... | ... | @@ -38,18 +38,21 @@ In this WiKi we provide examples on how to analyze and process FCS and imaging d |
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## 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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In this WiKi we provide a detailed explanation on which settings and parameters to use for the FCS calibration protocol and process the data for the fluorescent dye and fluorescent protein. At least the following 3 steps must be executed
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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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In this WiKi we provide a detailed explanation on which settings and parameters to use for the FCS calibration protocol and process the data for the fluorescent dye and fluorescent protein. At least the following 4 steps must be executed
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<a name=back></a>
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1. [Loading data into FA](#faload)
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2. [Compute correlation curves with FA](#facorr)
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3. [Compute correction factors with FA](#fafact)
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3. [Compute parameters for correction](#facorrpar)
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4. [Compute correction factors](#facorrfa)
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5. [Save FA result table ](#fares)
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### <a name=faload></a>Loading data into FA
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### [<img src='./images/up.png'>](#back) <a name=faload></a>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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... | ... | @@ -61,30 +64,65 @@ In this WiKi we provide a detailed explanation on which settings and parameters |
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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/import2_idx.png' width = "400px" > | 6. Specify a [session name](#sessionname) <br/> 7. 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. Use `Quick check` if data has already been loaded once
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### <a name=facorr></a>Compute correlation curves with FA
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FA computes auto- and cross-correlation functions given the raw data. Slow time varying trends in the photon-counts, such as photobleaching, are corrected by repeatidely calculating the correlation function in small time-windows where the slow trend can be neglected.
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### [<img src='./images/up.png'>](#back) 2. <a name=facorr></a>Compute correlation curves with FA
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FA computes auto- and cross-correlation functions given the raw data. FA corrects for slow time varying trends in the photon-counts, such as photobleaching, by calculating the correlation function in small time-windows where the slow trend can be neglected. Then all correlation functions are averaged.
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:--: | ---
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<img src = './images/FA/tab_correlate.png' width = "400px" > | 1. Switch to the page `Modify and correlate`
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<img src = './images/FA/correlate_idx.png' width = "400px" > | 2. Choose the correlation curves to compute. <br/> 3. Set the base frequency. For the fluorescent dye use 1.000.000 Hz (i.e. 1 us time interval). For a fluorescent protein 100.000 Hz is enough (i.e. 10 us time interval) <br/> 4. Set `Ch1 <> Ch2` for indipendently correct the channels <br/> 5. Check if `Autosave` is on <br/> 6. Click `Apply to` <br/> 7. Click on `Calculate all`
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### <a name=fafact> </a>Compute correction factors with FA
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Correction factors allow to correct the computed protein numbers in the effective volume for variations induced by background, cross-talk and photobleaching.
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###[<img src='./images/up.png'>](#back) 3. <a name=facorrpar> </a>Compute parameters for correction
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The [**Offset**](#offset) and [**Crosstalk**](#xtalk) parameters are used for computing the [correction factors](#facorrfa) in the next step. The crosstalk value is only needed in case of two color FCS (FCCS).
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Intensity corrections (for fluorescent proteins only)| |
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:--: | ---
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* <a name='offset'>**Offset**</a>: Average photon-count rate obtained from cells that do not express the fluorescent protein (WT cells). Alternatively the medium can also be measured, however this typically underestimate the background. The laser power and light-path settings must be the same as for the measurement of the fluorescent protein.
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* Acquire data from WT cells (5-10 cells)
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* Load data into FA and compute intensity corrections
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* Save data into result table (**.res* file)
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:---: | ----
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<img src = './images/FA/WT_correction_idx.png' width = "400px" > | [Import](#faload) FCS measurements from WT cells into FA. The [Modify and correlate](#facorr) step is not required <br/> 1. Change tab to `Intensity corrections` <br/> 2. Check that `Autosave` is on <br/> 3. Click `Apply to` <br/> 4. Click on `Calculate all`
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<img src = './images/FA/save_res.png' width = "400px" > | 5. Change tab to `Save, export and report` <br/> 6. Click on `Save all` to save single taces <br/>7. Click on `FA format` to save a summary result table (here 2c.res)
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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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* Acquire data from cells expressing only one fluorophore (5-10 cells)
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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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* Calculate all corrections and save *res* table
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* Compute crosstalk from Ch1 (to Ch2) from
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$\frac{Interval Ch2 - Offset Ch2}{Interval Ch1 - OffsetCh1}$
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:---: | ----
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<img src = './images/FA/xtalk_correction_idx.png' width = "400px" > | [Import](#faload) FCS measurements from WT cells into FA. The [Modify and correlate](#facorr) step is not required <br/> 1. Change tab to `Intensity corrections` <br/> 2. Check that `Autosave` is on <br/> 3. Click `Apply to` <br/> 4. Click on `Calculate all`
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<img src = './images/FA/save_res.png' width = "400px" > | 5. Change tab to `Save, export and report` <br/> 6. Click on `Save all` to save single taces <br/>7. Click on `FA format` to save a summary result table (here 2c.res)
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### [<img src='./images/up.png'>](#back) 4. <a name=facorrfa> </a> Compute correction factors
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Correction parameters are used to then compute correction computed protein numbers in the effective volume for bias induced by background, cross-talk, and photobleaching. The corrected values can be computed from the fitted values and correction factors stored in the result table. Correction factors need to be calculated for fluorescent proteins.
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Intensity corrections (for fluorescent proteins only)| |
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## Summary of typical parameters used for computing correlation functions and correction factors
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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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<img src = './images/FA/dye_fit_runA.PNG' width = "400px" > | 1. Set parameters and
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## Summary of parameters for Fluctuation Analyzer
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These parameters are suggestions and may be needed to be adapted depending on the system used.
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Parameters | Fluorescent dye | Flurescent protein
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:--- | :--- | :---
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Base freq. (Hz) | 1.000.000 | 100.000
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Composition | Ch1 <> Ch2 | Ch1 <> Ch2
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Model | two-component anomalous diffusion with triplet-like blinking | two-component anomalous diffusion with fluorescent protein-like blinking
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N | 10 | 10
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thetaT | 0.2 | 0.2
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tauT | 10 | **100**
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f1 | **1** | 0.5
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tauD1 | 20 | 500
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alpha1 | **1** | 1
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tauD2 | NA | 5000
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alpha2 | NA | 1
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kappa | 5.5 | 5.5
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offset | **0** | **0**
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## Fitting of data using Matlab
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... | ... | |