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In this WiKi we provide examples on how to analyze and process FCS and imaging data generated on Zeiss LSM microscopes using [microscopy pipeline constructor](#https://git.embl.de/politi/mypic) or
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In this WiKi we provide examples on how to analyze and process FCS and imaging data generated on Zeiss LSM microscopes using [microscopy pipeline constructor](#https://git.embl.de/politi/mypic) or
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[FCSRunner](#https://git.embl.de/politi/fcsrunner). The processed data can be used to generate a calibration curve for converting pixel fluorescence intensities to concentrations.
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[FCSRunner](#https://git.embl.de/politi/fcsrunner). The processed data can be used to generate a calibration curve for converting pixel fluorescence intensities to concentrations.
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[**Generate bleach corrected correlation functions using Fluctuation Analyzer 4G**](#gencorr)
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[**Fit correlation data with Fluctuation Analyzer**](#fitfa)
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[**Fit correlation data with Matlab**](#fitmatlab)
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## Generate bleach corrected correlation functions using FluctuationAnalyzer 4G
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## <a name = 'gencorr'> </a> Generate bleach corrected correlation functions using FluctuationAnalyzer 4G
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The [Fluctuation Analyzer 4G]( https://www.embl.de/~wachsmut/downloads.html) (FA) is a software tool for the interactive as well as automated processing of fluorescence auto- and cross-correlation spectroscopy (FCS/FCCS) data. It can read raw data, i.e., one-or two-channel photon streams, from various commercial suppliers of FCS/FCCS data acquisition equipment, organize such data in processing sessions by file-based management, calculate temporal auto- and cross-correlation functions, correct for photobleaching, cross-talk, and background signal and fit the data with appropriate model functions before saving the results. Refer to the manual of FA and the original article [Wachsmuth et al. (2015)]( http://europepmc.org/abstract/MED/25774713) for details.
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The [Fluctuation Analyzer 4G]( https://www.embl.de/~wachsmut/downloads.html) (FA) is a software tool for the interactive as well as automated processing of fluorescence auto- and cross-correlation spectroscopy (FCS/FCCS) data. It can read raw data, i.e., one-or two-channel photon streams, from various commercial suppliers of FCS/FCCS data acquisition equipment, organize such data in processing sessions by file-based management, calculate temporal auto- and cross-correlation functions, correct for photobleaching, cross-talk, and background signal and fit the data with appropriate model functions before saving the results. Refer to the manual of FA and the original article [Wachsmuth et al. (2015)]( http://europepmc.org/abstract/MED/25774713) for details.
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... | @@ -56,20 +59,14 @@ In this WiKi we provide a detailed explanation on which settings and parameters |
... | @@ -56,20 +59,14 @@ In this WiKi we provide a detailed explanation on which settings and parameters |
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4. [Compute correction factors and save table](#facorrfa)
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4. [Compute correction factors and save table](#facorrfa)
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See also [Typical values used for computing correlation functions and correction factors](#faval)
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> <p style='color:green'> **Apply to**: Throughout the different steps using fluctuation analyzer it is always required to click on `Apply to` for changes in settings to be active. </p>
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> **Apply to**: Throughout the different steps using fluctuation analyzer it is always required to click on `Apply to` for changes in settings to be active.
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Typical values used for the computation of the correlation curves and correction factors are:
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Parameters | Values |
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:--- | :---
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Base freq. (Hz) | 1.000.000 (dye) or 100.000 (protein)
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Composition | Ch1 <> Ch2
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Offset (kHz) | 1-5 kHz <br/> Must be measured
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Cross-talk (Ch1-Ch2), when using two color FCS | 0.04-0.05 (Ch1: mEGFP, Ch2: mCherry). <br/> Value depends on protein and optical settings. Must be measured.
