides all operations related to managing the custom model An example view of th
53. er Manual positions to upper left Additional actions with 2D Chart available through the local menu Local menu of 2D chart This local menu contains following items 1 Default view 2 Save graph 3 Copy graph to Clipboard 4 Export data 5 Graph configuration 6 Save settings as default 7 Load default settings 8 Reset default settings Local menu command Default view Resets a view of the current 2D Chart to the initial state Local menu command Save graph This menu command saves the current 2D Chart image to BMP file Local menu command Copy graph to Clipboard This menu command stores the current 2D Chart image to Clipboard Local menu command Export data Exports data for the dependences currently displayed in the 2D Chart Local menu command Graph configuration This menu command opens the 2D chart configuration dialog box for configuring the current 2D Chart Local menu command Save settings as default Saves current settings of the current 2D Chart as the default settings Local menu command Load default settings Loads and Applies the default settings to the current 2D Chart Local menu command Reset default settings Resets and Applies the default settings to the current 2D Chart 3D Graphics page This page is used to display characteristics and weighted residuals in three dimensional space The following components are related to the 3D Graphics pages e Toolbar e 3D Characteristics and Residuals box
54. es An example view of the 3D Graphics page is given below 59 FFSDP User Manual l FO Auto CR ie a aie Parameters 3D Graphics DO Graphics Characteristics Residuals 1 ix Uy Poh hog L ON L 1 BE fe be eee 3E 4 qe 1 174E 2 Toolbar of the 3D Graphics page This toolbar contains buttons for changing view of 3D Graphics page ejr 81 This toolbar has short Help Hints Help Hint is the pop up text that appears when the mouse pointer passes over a toolbar button Auto Button AutoSize Auto If this button is down the size of the graphical boxes that belong to the 3D Graphics page is automatically changed while the Experiment window resizes If this button is up the vertical size of the Characteristics box and Residuals box can be changed manually using the splitting line that lies between them Button Show Characteristics c If this button is down Characteristics box is visible 60 FFSDP User Manual Button Show Residuals R If this button 1s down Residuals box is visible ieu Button Rotate Mode E If this button is down 3D image rotation will be performed if the left mouse button is pressed and mouse is moved over the 3D Chart e Button Zoom Mode e If this button is down 3D image zooming will be performed 1f the left mouse button is pressed and mouse is moved over the 3D Chart Note
55. essssseeoccsssssseeccceccccoccccssssssssssececececcccocccsssssssscccccceccoccssssssssssecccceoccesssssssssscececesessssss 38 USING FFS DATA PROCESSOR noite iaa dais 39 meer nite FES Dala Processor NN m 39 VIA WOW emm M P M t 40 REISEN T 41 Mamona TP T 42 I quauis A 42 Configuration part of the Experiment window ee eee eee ee e e e eee ee eee ee ettet tete eese eese essa s eaae eee e esee esee e eese esses see eee eee eee esee 43 CIMT LE AOA DD S TREE D T T LR Dmm 44 Cont UE AONE TRE CV NET E mmm 45 Local menu ot the Config ration Eee VIO W ss A A iii 46 PA AMT Ves Es 0 NN OT T e UE O 47 PR A D a T OON pon O O E TE 47 Local ment or gross Uic RED o Oo o Em 48 A IOMA OA VUTERCIOW pi 48 WAT AIC COTS PU TERTII TE o II E 48 Toolbar OF Pris ari UU UU M 49 Linked parameters Web A a e E PE NEM FIM E A 50 Local menu of the Linked parameters TEC VIeW iiiiioieis vera rin RS el bas dee E Pea d RIA A a inacabada dicci n 51 PCG par BCLs MIS ON E E E rn PI o Oo REO TN MUS 52 Local ment ofthe Free parameters Osorio iia 52 cl e E E A E E A EEA E T E E co o ure NUES 53 Localmenu of Parameters setings Table ciclo 54 Alt parane EDS coen EEEE EET 55 B
56. eter on external parameter The fit parameters for this dependence can be taken from currently opened experiments that contain analysed data sets Note To choose the necessary page while working with FFS Data Processor you should click on the corresponding Tab Mesurements database page An example view of the Measurements database page of Experiment configuration dialog box is given below 63 Fa Experiment configuration Experiment 1 Measuremets database Simulator Fit parameter dependence In Database Sllas FFSMeasurementsD atab ase File groups Filter Search 22 10 1333 18 04 2005 Filter Search PINAOL SO 001 PINHOL30 00 2 PINHOL 30 005 PINHOLSO 004 PINHAOL50 005 PINHOL30 006 PINHOL 30 007 PINHOL30 008 PINHOL 30 005 FFSDP User Manual Autocorrelation Autocorrelation Autocorrelation Autocorrelation Autocorrelation Autocorrelation Autocorrelation Autocorrelation Autocorrelation To be analyzed Ss PINHOL30 007 M_PINHOL30 007 Triplet State Ss PINHOL30 008 M_PINHOL30 008 Triplet State Ss PINHOL30 009 M_PINHOL30 009 Triplet State Ss PINHOL30 010 M_PINHOL30 010 Triplet State E Hide Source Data DataBase Bellow the brief description of main components of Measurements database page is given Alias Shows the alias associated with Measurements Database In Database box In Database box is used for displaying and choosing the measured data from t
57. ets in the Data Sets list Button Clear AII This button unselects all data sets 1n the Data Sets list Parameters groups group box This group box provides a possibility to export the data related to parameters groups The following information about the parameters groups can be exported Names Values Included parameters Arrays to be saved group box This group box is used to choose the arrays of values to be saved Check the corresponding check box to export the certain array The following arrays of values are available for the export X axis values Experimental data measured or simulated data gt Theoretical data the data calculated with chosen theoretical model Residuals Check all Button Check al selects all arrays Clear all Button unselects all arrays Button Export This button executes the export procedure Button Help This button opens the help window that describes how to work with Export data dialog box Button Close This button finishes work with Export data dialog box 73 FFSDP User Manual Create and save new template dialog box This dialog box is used to create and save a new template An example view of the Create and save new template dialog box is given below Pate and save new template a Template name Template 3 Previously saved templates Filter 25 Template 1 26 Template 2 Database Help Save Cancel Bellow the bri
58. f Enzyme Kinetics Biophys J 83 1671 1681 Oleg Krichevsky and Gregoire Bonnet 2002 Fluorescence correlation spectroscopy the technique and its applications Rep Prog Phys 65 251 297 Samuel T Hess Shaohui Huang Ahmed A Heikal Watt W Webb 2002 Biological and Chemical Applications of Fluorescence Correlation Spectroscopy A Review Biochemistry 41 697 704 Petra Schwille and Elke Haustein Fluorescence Correlation Spectroscopy A Tutorial for the Biophysics Textbook Online 2002 Wu B and J D Muller 2005 Time Integrated Fluorescence Cumulant Analysis in Fluorescence Fluctuation Spectroscopy Biophys J 89 2721 2735 Kask P K Palo D Ullmann and K Gall 1999 Fluorescence intensity distribution analysis and its application in biomolecular detection technology Proc Natl Acad Sci USA 96 13756 13761 Palo K U Mets S Jager P Kask and K Gall 2000 Fluorescence intensity multiple distribution analysis concurrent determination of diffusion times and molecular brightness Boiphys J 79 2858 2866 Perroud T D B Huang M I Wallace and R N Zare 2003 Photon Counting Histogram for One Photon Excitation ChemPhysChem 4 1121 1123 Huang B T D Perroud and R N Zare 2004 Photon Counting Histogram One Photon Excitation ChemPhysChem 5 1523 1531 Chen Y J D Muller P T So and E Gratton 1999 The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy Biophis J 77 553 567 Victo
