Time-shifted mean-segmented Q data of a luminal protein measured at the nuclear envelope by fluorescence fluctuation microscopy

Published: 25-11-2019| Version 2 | DOI: 10.17632/jcknwhy4r8.2
Siddarth Reddy Karuka,
Jared Hennen,
Kwang-Ho Hur,
Joachim Mueller


The data presented here are acquired by focusing the two-photon excitation spot at the nuclear envelope of a living U2OS cell expressing luminal EGFP. This dataset contains the fluorescence intensity fluctuations due to protein diffusion. Superimposed on this signal is a slow fluctuation process due to local volume changes caused by undulations in the distance separating the two membranes of the nuclear envelope. The combination of both fluctuation sources provides a challenging case for traditional fluorescence fluctuation analysis. The data were analyzed using the recently developed tsMSQ method, which has been shown to provide an accurate separation of both fluctuation sources. - 'trace.dat' contains photon count data that were collected for ~60 s with a sampling frequency of 20 kHz. It was recorded by a Flex04-12D data acquisition card (correlator.com, Bridgewater, NJ) and contains 16-bit unsigned integer data in binary format from two single photon counting detectors collected at 20kHz. The data points alternate between each detector. For example, the first three data corresponding to first detector are stored at positions 1,3 and 5 of the data record, whereas the corresponding data of the second detector are found at positions 2,4 and 6. Only the fluctuation data of the second detector were used in this publication. The data from the second channel were also saved in csv format as 'trace.csv' to provide easy access for other analysis programs. - 'example_script.pro' is an IDL (Interactive Data Language) program that performs time-shifted mean-segmented Q analysis on this dataset. - All other files are supporting files called by 'example_script.pro'. - 'mpfit' and 'mpfitfun' packages are required and can be downloaded from http://cow.physics.wisc.edu/~craigm/idl/down/mpfit.pro & http://cow.physics.wisc.edu/~craigm/idl/down/mpfitfun.pro - Open-source GDL (GNU Data Language) can also be used in lieu of proprietary IDL. ‘Running_on_GDL.txt’ is a text file containing information on the environment that was used to successfully run 'example_script.pro' on GDL.