Development of time-integrated multipoint moment analysis for spatially resolved fluctuation spectroscopy with high time resolution

Doogie Oh, Alexandra Zidovska, Yangqing Xu, Daniel J. Needleman

    Research output: Contribution to journalArticle

    Abstract

    Spatial gradients in the behaviors of soluble proteins are thought to underlie many phenomena in cell and developmental biology, but the nature and even the existence of these gradients are often unclear because few techniques can adequately characterize them. Methods with sufficient temporal resolution to study the dynamics of diffusing molecules can only sample relatively small regions, whereas methods that are capable of imaging larger areas cannot probe fast timescales. To overcome these limitations, we developed and implemented time-integrated multipoint moment analysis (TIMMA), a form of fluorescence fluctuation spectroscopy that is capable of probing timescales down to 20 μs at hundreds of different locations simultaneously in a sample. We show that TIMMA can be used to measure the diffusion of small-molecule dyes and fluorescent colloids, and that it can create spatial maps of the behavior of soluble fluorescent proteins throughout mammalian tissue culture cells. We also demonstrate that TIMMA can characterize internal gradients in the diffusion of freely moving proteins in single cells.

    Original languageEnglish (US)
    Pages (from-to)1546-1554
    Number of pages9
    JournalBiophysical journal
    Volume101
    Issue number6
    DOIs
    StatePublished - Sep 21 2011

    ASJC Scopus subject areas

    • Biophysics

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