59. f parameters linked together are kept equal to each other Each parameter can be fixed into the predefined value For each parameter the range of admissible values can be set by defining the constraints Sequential fit The parameters of the models attached to the characteristics can be estimated by a sequential fitting procedure based on the Marquardt Levenberg non linear method of least squares 9 10 In the sequential analysis several measured and or simulated characteristics are fitted one by one Each parameter can be fixed into the predefined value For each parameter the range of admissible values can be set by defining the constraints 22 FFSDP User Manual Initial guesses For correlation functions the initial guesses for parameters are generated by the phase plane method For other characteristics method of moments is used To find out more about how to generate initial guesses for parameters while working with FFS Data Processor see button Initial guesses of the Parameters page toolbar or command Initial guesses of the Parameters settings table local menu Quality of fit The quality of fit is judged by criterion and visual inspection of the residuals between experimental and fitted curves Exhaustive search Error estimation of the recovered parameters can be performed by the exhaustive search method 12 see button CI Analysis of the Configuration toolbar In this method the examined parameter is fixed at a nu
60. g box Press the button on the right side of the property value box to open this dialog box 3 Points count indicates the total number of points in the measured simulated characteristic This 4 property is read only if Data Set contains measured data otherwise it is changeable Start analysis at defines the initial point for analysis The characteristic contained in the Data Set will be analyzed starting at this point Minimum possible value is 0 Maximum possible value is Points count 1 5 End analysis at defines the end point for analysis Minimum possible value equals to the Start analysis at Maximum possible value is Points count 1 Default value is Points count 1 Start criterion at specifies the initial point for the Chi Square calculation Minimum possible value equals to the Start analysis at Maximum possible value is Points count 1 Default value equals to the Start analysis at Data Source Data Source Object is used to prepare the source data for the analysis Data Source can be Measured or Simulation Measured Data Source is used for preparation of measured data Simulation Data Source is used to generate data on the bases of Model and Noise Data Source icon 1s following Gel Data Source properties This object does not have any properties Model FFS technique 1s aimed to the investigations of the intensity fluctuations of fluorescent molecules excited by a tightly focused laser beam The
61. he Measurements Database This box contains Files groups list and Files list The Files group list displays file groups available from the Database the Files list displays files contained in the selected files group To add measured data to the current experiment double click left mouse button on the selected file or the file group If the item from the Files list was double clicked the data set with the same name will be added to the For analysis table If the item from the File groups list was double clicked the procedure described above will be repeated for every file related to this group Files group list The Files group list displays file groups available from the Measurements Database Files list The Files list displays files contained in the files group selected in the Files group list 64 FFSDP User Manual Button Filter This button is used for filtering data in the corresponding list After this button is pressed Filter dialog box will be displayed This dialog box is used for creating and applying the filter to the corresponding list Button Search This button displays a search box for the corresponding list If you want to find quickly the necessary item you should type the first few characters of its name and press Enter In this case the application will automatically select for you the first item that has the name with this few characters at the beginning To close the search box press key Esc Simulator page S
62. he object currently selected in the Configuration treewiev An example view of the Parameters table is given below Properties Parameters CE trip trip T1 6 S98E 2 Note To choose the Properties table you should click on the properties tab Properties table This table is used to display the names and the values of the properties related the object currently selected in the Configuration treewiev An example view of the Properties table is given below 47 FFSDP User Manual Properties Parameters Hame Value AAA External parameters Points Court 192 Start analysis at b End analysis at 131 Stark criterion at b Note To choose the Parameters table you should click on the parameters tab Local menu of the Properties table This local menu contains following items 1 Apply to all similar objects Local menu command Apply to all similar objects This menu command allows to set the value of the currently selected property to the same properties of all similar objects within the current experiment Analysis information window This window monitors the information about the analysis progress An example view of the Analysis Information window is given below Analysis information Analysis is running Chi final value 1 5 7 3E 2 finish with difference of criterion values Final Chi 2 criterion value The new information in this window appears in the following cases after Chi square
63. ical models are represented by the corresponding Model Objects Model Object can belong either to the Data Set or to the Simulation Data Source In the first case Model is used to generate the theoretical characteristic In the second case it 1s used to generate noise free source characteristic Model icon is following mM Now the following models are available to be used with FFS Data Processor Characteristics Models Correlation function Pure Diffusion 2D 3D free diffusion Triplet State 2D 3D anomalous diffusion Conformational 2D 3D confined diffusion Protonation TIR FCS are supported FCS flow Custom Coincidence values histogram Gaussian Custom Comulants FFC Custom Photon counting distribution PCH Gaussian Custom Identifiability All models mentioned above except Custom model and PCH with FIDA like polynomial brightness profile approximation are totally identifiable 1 e all parameters of the models can be uniquely determined on the basis of only one measured characteristic PCH with FIDA like polynomial brightness profile approximation at fixed background can have up to 3 equvalent sets of parameters each of them can be used 27 FFSDP User Manual Custom model Custom model is designed to perform the data analysis with user defined mathematical description The special Script programming language was developed for writing the user defined models Before constructing new model some fit parameters should
