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# Generate bleach corrected correlation functions using FluctuationAnalyzer 4G
# I. Generate bleach corrected correlation functions using Fluctuation Analyzer 4G (FA)
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.
The [FA]( https://www.embl.de/~wachsmut/downloads.html) is a software for the interactive as well as automated processing of fluorescence auto- and cross-correlation spectroscopy (FCS/FCCS) data. Refer to the manual of FA and the original article [Wachsmuth et al. (2015)]( http://europepmc.org/abstract/MED/25774713) for details.
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
In this WiKi we provide a detailed explanation on which settings and parameters to use for the FCS-Calibration protocol. At least the following 4 steps must be executed
<a name=back></a>
1. [Loading data into FA](#faload)
[**1. Loading data into FA**](#faload)
2. [Compute correlation curves with FA](#facorr)
[**2. Compute correlation curves with FA**](#facorr)
3. [Compute offset and crosstalk parameters](#facorrpar)
[**3. Compute offset and crosstalk parameters**](#facorrpar)
4. [Compute correction factors and save table](#facorrfa)
[**4. Compute correction factors and save table**](#facorrfa)
See also [Typical values used for computing correlation functions and correction factors](#faval)
> **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.
> **Apply to**: Throughout the different steps using FA it is always required to click on `Apply to` for changes in settings to be active.
......@@ -23,26 +24,26 @@ See also [Typical values used for computing correlation functions and correction
## [<img src='./images/up.png'>](#back) <a name=faload></a>1. Loading data into FA
**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)`
**FCS raw data**: FA requires raw photon-counting data. For Zeiss LSM with ZEN (black edition) set the `Confocor Options` in the `Maintain` tab. When saving the FCS recording manually be sure to save as `Fcs files with raw data (*.fcs)`
<div align = "center" > <img src = './images/FA/save_raw_data.PNG' ><img src = './images/FA/save_as_type.PNG' > </div>
<a name='sessionname'></a>**Session name** : Alphanumeric identifier used for re-loading processed data. Typically use **1c** when fitting one component model (e.g. fluorescent dye) or **2c** when fitting a two component model (e.g. fluorescent protein)
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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
<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
**Import raw data** | **Usage**
:--- | :---
<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 <br/> should be Ch1 <br/> FA\_Ch1/2 = None if only one channel has been acquired <br/> 2a. With APD FA\_Ch1 = Zeiss\_Ch2 and FA\_Ch2 = Zeiss\_Ch1.<br/> 2b. With GaAsP FA\_Ch1 = Zeiss\_ChS1 and FA\_Ch2 = Zeiss\_ChS2 <br/> 3. Select file path <br/> 4. To process subdirectories click on **Include subdirectories** <br/> 5. Add data to FA
<img src = './images/FA/import2_idx.png' width = "400px" > | 6. Specify a session name. <a name='sessionname'></a> This is a alphanumeric <br/> identifier used for re-loading processed data.<br/> Use **1c** or **2c** when fitting one or two component model <br/> 7. Click on `Check files` <br/> 8. Click on `Quick check` if data has already been loaded once
## [<img src='./images/up.png'>](#back) 2. <a name=facorr></a>Compute correlation curves with FA
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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:--: | ---
**Modify and correlate**| **Usage**
:--- | :---
<img src = './images/FA/tab_correlate.png' width = "400px" > | 1. Switch to the page `Modify and correlate`
<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` all <br/> 7. Click on `Calculate all`
<img src = './images/FA/correlate_idx.png' width = "400px" > | 2. Choose the correlation curves to compute <br/> 3. Set **Base freq. [Hz]** for the fluorescent dye 1.000.000 Hz <br/> For a fluorescent protein 100.000 Hz <br/> 4. Set `Ch1 <> Ch2` for indipendently correct the channels <br/> 5. Set `Autosave` to on <br/> 6. Click `Apply to` all <br/> 7. Click on `Calculate all`
......@@ -54,16 +55,16 @@ The [**Offset**](#offset) and [**Crosstalk**](#xtalk) parameters are used for co
* <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.
