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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
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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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* 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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* Compute crosstalk from Ch1 (to Ch2) from $`\frac{\text{IntervalCh2} - \text{OffsetCh2}}{\text{IntervalCh1} - \text{OffsetCh1}}`$
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```math
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\frac{\text{IntervalCh2} - \text{OffsetCh2}}{\text{IntervalCh1} - \text{OffsetCh1}}
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