64. ies table and Parameters table respectively Triplet State model For Triplet State model 8 kinetic term inneties T in general formula is defined by the following equation Fs c5 A kinetics x zr Lud Feo 1 Foy and 77 are respectively the fractional population and relaxation time of the triplet state where F sip En Triplet State model properties 1 IG type determines how the initial guesses of all model parameters are generated See list of IG type values for details 2 ComponentsCount defines number of fluorescent components 3 Background correction determines if background correction is taken into account If this property is true the Data Set associated with the model should contain the external parameter BG ratio 4 Brightness correction determines if different quantum efficiency of each component is taken into account If this property is true the Data Set associated with the model should contain the external parameters Component i brightness where i 1 2 n n number of fluorescent components See brightness correction for more details 5 Motion type specifies the motion term motion t in the model equation see general formula Triplet State model parameters 1 Ginf is the level of autocorrelation function when T ca 2 N is the average number of fluorescent molecules in the detection volume exists only if brightness correction is not performed 19 FFSDP User Manual 3 F t
65. imulator page is used to create and include to the current experiment a new Simulation Data Set This box contains three combobox lists named characteristic model and noise Characteristic combobox list 1s used to choose type of the characteristic that will be simulated The model type and the noise type that were chosen in the corresponding comboboxes will be used as default source model and source noise for new Simulation Data Sets that will be created To create and add a new simulation data set to the current experiment click the Add simulation data set button An example view of the Simulator page of Experiment configuration dialog box is given below 40 Experiment configuration Experiment 1 Measuremets database Simulator Fit parameter dependence Characteristic Autocorrelation Model Pure Diffusion Noise Marmal hal Add simulation data set To be analyzed Analysis model name Analysis model type De Datas et I DataSet 1 Pure Diffusion PESCE Pure Diffusion Hide Source Data DataBase Cancel Help Bellow the brief description of main components of Simulator page is given 65 FFSDP User Manual Characteristic combobox list This combobox list is used to choose type of the characteristic that will be simulated Model combobox list This combobox list is used to chose model type The model of this type will be used as default source model for new Simulation Data Sets that will be
66. in the current experiment to the text file Local menu command Apply Template This menu command opens the Load and apply an existing template dialog box for applying the previously saved template to the current experiment Local menu command Save Template This menu command opens the Create and save new template dialog box for creating a new template and saving it to the Analysis Database Local menu command Simulate This menu command executes the simulation procedure to prepare the source data in the Simulation Data Sets Local menu command Execute This menu command executes the fit 46 FFSDP User Manual Local menu command Stop This menu command interrupts the fit Local menu command Calculate Chi square This menu command calculates Chi square for the current values of fit parameters Also theoretical curves and residuals curves will be built and displayed in the corresponding graphical windows Local menu command Show properties If this menu item is checked properties and parameters tables are shown otherwise they are hidden Local menu command CI Analysis If this menu item is checked confidential intervals for the estimated parameters will be calculated after execution of the fit Local menu command Configuration This menu command opens the Configuration dialog box for configuring the current experiment Parameters table This table is used to display the names and the values of the parameters related t
67. inke POAC E ae E TO TOO DE EEE EE O 56 la T I C I 56 Toolbar OF he2 D Graphics PA Ce erstes 57 21 Characteristics and Residuals charis csi veunnssSuuienseedtineduguyanensesnsedaneneauteouseaubboernaneness 58 bocolar D CO OR 59 3D Graphics Ppa iE AO NN UI aa r a 59 dro logo BA Crt Ap MICS PITE OT mmm 60 3D Characteristics and Residuals charts 2c sccnscacuenatouduccnncaescwainemasowedsncdeunssctsiesadsetseoabiiedadaadiaenbnutdiadantaniedsawahswehseadehisewadcubiednsiedanet 61 Bore ICRU OU BION MORET E Ms 62 Experiment configuration dialog DOX nora esye eve Y oe p ER iia ER EP deenen e eae AAA e bn eris doen s VAN Va Us Ve Vas AA ENEE aE 63 Mesurements AED SNB EO m 63 Simulator page A UREAN EErEE AA E EE ENEE EEEE E EAEE EAE UNSERES NAET EKEREN 65 Fit parameter de pene MCS pane ns codi 66 FFSDP User Manual POr ARTIS ADS estaca RR TEE 68 Button Hide Show SOURCE Dita titan ld 68 Duttom DAA do 68 O O ee eaert ee eee e 68 Dutton c ANC trea hice ida detto E ee ketene ceeds ta Sada MEUM EE 68 Dutton HA lp d e IE ies MEM E 68 Local menu of Configuration dialog box eeeesessisssisssssssssseeeeen nennen eene nee nsesene ei irr nnn nnn nn nere sess sss sss sen err r nnne eren 68 Open an existing experiment dialog DOX eee eee eee eon ener e root erre reete eere eene eoa Pee epo Pee eee e eee eee ee
68. ity restore dialog box is used to restore the custom model integrity Custom model integrity means that custom model contents with given subtype must exactly correspond to the custom model contents with same subtype stored in the Custom model database An example view of the Custom model integrity restore dialog box is given below 38 FFSDP User Manual Custom model integrity restore E inl x Custom model Custom model is different than custom model with the same subtype stored in the Custom Models database Lk What would you like to do Use the following custom model with same contents Custom model old f Save with new subtype Custom model new C Dvenwrite Any time when the custom model integrity 1s broken it can be restored by one of the following ways 1 By using custom model with same contents but different subtype This option is available in the case if Custom model database contains records with same contents as in current custom model but with different subtype names 2 By saving current custom model contents to Custom model database with new subtype 3 By overwriting custom model contents with same subtype currently stored in the Custom model database One of the ways mentioned above can be chosen by selecting the corresponding radio button Button Ok finishes work with the Custom model integrity restore dialog box and restores the experiment integrity Button Help opens the help that des
69. lent to the name of the target parameter group Local menu command Find Model This menu command finds and selects the model in the Configuration treeview which contains the parameter currently selected in the Linked parameters treeview 51 FFSDP User Manual Local menu command View on graph This menu command opens Fit parameter vs data set number view window Submenu command Position This submenu contains local menu commands that are used for changing the position of the selected parameter within the given parameter group or the position of any parameter group within the current experiment This submenu contains the following commands Move Up moves the selected parameter or parameter group one position up Move Down moves the selected parameter or parameter group one position down Place First places the selected parameter or parameter group to the first position in the list Place Last places the selected parameter or parameter group to the last position in the list Local menu command New Group This menu command creates new parameter group within the current experiment Free parameters list Free parameters list displays all free unlinked parameters within the experiment An example view of the Free parameters list is given below The parameters that are displayed in this list are sorted by name To change the sorting direction of the parameters contained in the Free parameters list click on the list hea
