Fluorescence fluctuation microscopy is a widely used method to determine the mobility and oligomeric state of proteins in the live cell environment. Existing analysis methods rely on statistical evaluation of data segments with the implicit assumption that no significant signal fluctuations occur on the time scale of a data segment. Recent work on extending fluorescence fluctuation methods to the nuclear envelope of living cells identified a slow fluctuation process that is associated with the undulations of the nuclear membranes, which lead to intensity fluctuations due to local volume changes at the nuclear envelope. This environment violates the above-mentioned assumption and is associated with biased evaluation of fluorescence fluctuation data by traditional analysis methods, such as the autocorrelation function. This challenge was overcome by the introduction of the time-shifted mean-segmented Q function, which relies on a sliding scale of data segment lengths. Here, we share experimental fluorescence fluctuation data taken at the nuclear envelope and demonstrate the calculation of the time-shifted mean-segmented Q function from the raw data. The data and analysis should be valuable for researchers interested in fluorescence fluctuation techniques and provides an opportunity to examine the influence of slow fluctuations on existing data analysis methods. The data is related to the research article titled “Protein oligomerization and mobility within the nuclear envelope evaluated by the time-shifted mean-segmented Q factor” .
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health ( GM064589 ).
© 2019 The Authors
- Fluorescence fluctuation spectroscopy
- Nuclear envelope
- Protein assembly
- Protein mobility
- Two-photon microscopy