Autocorrelation function of finite-length data in fluorescence correlation spectroscopy

John Kohler, Kwang Ho Hur, Joachim Dieter Mueller

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

The experimental autocorrelation function of fluorescence correlation spectroscopy calculated from finite-length data is a biased estimator of the theoretical correlation function. This study presents a new theoretical framework that explicitly accounts for the data length to allow for unbiased analysis of experimental autocorrelation functions. To validate our theory, we applied it to experiments and simulations of diffusion and characterized the accuracy and precision of the resulting parameter estimates. Because measurements in living cells are often affected by instabilities of the fluorescence signal, autocorrelation functions are typically calculated on segmented data to improve their robustness. Our reformulated theory extends the range of usable segment times down to timescales approaching the diffusion time. This flexibility confers unique advantages for live-cell data that contain intensity variations and instabilities. We describe several applications of short segmentation to analyze data contaminated with unwanted fluctuations, drifts, or spikes in the intensity that are not suited for conventional fluorescence correlation analysis. These results demonstrate the potential of our theoretical framework to significantly expand the experimental systems accessible to fluorescence correlation spectroscopy.

Original languageEnglish (US)
Pages (from-to)241-253
Number of pages13
JournalBiophysical journal
Volume122
Issue number1
DOIs
StatePublished - Jan 3 2023

Bibliographical note

Funding Information:
We thank Jared Hennen and Isaac Angert for providing SS-EGFP and fluorescein isothiocyanate-dextran data, respectively. This work was supported by National Institutes of Health grants GM098550 and AI150468 . J.K. was supported by National Institutes of Health grant T32 AI083196 . The Minnesota Supercomputing Institute (MSI) at the University of Minnesota provided resources that contributed to these results.

Funding Information:
We thank Jared Hennen and Isaac Angert for providing SS-EGFP and fluorescein isothiocyanate-dextran data, respectively. This work was supported by National Institutes of Health grants GM098550 and AI150468. J.K. was supported by National Institutes of Health grant T32 AI083196. The Minnesota Supercomputing Institute (MSI) at the University of Minnesota provided resources that contributed to these results. The authors declare no competing interests.

Publisher Copyright:
© 2022 Biophysical Society

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural

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