70. mber of particular values in a predetermined range while other parameters are allowed to adjust to the minimum of 2 Thus the dependence of the values on the particular parameter 1s observed Analysis stops when the calculated value of becomes higher than the level obtained from the statistical F test for that particular confidential probability and number of degrees of freedom 9 The value of the examined parameter obtained by the procedure described above 1s taken as the border of the confidential interval To set confidential probability use property CI Probability of the Experiment Object Standard errors Error estimation of the recovered parameters can be performed by calculating asymptotic standard errors 14 see button CI Analysis of the Configuration toolbar Confidential interval for the parameter pj j 0 1 is calculated according to the following equation PE F P TEN min y P Py bee fy Tmin Ey In this equation l pj the value of estimated parameter 2 Ci the jj element of the inverted Marquardt matrix obtained after the analysis 3 a 1 fis confidential probability 0 lt K1 4 tah the upper percentage point of the f distribution Student distribution with v n p 1 degrees of freedom 71 is the number of experimental points and p is the number of estimated parameters It can be calculated from the definitions of percentage point of Student distribution and its distrib
71. me Empty Delete Expanded Free Link To Find Model View on graph Position New Group b Ed ee Local menu command Rename This menu command allows to rename parameter group selected in Linked parameters treeview After you have chosen this menu command the inplace editor will be displayed instead of the selected treeview node Local menu command Empty This menu command empties the parameter group currently selected in the Linked parameters treeview and moves all its parameters to the Free parameters list Execution of this command is equivalent to the unlinking the parameters contained in this group Local menu command Delete This menu command deletes the parameter group currently selected in the Linked parameters treeview and moves all its parameters in the Free parameters list Execution of this command is equivalent to the unlinking the parameters contained in this group and deleting the current parameter group Local menu command Expanded If this menu item is checked the parameters related to the current parameter group are visible Local menu command Free for parameters only This menu command unlinks the selected parameter in the Linked parameters treeview e g moves it to the Free parameters list Local submenu Link To This submenu contains local menu commands that provide the ability to relink the selected parameter or parameter group to any other parameter group The name of the submenu command is equiva
72. mmand View on graph This menu command opens Fit parameter vs data set number view window Parameters settings table This table displays the settings of the parameters and parameter groups related to the current experiment An example view of the Parameters settings table is given bellow Parameters settings P F1 1 O00 0 000 E aun false false TSModelt Triplet State P F1 1 000 Don 1 000 false false TSModel2 Triplet State PP Ftrip Group 0 146 o Or 1 B00 false falze P tu B7B 1 OOOE 3 100 000 false false TSModelt Triplet State E N a 401 1 OOOE 3 100 000 false false TSModel2 rTriplet State pP T1 Group 6 S96E 2 1 000E 3 10 000 false false PP Ttrip s Group z 2B54E 3 1 OOUE 6 1 000 false false Wa a s Group gd 565 0 500 20 000 false falze Parameters settings table contains the following fields Field Name Description Name Contains the name of the parameter or parameter group It can be changed only for parameter groups Value Contains the value of the parameter or parameter group It can vary from the minimum value contained in the field Minimum to the maximum value contained in the field Maximum Minimum Contains the minimum possible value of the parameter or parameter group Maximum Contains the maximum possible value of the parameter or parameter group Fixed Indicates whether the value of the current parameter or parameter group should be optimized during the an
73. molecules of i th brightness component 3 qi is the mean number of photons detected in a time interval brightness cpsm of i th brightness component 4 Tj 1s the translational diffusion relaxation time of molecules of the i th diffusion component exists only if Motion correction property is true 5 a is the structural parameter exists only if Motion correction property is true 6 F trip is the fractional population of the triplet state exists only if Process correction property is Triplet State 7 Trip 18 the relaxation time of the triplet state exists only if Process correction property is Triplet State 8 Tar is the dead time exists only if Dead time correction property is true 9 Fit parameters of selected profile FFSDP User Manual To find out more information about selecting the model and specifying related properties and parameters see Experiment configuration box Properties table and Parameters table respectively 3D Gaussian profile E E E PSP z ezp C ian ae E a Sp 2 Out of focus correction 3D Gaussian 1 st corr 3D Gaussian 2 nd corr To improve the model Perroud and Huang 23 24 introduced additional fitting parameters Fx defined as the relative difference between the integral of the k th power 7 of the normalized to unity actual brightness profile function PSF r and that of its 3D Gaussian approximation zo M L Yond Ton 3 Fk can be also treated as an out of focus emis
74. n example view of the External parameters dialog box is given below External parameters Ei Add Temperature 20 RepeatNumber 5 Delete MumChannels 192 Duration 247 Create new TimeStep E P SamplePosx 46 409 SamplePosy 37 337 IrderNMumber OF Cancel Bellow the brief description of main components External parameters dialog box 1s given Lr Table External parameters This table 1s used for displaying external parameters within the Data Set 79 FFSDP User Manual It contains the following fields Name displays the name of the external parameter contained in the current Data Set Value displays the value of the external parameter Button Add This button adds empty external parameter to the current Data Set which must be filled in Button Delete This button deletes the external parameters selected in the External parameters table Button Create new This button opens Parameters form Button Ok This button finishes work with the External parameters dialog box and applies all changes to the current Data Set Button Cancel This button finishes work with the External parameters dialog box without applying any changes Button Help This button opens the help window that describes how to work with External parameters dialog box Terms FCS FCS Fluorescence Correlation Spectroscopy FFS FFS Fluorescence Fluctuation Spectroscopy Properties Properties are the
