Abstract
The multiplexing of fluorescence correlation spectroscopy (FCS), especially in imaging FCS using fast, sensitive array detectors, requires the handling of large amounts of data. One can easily collect in excess of 100,000 FCS curves a day, too many to be treated manually. Therefore, ImFCS, an open-source software which relies on standard image files was developed and provides a wide range of options for the calculation of spatial and temporal auto- and cross-correlations, as well as differences in Cross-Correlation Functions (ΔCCF). ImFCS permits fitting of standard models to correlation functions and provides optimized histograms of fitted parameters. Applications include the measurement of diffusion and flow with Imaging Total Internal Reflection FCS (ITIR-FCS) and Single Plane Illumination Microscopy FCS (SPIM-FCS) in biologically relevant samples. As a compromise between ITIR-FCS and SPIM-FCS, we extend the applications to Imaging Variable Angle-FCS (IVA-FCS) where sub-critical oblique illumination provides sample sectioning close to the cover slide.
Highlights
The image file directory” (IFD) are read until the value of “0” is reached in order to determine the number of frames (n) in the particular tiff file
The Graphics Device Interface (GDI) contains the PtInRegion() function which determines whether the pixels lie within the drawn polygon
The cursor movement is intercepted by a “hook function” which determines the position of the cursor on the image
Summary
The function calculating the correlation described above was programmed in C++ using Microsoft Visual Studio .net 2003(Redmond, WA, USA). This program is controlled using Igor Pro 6.0 as front end by reprogramming the above program using the external operation (XOP) tool kit provided by Igor Pro. XOPs were used instead of using Igor procedures because XOPs in C++ were much faster in performing “number-crunching operations” [1]. The source code is available with the package and the user is free to modify it. The user can include newer fitting models. The null character has to be manually added to the name of the tif file while being passed to the correlation function for calculation
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