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### [<img src='./images/up.png'>](#back) <a name=faload></a>Loading data into FA
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### [<img src='./images/up.png'>](#back) <a name=faload></a>1. Loading data into FA
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**FCS raw data**: FA requires the raw photon-counting data. For Zeiss LSM with ZEN (black edition) to save raw data use the `Confocor Options` menu in the `Maintain` tab. When saving the fcs recording be sure to save as type `Fcs files with raw data (*.fcs)`
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**FCS raw data**: FA requires the raw photon-counting data. For Zeiss LSM with ZEN (black edition) to save raw data use the `Confocor Options` menu in the `Maintain` tab. When saving the fcs recording be sure to save as type `Fcs files with raw data (*.fcs)`
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... | @@ -81,7 +78,7 @@ Cross-talk (Ch1-Ch2), when using two color FCS | 0.04-0.05 (Ch1: mEGFP, Ch2: mCh |
... | @@ -81,7 +78,7 @@ Cross-talk (Ch1-Ch2), when using two color FCS | 0.04-0.05 (Ch1: mEGFP, Ch2: mCh |
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:---: | ----
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:---: | ----
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<img src = './images/FA/import1_idx.png' width = "400px" > <br/> Import settings APD/Confocor and GaAsP <br/> <img src = './images/FA/load_APD.png' width = "200px" > <img src = './images/FA/load_GaAsP.png' width = "200px" > | 1. Choose appropriate file format <br/> 2. Click on import settings. The channel with the lowest wavelength should be Ch1. <br/> FA_Ch1/2 = None if only one channel has been acquired <br/> 2a. With APD FA_Ch1 = Ch2 and FA_Ch2 = Ch1.<br/> 2b. With GaAsP FA_Ch1 = ChS1 and FA_Ch2 = ChS2 <br/> 3. Select file path. <br/> 4. Click if data in all subdirectories need to be processed. <br/> 5. Add data to FA
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<img src = './images/FA/import1_idx.png' width = "400px" > <br/> Import settings APD/Confocor and GaAsP <br/> <img src = './images/FA/load_APD.png' width = "200px" > <img src = './images/FA/load_GaAsP.png' width = "200px" > | 1. Choose appropriate file format <br/> 2. Click on import settings. The channel with the lowest wavelength should be Ch1. <br/> FA_Ch1/2 = None if only one channel has been acquired <br/> 2a. With APD FA_Ch1 = Ch2 and FA_Ch2 = Ch1.<br/> 2b. With GaAsP FA_Ch1 = ChS1 and FA_Ch2 = ChS2 <br/> 3. Select file path. <br/> 4. Click if data in all subdirectories need to be processed. <br/> 5. Add data to FA
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<img src = './images/FA/import2_idx.png' width = "400px" > | 6. Specify a [session name](#sessionname) <br/> 7. Click on `Check files` <br/> 8. Use `Quick check` if data has already been loaded once
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<img src = './images/FA/import2_idx.png' width = "400px" > | 6. Specify a [session name](#sessionname) <br/> 7. Click on `Check files` <br/> 8. If data `Quick check` if data has already been loaded once
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... | @@ -118,7 +115,7 @@ The [**Offset**](#offset) and [**Crosstalk**](#xtalk) parameters are used for co |
... | @@ -118,7 +115,7 @@ The [**Offset**](#offset) and [**Crosstalk**](#xtalk) parameters are used for co |
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* <a name='xtalk'>**Crosstalk (Ch1 to Ch2)**</a>: In case that two fluorescent proteins are measured the value of the crosstalk is measured from cells expressing just one of the two fluorophores.
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* <a name='xtalk'>**Crosstalk (Ch1 to Ch2)**</a>: This parameter is required when two fluorophores are measured, e.g. to quantify the cross-correlation. To quantify the crosstalk cells expressing each one of the two fluorophores are measured.
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* Acquire data from cells expressing only one fluorophore (5-10 cells)
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* Acquire data from cells expressing only one fluorophore (5-10 cells)
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* Load data into FA
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* Load data into FA
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* Change to `Intensity corrections` and enter the offset values for Ch1 and Ch2
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* Change to `Intensity corrections` and enter the offset values for Ch1 and Ch2
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... | @@ -144,12 +141,19 @@ The procedure is similar as for the Offset and Crosstalk value computation: |
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* Click on `Calculate all`
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* Click on `Calculate all`
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* Save results to *res* file
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* Save results to *res* file
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### [<img src='./images/up.png'>](#back) 5. <a name=faval> </a> Typical values used for computing correlation functions and correction factors
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Typical values used for the computation of the correlation curves and correction factors are:
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Parameters | Values |
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:--- | :---
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Base freq. (Hz) | 1.000.000 (dye) or 100.000 (protein)
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Composition | Ch1 <> Ch2
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Offset (kHz) | 1-5 kHz <br/> Must be measured
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Cross-talk (Ch1-Ch2), when using two color FCS | 0.04-0.05 (Ch1: mEGFP, Ch2: mCherry). <br/> Value depends on protein and optical settings. Must be measured.
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## Fitting data using FA
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## Fitting data using FA
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Using FA the user can freely decide which model to use and which parameters are fitted or not for up to two consecutive fitting settings (Run A and Run B). For the fitting of the fluorescent dye and fluorescent protein we use Run A an Run
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Using FA the user can freely decide which model to use and which parameters are fitted or not for up to two consecutive fitting settings (**RunA** and **RunB**). Typically one uses different models and initial values when fitting FCS traces from a [fluorescent protein](#fitprot) or a [fluorescent dye](#fitdye).