75. naging the experiments The File menu contains the following commands Commands Description New Creates a new experiment Open Opens an Open an existing experiment dialog box for opening or finding an experiment Close Closes the active experiment without exiting the application Save Saves the active experiment In the case if active experiment is saved for a first time Save experiment as dialog box is opened Save As Opens Save experiment as dialog box for saving the active experiment with a different name Exit Finishes work with FFS Data Processor View menu Use commands of the View menu for managing the experiments The View menu contains the following commands Commands Description CVH separation Shows a Separation of coincidence values histograms window Tools menu The Tools menu contains the following commands Commands Description Experimental configuration Opens the Configuration dialog box for configuring the currently active experiment Measurements Database Opens the Measurements Database Analysis Database Opens the Analysis Database Window menu Use commands of this menu for managing the view of opened experiment windows and switching between them The Window menu contains the following commands Commands Description Cascade Resizes and positions all windows in an overlapping pattern Tile Resizes and positions all windows in a non overlapping pattern 4 FFSDP User Manual Arrange Icons Arranges i
76. nce of Autofluorescence Biophys J 75 2547 2557 8 Widengren J Rigler R and Mets U 1994 Triplet state monitoring by fluorescence correlation spectroscopy J Fluorescence 4 255 258 83 10 IT 12 15 14 15 16 17 18 19 20 21 22 25 24 25 26 27 FFSDP User Manual Bevington P R Data Reduction and Error Analysis for the Physical Sciences McGraw Hill New York 1969 Grinvald A and I Z Steinberg 1974 On the analysis of fluorescence decay kinetics by the method of least squares Anal Biochem 59 583 598 E G Novikov A van Hoek A J W G Visser and J W Hofstraat 1999 Linear algorithms for stretched exponential decay analysis Opt Commun 166 189 198 Beechem J M Gratton E Ameloot M Knutson J R Brand L In Topics in Fluorescence Spectroscopy Vol 2 ed J R Lakowicz Kluwer Academic Publishers New York 2002 p 241 Johnson M L Faunt L M 1992 Parameter Estimation by Least Squares Methods Methods Enzymol 210 1 37 Martin Straume Susan G Frasier Cadoret and Michael L Johnson In Topics in Fluorescence Spectroscopy Vol 2 ed J R Lakowicz Kluwer Academic Publishers New York 2002 p 177 J D Muller 2004 Cumulant Analysis in Fluorescence Fluctuation Spectroscopy Biophys J 86 3981 3992 Katrin G Heinze Markus Rarbach Michael Jahnz and Petra Schwille 2002 Two Photon Fluorescence Coincidence Analysis Rapid Measurements o
77. ness correction determines if different quantum efficiency of each component is taken into account If this property is true the Data Set associated with the model should contain the external parameters Component i brightness where i 1 2 n n number of fluorescent components See brightness correction for more details 5 Motion type specifies the motion term motion t in the model equation see general formula Protonation model parameters 1 Ginf is the level of autocorrelation function when T ca 2 N is the average number of fluorescent molecules in the detection volume exists only if brightness correction is not performed 3 P is the pre exponential factor associated with the external protonation 4 P is the pre exponential factor associated with the internal protonation 5 taul is the decay constant associated with the external protonation 6 tau2 is the decay constant associated with the internal protonation 7 Fit parameters of selected motion term motion T To find out more information about selecting the model and specifying related properties and parameters see Experiment configuration dialog box Properties table and Parameters table respectively FCS flow model For FCS flow model 19 kinetic term ianetics T in general formula is defined by the following equation Genie Js Aj zb mena LE 21 FFSDP User Manual where C motion t is the motion term in general formula zy is th
78. placed by Nj the number of particles for i th component For more information see brightness correction 2 Tj 1s the translational diffusion relaxation time of molecules of the i th fluorescent component 3 alpha is the anomality factor 15 FFSDP User Manual Anomalous 3D Diffusion term motion T E gt i 1 where and 7i id are respectively fraction and translational diffusion relaxation time of molecules of the i th fluorescent component a is the structural parameter denotes the anomality factor Fit parameters added to the model by Anomalous 3D Diffusion term 1 F is the contribution of molecules of the i th fluorescent component In the case if brightness of the particles should be taken into account F 1s replaced by Nj the number of particles for i th component For more information see brightness correction 2 Tj 1s the translational diffusion relaxation time of molecules of the i th fluorescent component 3 alpha is the anomality factor 4 a is the structural parameter Confined 2D Diffusion term Emi 0 2 5 g al Z5 where gy 1 E Jf i Zhi x erf Y 1 a RUNNY a 53 Jm erf Yi K i 0 689 0 34 03705f F EA P Pi and 7 id are respectively fraction and translational diffusion relaxation time of molecules of the i th fluorescent component denotes the distance in lateral direction at which the intensity of the exciting laser beam is dropped by e Gy is th
79. r V Skakun Mark A Hink Anatoli V Digris Ruchira Engel Eugene G Novikov Vladimir V Apanasovich Antonie J W G Visser Global Analysis of Fluorescence Fluctuation Data Eur Biophys J 34 2005 323 334 V V Skakun E G Novikov V V Apanasovich H J Tanke A M Deelder O A Mayboroda Initial Guesses Generation for Fluorescence Intensity Distribution Analysis Eur Biophys J 35 5 2006 410 423 84
80. r name s Link To Find Model View on graph Initial guesses Export Parameters Optimize View Roe IIS bw us Local menu command Rename Allows to rename the parameter group currently selected in the Parameters settings table Local menu command Empty This menu command empties the parameter group currently selected in the Parameters settings table and moves all its parameters to the Free parameters list Execution of this command i1s equivalent to the unlinking the parameters contained in this group Local menu command Delete This menu command deletes the parameter group currently selected in the Parameters settings table and moves all its parameters in the Free parameters list Execution of this command is equivalent to the unlinking the parameters contained in this group and deleting the current parameter group 54 FFSDP User Manual Local menu command Link All This menu command creates new parameter group with name parameter name s Group and moves all parameters with the name parameter name to this newly created parameter group Note parameter name is the name of the currently selected parameter Local submenu Link To This submenu contains local menu commands that provide the ability to link the selected parameter to any existing parameter group The name of the submenu command is equivalent to the name of the target parameter group Local menu command Find Model This menu command finds and selec
