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# Wiki FCSAnalyze tools
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# Wiki FCSAnalyze tools
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## Fluctuation Analyzer
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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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## Generate bleach corrected correlation functions using FluctuationAnalyzer 4G (FA)
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The [Fluctuation Analyzer 4G]( https://www.embl.de/~wachsmut/downloads.html) 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. Below we provide a detailed explanation of on which settings and parameters to use for the FCS calibration protocol.
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Loading of data |
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:---: | ----
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<img src = './images/FA/import1_idx.png' width = "400px" > <br/> Import settings APD/Confocor <br/> <img src = './images/FA/load_APD.png' width = "400px" ><br/> Import settings GaAsP <br/><img src = './images/FA/load_GaAsP.png' width = "400px" > | 1. Choose appropriate file format <br/> 2. Click on import settings. The channel with the lowest wavelength should be Ch1. <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.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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> <a name = 'sessionname'>**Session name** </a> This name is used to reload the data. Typically use **1c** when fitting one component model (e.g. fluorescent dye) or **2c** when fitting a 2 component model (e.g. fluorescent protein).
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## Fitting of data using FA
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## Fitting of data using Matlab
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The Fluctuation Analyzer 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. Please download Fluctuation Analyzer 4G and the user guide from https://www.embl.de/~wachsmut/downloads.html. A datailed explanation on which settings and parameters to use for the FCS calibration protocol is given [here](./fluctuationanalyzer.md). |
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