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In the parameter choice section the fate of a parameter can be changed by clicking on the boxes below a parameter.
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In the parameter choice section the fate of a parameter can be changed by clicking on the boxes below a parameter.
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... | @@ -158,15 +162,45 @@ In the parameter choice section the fate of a parameter can be changed by clicki |
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* **Link/Change**: This is used for specifying changes in the fitting state of a parameter between RunA and RunB.
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* **Link/Change**: This is used for specifying changes in the fitting state of a parameter between RunA and RunB.
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Alternatively the user can use the matlab workflow that combines several steps of the fitting. A summary of initial parameter values is given below. Note that the parameters for the fluorescent dye and protein are not the same.
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Alternatively the user can use the matlab workflow that combines several steps of the fitting. A summary of initial parameter values is given below. Note that the parameters for the fluorescent dye and protein are not the same.
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### Fluorescent protein
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**RUN A** <br/><img src = './images/FA/protein_runA.png' width = "400px" >**RUN B** <br/><img src = './images/FA/protein_runB.png' width = "400px" > | 1. Set `Weighted fit`, `Initial guess` on. To perform RUNB the `2nd run` needs to be on. This may need to be reset after a run <br/> 2. Verify that model is *two-component anomalous diffusion with fluorescent protein-like blinking* for all measurements <br/> 3. Set the parameter values and fitting options as shown in the table <br/> 4. Verify that `Autosave` is on <br/> 5. Click `Apply to` all <br/> 6. Switch to settings for RUNB <br/> Change parameter table and values Click on `Fit all`
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### <a name=fitprot></a> Fluorescent protein
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1. Start fluctuation analyzer, [load data](#faload), and [compute correlations] (#facorr). Set `Weighted fit`, `Initial guess` on. To perform RUNB the `2nd run` needs to be on.This may need to be reset after a fit has been peformed
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**RUN A** <br/><img src = './images/FA/protein_runA_idx.png' >
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2. Verify that model is *two-component anomalous diffusion with fluorescent protein-like blinking* for all measurements
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3. Set the parameter values and fitting options as shown in the table
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4. Change kappa according to value appropriate for objective and microscope settings as determined using the fluorescent dye
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5. Verify that `Autosave` is on
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6. Click `Apply to` all
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**RUN B** <br/><img src = './images/FA/protein_runB_idx.png' >
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7. Switch to settings for RUNB
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8. Set the parameter values and fitting options as shown in the table for RUNB
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9. Click `Apply to` all
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10. Click `Fit all`
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Set the model *two-component anomalous diffusion with fluorescent protein-like blinking*
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### <a name=fitdye></a> Fluorescent dye
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1. Start fluctuation analyzer, [load data](#faload), and [compute correlations](#facorr) using the highest frequency of 1.000.0000 Hz. Set `Weighted fit`, `Initial guess` on. To perform RUNB the `2nd run` needs to be on. This may need to be reset after a fit has been peformed
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**RUN A** <br/><img src = './images/FA/dye_runA.png' >
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2. For detectors with after-pulsing (e.g. APD's) set the lower fit boundary to 2 μs
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3. Set the upper fit boundary to a value suitable for a fast-diffusing molecule (~ 10230 μs )
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4. Change the model to *two-component anomalous diffusion with triplet-like blinking*. Press `Apply to` all in the `Change model ...` menu
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5. Set the parameters for RoundA as shown
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6. Verify that `Autosave` is on
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6. Click `Apply to` all
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**RUN B** <br/><img src = './images/FA/dye_runB.png' >
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7. Switch to settings for RUNB
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8. Set the parameter values and fitting options as shown in the table for RUNB
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9. Click `Apply to` all
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10. Click `Fit all`
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Parameters | Round A | Round B
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Parameters | Round A | Round B
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:--- | :--- | :---
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:--- | :--- | :---
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Model | two-comp. anomalous diffusion with triplet-like blinking |
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Model | two-comp. anomalous diffusion with triplet-like blinking |
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... | @@ -193,15 +227,9 @@ tauD2 | NA | 5000 |
... | @@ -193,15 +227,9 @@ tauD2 | NA | 5000 |
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alpha2 | NA | 1
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alpha2 | NA | 1
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kappa | 5.5 | 5.5
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kappa | 5.5 | 5.5
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offset | **0** | **0**
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offset | **0** | **0**
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### Fitting of the fluorescent dye
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### Fit of fluorescent dye data
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### Fitting of the fluorescent dye
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For the fitting of autocorrelation curves to a physical model
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Fit dye data | |
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:---: | ---
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<img src = './images/FA/dye_fit_runA.PNG' width = "400px" > | 1. Set parameters and
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... | | ... | |