81. ration Example Var X y Z Semicolon The semicolon is a statement terminator Any legal Script programming language expression 1s followed by a semicolon Numeric expression Any expression that can be evaluated as a number Elements of an expression can include any combination of variables built in functions and operators that result in a number Expression An expression is a sequence of operators operands and punctuators that specifies a computation Syntax variable part numeric expression Where variable part is any defined internal variable Note The script must contain at least one expression with result variable as variable part Parameters Parameters are the values that can be estimated during the analysis Custom model configuration dialog box This dialog box provides all operations related to managing the custom model An example view of the Custom model configuration dialog box is given below 32 FFSDP User Manual Fa Custom model configuration a Alias FCS Analysis atab ase 7 x i Fit parameters Formula Description Or a Subtype Initial settings Cancel Conformational Madel 1 Name value Minimum Maxmum Fixed A Model 2 In ase Help false false false false false d Filter Search 5 Delete Notes To choose the necessary page while working with Custom model configuration dialog box you should click on the corresponding
82. rip is the fractional population of the triplet state 4 T trip 18 the relaxation time of the triplet state 5 Fit parameters of selected motion term motion t To find out more information about selecting the model and specifying related properties and parameters see Experiment configuration dialog box Properties table and Parameters table respectively Conformational model For Conformational model 3 4 kinetic term A amp kmetics in general formula is defined by the following equation 0C A kinetics 7 1 Ae where A is the pre exponential factor Pis the stretch parameter Tconf is the characteristic time of conformational relaxation Conformational model properties 1 IG type determines how the initial guesses of all model parameters are generated See list of IG type values for details 2 ComponentsCount defines number of fluorescent components 3 Background correction determines if background correction is taken into account If this property is true the Data Set associated with the model should contain the external parameter BG ratio 4 Brightness correction determines if different quantum efficiency of each component is taken into account If this property is true the Data Set associated with the model should contain the external parameters Component i brightness where i 1 2 n n number of fluorescent components See brightness correction for more details 5 Motion type specifies the motion
83. ror will occur Return Value On success fact returns the factorial of x erf Syntax erf x Description Calculates the error function of x e 1 30 erf x feat l Return Value erf returns the error function of x erfe Syntax erfc x Description Calculates the complementary error function of x e 1 ertolx me at Return Value erfc returns the complementary error function of x Gamma Syntax Gamma x Description Calculates the gamma function of x e 1 Gamma x teat i Return Value Gamma returns the gamma function of x logGamma Syntax logGamma x Description Calculates the natural logarithm of the gamma function of x Return Value logGamma returns the natural logarithm of the gamma function of x Punctuators The Script programming language punctuators also known as separators are Parentheses There are two cases when open and close parentheses are used to indicate function calls and function parameters Example func function call no arguments to group expressions and change operator precedence 31 FFSDP User Manual FFSDP User Manual Example d 2c a b override normal precedence Comma The comma punctuator is used for e separation of the elements of a function argument list Example func 1 call function with two arguments separation of the different variables while making variable decla
84. s 80 Structural paramet ERE DL IRR AR 80 Parameter SEOU D 2 ied E eiecit d eU cH 1 OEDIPUS T T T RT 1 Local MENU ec 1 ADE NM MG ees M em 1 PX PE MINIC MET OPCE CS uste init ta danita 81 Data 81 Data o ec sc O T est MEI i a O LO DD E 82 Data UC onus Kali uie uel I udi EA D ud M Ai I udin 82 Data Source propete usted lil Saco e O00 Ue elo oo dae SD a S eS Lon Ese UE O as ade oc d008e 82 A O 82 aO FS E rr E O A EAE PB A EE A A PE A O PTE 83 REFERENCES TO LITERATURE ci 83 FFSDP User Manual Theoretical background Characteristics Following characteristis can be analysed in FFSDP 1 Autocorrelation function 2 Crosscorrelation function 3 Fluorescence factorial comulants 4 Coincidence values histogram 5 Photon Counting Distribution Models FFS technique is aimed to the investigations of the intensity fluctuations of fluorescent molecules excited by a tightly focused laser beam These fluctuations may arise from translational and rotational diffusion chemical reactions deexcitation of the triplet state conformational and structural changes etc 1 2 3 4 5 15 16 26 To take into account any phenomenon mentioned above while analyzing the FFS data the appropriate mathematical model should be chosen In the FFS Data Processor the mathemat
85. se fluctuations may arise from translational and rotational diffusion chemical reactions deexcitation of the triplet state conformational and structural changes etc 1 2 3 4 2 15 16 To take into account any phenomenon mentioned above while analyzing the FFS data the appropriate mathematical model should be chosen In the FFS Data Processor the mathematical models are represented by the corresponding Model Objects Model Object can belong either to the Data Set or to the Simulation Data Source In the first case Model is used to generate the theoretical characteristic In the second case it 1s used to generate noise free source characteristic Model icon is following M Now the following models are available to be used with FFS Data Processor Characteristics Models Correlation function Pure Diffusion Triplet State Conformational 82 FFSDP User Manual Protonation FCS flow Custom Coincidence values histogram Gaussian Custom Cumulants FFC Custom Photon counting distribution PCH Gaussian Custom Identifiability All models mentioned above except Custom model are totally identifiable 1 e all parameters of the models can be uniquely determined on the basis of only one measured characteristic Noise Noise Object adds noise to the correlation function curve generated by Model for Simulation Data Set To find out more about noise generation see the description of the Simulator Noise icon is following H
86. sion ratio In the most cases only the first order correction all Fx equal zero except F1 1s sufficient to get the best fit to the experimental data The second order correction F 1 and F2 are different from zero is rarely needed We use normalization to the effective volume Fa Ki i3 in order to relate N obtained by both PCH and FCS Single molecule PCD with out of focus correction takes the form 24 1 4 qm Nee ae poQuQ g E Ry moor LEA 4 Specific PCH model parameters 1 Fc1 is first order correction parameter 2 Fc2 is second order correction parameter for 3D Gaussian 2 nd corr profile only Polynomial profile PSF r is approximated by an exponential function of one argument with polynomial transformation of volume unit further simply FIDA like polynomial approximation 21 22 dy llo l ES x i in g 3s Ay Gba tds j PSP r 5 where ai a2 are instrumental parameters and PSFo is the value of PSF r at r equal to 0 PSFo and Ao are calculated from a system of normalization equations PSF r dV 1 PSF r dV 1 6 FFSDP User Manual The total PCD is calculated by Eq 1 and 2 taking into account Eq 5 6 Specific PCH model parameters 1 a1 is the brightness profile parameter 2 a2 is the brightness profile parameter FFC model The FFC model is used to analyse a set of Fluorescence Factorial Cumulants 15 20 It is defined by the following formula