* Acquire data from WT cells (5-10 cells)
* Load data into FA and compute intensity corrections
* Save data into a tab delimited result table (**.res* file)
* Load *res* file into a spreadseet and compute average of the column named *Interval Ch1* (for `Offset [Khz] Ch1`) and/or *Interval Ch2* (for `Offset [Khz] Ch2` )
* Compute background photon counts using [FCSFitM](./Fcsfitm)
Alternatively you can use FA
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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` all <br/> 4. Click on `Calculate all`
<img src = './images/FA/save_res.png' width = "400px" > <br/> <img src = './images/FA/res_file_interval.png'> | 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)<br/> 8. Compute offset values from averages of columns named *Interval Ch1/2* in the *res* file
**Intensity corrections Offset**| **Usage**
:--- | :---
<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. Set `Autosave` to on <br/> 3. Click `Apply to` all <br/> 4. Click on `Calculate all`
<img src = './images/FA/save_res.png' width = "400px" > <br/> <img src = './images/FA/res_file_interval.png'> | 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**)<br/> 8. Compute `Offset [Khz] Ch1,2` from averages of columns named `Interval Ch1,2` in the *res* file
......@@ -73,15 +74,19 @@ The [**Offset**](#offset) and [**Crosstalk**](#xtalk) parameters are used for co
* <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.
* Acquire data from cells expressing only one fluorophore (5-10 cells)
* Load data into FA
* Change to `Intensity corrections` and enter the offset values for Ch1 and Ch2
* Calculate all corrections and save *res* table
* Compute crosstalk from Ch1 (to Ch2) from average of columns in *res* file $`\frac{\text{IntervalCh2} - \text{OffsetCh2}}{\text{IntervalCh1} - \text{OffsetCh1}}`$
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:---: | ----
<img src = './images/FA/xtalk_correction_idx.png' width = "400px" > | [Import](#faload) FCS measurements from cells expressing a single fluorophore. Proceed [Modify and correlate](#facorr) <br/> 1. Change tab to `Intensity corrections` <br/> 2. Enter previously estimated Offset value for Ch1 <br/> 3. For two color FCS enter Offset value for Ch2 <br/> 4. Click on `Apply to` all <br/> 5. Click on `Calculate all`
**Intensity corrections crosstalk** | **Usage**
:--- | :---
<img src = './images/FA/xtalk_correction_idx.png' width = "400px" > | [Import](#faload) FCS measurements from cells expressing a single fluorophore. Proceed [Modify and correlate](#facorr) <br/> 1. Change tab to `Intensity corrections` <br/> 2. Enter previously estimated Offset values for Ch1 <br/> 3. For two color FCS enter Offset value for Ch2 <br/> 4. Click on `Apply to` all <br/> 5. Click on `Calculate all`
Compute crosstalk from Ch1 (to Ch2) from average of columns in *res* file
$`\frac{\text{IntervalCh2} - \text{OffsetCh2}}{\text{IntervalCh1} - \text{OffsetCh1}}`$
## [<img src='./images/up.png'>](#back) 4. <a name=facorrfa> </a> Compute correction factors and save table
Correction factors are used to correct the protein numbers for bias induced by background, cross-talk (for 2 color FCS), and photobleaching. The corrected values can be calculated from the fitted values and correction factors stored in the result table.
......@@ -92,7 +97,7 @@ The procedure is similar as for the Offset and Crosstalk value computation:
* Switch to the `Intensity corrections` tab
* Enter the `Offset [kHz]` for Ch1 (and Ch2 for two color FCS)
* Enter the `Crosstalk` value in case of two color FCS
* Click `Appky to`
* Click `Apply to`
* Click on `Calculate all`
* Save results to *res* file
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