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# I. Generate bleach corrected correlation functions using Fluctuation Analyzer 4G (FA)
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.
The [FA]( https://www.embl.de/~wachsmut/downloads.html) is a software for the interactive as well as automated processing of fluorescence correlation and cross-correlation spectroscopy (FCS and 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. 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)
......@@ -14,7 +14,7 @@ In this WiKi we provide a detailed explanation on which settings and parameters
[**4. Compute correction factors and save table**](#facorrfa)
See also [Typical values used for computing correlation functions and correction factors](#faval)
[**5. Typical values used for computing correlation functions and correction factors**](#faval)
> **Apply to**: Throughout the different steps using FA it is always required to click on `Apply to` for changes in settings to be active.
......@@ -24,7 +24,8 @@ 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 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)`
**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, make sure to save them 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>
......@@ -32,18 +33,18 @@ See also [Typical values used for computing correlation functions and correction
**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
<div align= center> <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 = "300px" > <img src = './images/FA/load_GaAsP.png' width = "300px" > </div>| 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 = 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 an alphanumeric <br/> identifier used for reloading processed data.<br/> Use **1c** or **2c** when fitting a 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.
FA computes correlation functions based on 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 in which the slow trend can be neglected. Then all correlation functions are averaged.
**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 **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`
<img src = './images/FA/correlate_idx.png' width = "400px" > | 2. Choose the correlation curves to be computed <br/> 3. Set **Base freq. [Hz]** for the fluorescent dye 1.000.000 Hz. For a fluorescent protein set 100.000 Hz <br/> 4. Set `Ch1 <> Ch2` to independently correct the channels <br/> 5. Set `Autosave` to on <br/> 6. Click `Apply to` all <br/> 7. Click on `Calculate all`
......@@ -53,43 +54,42 @@ FA computes auto- and cross-correlation functions given the raw data. FA correct
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).
* <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.
* <a name='offset'>**Offset**</a>: Average photon count rate obtained from cells that do not express the fluorescent protein (WT cells). The culture medium can also be measured. However this typically underestimates 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)
* Compute background photon counts using [FCSFitM](./Fcsfitm)
Alternatively you can use FA
Alternatively you can use FA according to:
**Intensity corrections Offset**| **Usage**
**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
<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 traces <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
* <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.
* <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 each expressing one of the two fluorophores are measured.
* Acquire data from cells expressing only one fluorophore (5-10 cells)
* Load data into FA
**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`
<img src = './images/FA/xtalk_correction_idx.png' width = "400px" > | [Import](#faload) FCS measurements from cells expressing a single fluorophore. Proceed with [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
Compute crosstalk from Ch1 (to Ch2) from the 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.
Correction factors are used to correct the protein numbers for bias induced by background, crosstalk (for 2 color FCS), and photobleaching. The corrected values can be calculated from the fitted values and correction factors stored in the result table.
The procedure is similar as for the Offset and Crosstalk value computation:
......@@ -109,7 +109,7 @@ Parameters | Values |
Base freq. (Hz) | 1.000.000 (dye) or 100.000 (protein)
Composition | Ch1 <> Ch2
Offset (kHz) | 1-5 kHz <br/> Must be measured
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.
Crosstalk (Ch1-Ch2) if 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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