87. ss correction is false and Ni qi in the case if brightness correction is true are fit parameters where i 2 n n number of fluorescent components Pure Diffusion model For Pure Diffusion model 1 2 6 7 kinetic term amp xinetics t in general formula is defined by the following equation A netos T 1 Pure Diffusion model properties 1 IG type determines how the initial guesses of all model parameters are generated See list of IG type values for details 2 ComponentsCount defines number of fluorescent components 3 Background correction determines if background correction is taken into account If this property is true the Data Set associated with the model should contain the external parameter BG ratio 18 FFSDP User Manual 4 Brightness correction determines if different quantum efficiency of each component is taken into account See brightness correction for more details 5 Motion type specifies the motion term motion t in the model equation see general formula Pure Diffusion model parameters 1 Ginf is the level of autocorrelation function when T ta 2 N is the average number of fluorescent molecules in the detection volume exists only if brightness correction is not performed 3 Fit parameters of selected motion term Emotion iT To find out more information about selecting the model and specifying related properties and parameters see Experiment configuration dialog box Propert
88. ssor An example of the Separation of coincidence values histograms window is given on the following figure Fa Separation of coincidence values histograms A Option Experiment 2 Data set 1 Data set 2 Experiment 2 Coincid_002 Experiment 1 Commend O01 Separation quality 2 56 1 O Data set 1 source B Data set 2 source Data set 1 theory Data set 2 theory Bellow the brief description of main components Separation of coincidence values histograms window is given 78 FFSDP User Manual Experiments list Contains all expeiments currently opened in FFS Data Processor Options group box Allows specify data sets containing coincidence values histograms that will be used for calculating the separation quality 2D Chart Displays coincidence values histograms from selected data sets and allows visually estimate the separation quality of them Separation quality window Displays separation quality value This value is calculated according to the following equation o 2KK0 K T try Fu rm sep where o xi and o x are the standard deviations Ki and Ko are mean values of the coincidence values Ki n and K2 n for two samples External parameters dialog box This dialog box is used to view and manage the extenal parameters of the current Data Set It provides the ability to add remove and edit the external parameters within the Data Set and create new external parameters A
89. ssor you should click on the corresponding Tab Configuration part of the Experiment window This part of the Experiment window provides the quick access to the properties and parameters of the objects included in the current experiment and is responsible for displaying the information about the analysis progress An example view of the Experiment window Configuration part is given below 43 FFSDP User Manual gt eo amp D oc x CH Ew nM RhaodBc 2 10 Properties Parameters El Y Bg RH amp G10 001 EE tal Source measure Po ew M Model TEMoader Pcs Configuration Ei y Da RHEG10 002 CI Probability Pew tl SOURCE measure wf MI Model TSModel2 Analysis information Done Final Chi 2 criterion value Configuration toolbar Configuration toolbar contains buttons for experiment configuration analysis execution and exporting the data bizmoperm os Button Export data This button opens Export data dialog box that allows exporting the data from any Data Set within the current experiment to the text file oT Button Apply Template e This button opens the Load and apply an existing template dialog box for applying the previously saved template to the current experiment Button Save Template E This button opens the Create and save new template dialog box for creating a new template and saving it to the Analysis Database Button Simulate a This button executes the simulation procedures
90. te value of a number Return Value abs returns the absolute value of x acos Syntax acos x Description Calculates the arc cosine Arguments to acos must be in the range 1 to 1 Otherwise a runtime error will occur Return Value acos of an argument between 1 and 1 returns a value in the range 0 to pi asin Syntax asin x Description Calculates the arc sine Arguments to asin must be in the range 1 to 1 Otherwise a runtime error will occur Return Value asin of an argument between 1 and 1 returns a value in the range pi 2 to pi 2 atan Syntax atan x Description Calculates the arc tangent Return Value atan of x returns a value in the range pi 2 to pi 2 28 FFSDP User Manual COS Syntax COS X Description Calculates the cosine of a number The angle is specified in radians Return Value cos returns a value in the range 1 to 1 exp Syntax exp x Description Calculates the exponential e to the x Return Value exp returns e to the x The constant e is approximately 2 718282 If the value of x exceeds 709 782712893 a runtime error occurs Note The exp function complements the action of the log function and is sometimes referred to as the antilogarithm log Syntax log x Description Calculates the natural logarithm of x If the argument x passed to this function is 0 or less than O runtime error occurs Return Value On success log returns the value calc
91. tial guesses of all model parameters are generated See list of IG type values for details 7 FFSDP User Manual IG type Description None Generation of the initial guesses 1s not performed in any cases Method of moments Generation of the initial guesses is performed by method of moments Phase plane method Generation of the initial guesses is performed by phase plane method User defined The values defined by user will be used as initial guesses In the case of Custom model these values can be set via Custom model configuration dialog box Predefined values The predefined values will be used as initial guesses These values are specific for each type of the model and do not depend on the data of characteristic associated with the model 2 ComponentsCount defines a number of molecular components with different brightness 3 Dead time correction determines if dead time correction accordingly to Hillesheim at al 2003 is taken into account 4 Process correction determines if process correction due to triplet state intercrossing is taken into account 5 Motion correction determines if motion correction due to diffusion is taken into account 6 Profile type specifies the type of PSF x y z The following profile types are available 3D Gaussian 3D Gaussian 1 st corr 3D Gaussian 2 nd corr Polynomial Common PCH model parameters 1 Bg is the mean background count rate of detector 2 Ni is the mean number of
92. ts model in the Configuration treeview which contains the parameter currently selected in the Parameters settings table Local menu command View on graph This menu command opens Fit parameter vs data set number view window Local menu command Initial guesses This menu command sets initial guesses to all parameters and groups within the current experiment Initial guess for every parameter depends on the value of the property Auto Init of model that contains this parameter Initial guess for every parameter depends on the value of the IG type property of model that contains this parameter Local menu command Export parameters This menu command executes the export procedure that exports all values from Parameters settings table to the standard MS Excel file After pressing this button Save dialog box will be opened in which the path and the name of the target MS Excel file can be specified Local menu command Optimize View This menu command automatically sets most suitable width of Parameters setting table columns Unlinking parameters To unlink any parameter and make it free you can follow one of the ways listed bellow 1 Select the parameter you want to unlink in Linked parameters treeview drag it and drop on the Free parameters list 2 Select the parameter you want to unlink in Linked parameters treeview press right mouse button to display the Linked parameters treeview local menu and select the Free local menu command
93. u commands Link To Link all parameter name s of Free parameters list or Parameters settings table as an alternative way for linking parameters 2D Graphics page This page is used to display characteristics and weighted residuals in two dimensional space The following components are related to the 2D Graphics pages Toolbar 2D Characteristics and Residuals boxes An example view of the 2D Graphics page is given below 56 FFSDP User Manual Auto CR Parameters 3D Graphics D Graphics Characteristics Toolbar of the 2D Graphics page This toolbar contains buttons for changing view of 2D Graphics page sol R This toolbar has short Help Hints Help Hint is the pop up text that appears when the mouse pointer passes over a toolbar button Button AutoSize Auto If this button is down the size of the graphical boxes that belong to the 2D Graphics page is automatically changed while the Experiment window resizes If this button is up the vertical size of the Characteristics box and Residuals box can be changed manually using the splitting line that lies between them Button Show Characteristics c If this button is down Characteristics box is visible 57 FFSDP User Manual Button Show Residuals R If this button is down Residuals box is visible 2D Characteristics and Residuals charts 2D Characteristics chart displays measured green and theoretical red characteristics in two dimensional spa
94. ulated In x log10 Syntax log10 x Description Calculates the base ten logarithm of x If the argument x passed to this function is O or less than 0 runtime error occurs Return Value On success log 10 returns the calculated value log base ten of x pow Syntax pow x y Description Calculates x to the power of y If the result of this function is more than 1 75 102 the overflow runtime error will occur If the argument x passed to pow is real and less than 0 and y is not a whole number or you call pow 0 0 29 FFSDP User Manual runtime error will occur Return Value On success pow returns the value calculated of x to the power of y sin Syntax sin x Description Calculates the sine of a value The angle is specified in radians Return Value sin returns a value in the range 1 to 1 sqrt Syntax sqrt x Description Calculates the positive square root If x 1s positive the result is positive If x 1s negative runtime error will occur Return Value On success sqrt returns the square root of x tan Syntax tan x Description Calculates the tangent Angles are specified in radians Return Value tan returns the tangent of x sin x cos x fact Syntax fact x Description Calculates the factorial of x rounded to the nearest lesser integer If x 1s negative runtime error will occur If the result of this function is more than 1 75 io the overflow runtime er
95. ution function Sou Ld the value of 2 obtained after the analysis To set confidential probability use property CI Probability of the Experiment Object 23 FFSDP User Manual Simulator Simulation tools are aimed to investigate the performance of the fitting procedures with respect to the particular type of characteristic Simulation consists of the numeric generation of the corresponding characteristic distorted by statistical noise see Simulator box of the Experiment configuration dialog box The characteristics of statistical noise are strongly dependent on the experimental methods and apparatus tools In the case of FCS measurements for example expressions describing statistics of the obtained correlation function are known only for a limited number of particular cases That is why we assume that value in each sampling interval or channel is the random value with Gaussian probability function with mean value equal to the true value of correlation function and empirically derived standard deviation o f at 04 7 where a y and f are the adjusted parameters parameters of simulator To find out more about how to chose the noise and how to input corresponding parameters in the interface see Experiment configuration box and Parameters table respectively Using custom model Script programming language The following topics provide a formal definition of Script programming language which is used for creating custom model
96. v Scientific Software Technologies Center FFS Data Processor Global analysis software in fluorescence fluctuation spectroscopy User Manual Main Application SSTC contacts www SSTCenter com info O sstcenter com FFSDP User Manual THEORETICAL BACKGROUND ju cece cece eeeecece cece eee ececececeeeeeeeeeesesesaeaeaeeeeeeesesaseseueeeeeeeusaeeeeees 6 Ati AAA II o 5 AN VENEA AEA T 6 A o E A A A 6 C a E EAEE EAA AAEN EAEE AAE E AE EANES 7 Ss APS A 7 PECO E EEEE AAEE AEAEE EAE AAAA AAEE A AET 10 ENS e SA A 13 CotrelatonTincton models ardor cid dde 14 A II n 22 iren iR ET 22 vive SINE CE oma 22 Enid Os SG EE dete o d OO PO ME iei mM i E M mU nM A M NIU 23 Burner M 23 Je E CREBRA Sc 6 a NANO To 23 ZUG FETA 6 EPEOES octo decet Rese Ge LM E IC MM LM LM UI ED ene ee 23 IIS A III SC Pe ee eT A 24 USING CUSTOM MODEL ua 24 Script programimin AMA sssaaa aE aeaaeae aaa EEE enee NEn ae 24 DA O e o o oo o E E eE PEE UE E E E OC ER 24 Ee 4er RR 24 KE Wy A A e e o UE O ei EE o EE o A 25 Varable dente tail ote bits 25 o RETO e E 25
97. w 2 Save graph 3 Copy graph to Clipboard 4 Graph configuration 5 Save settings as default 6 Load default settings 7 Reset default settings Local menu command Default view Resets a view of the current 3D Chart to the initial state Local menu command Save graph Saves the current 3D Chart image to BMP file Local menu command Copy graph to Clipboard Stores the current 3D Chart image to Clipboard 62 FFSDP User Manual Local menu command Graph configuration Opens the 3D chart configuration dialog box for configuring the current 3D Chart Local menu command Save settings as default Saves current settings of the current 3D Chart as the default settings Local menu command Load default settings Loads and Applies the default settings to the current 3D Chart Local menu command Reset default settings Resets and Applies the default settings to the current 3D Chart Experiment configuration dialog box This dialog box is used to change the configuration of the current experiment It provides the ability to add and remove the Data Sets within the experiment and select the model that will be employed to analyze the data from the given Data Set The Experiment configuration dialog box consists of three pages e Mesurements database page is used to add data sets from database e Simulator page to add simulation data sets e Fit parameter dependence IS used to create data sets that contain dependence of some fit param

- Scientific Software Technologies Center

Contents

Download Pdf Manuals

Related Search

Related Contents