Fluorescence fluctuation spectroscopy (FFS) encompasses a bevy of techniques that involve analyzing fluorescence intensity fluctuations occurring due to fluorescently labeled molecules diffusing in and out of a microscope's focal region. Statistical analysis of these fluctuations may reveal the oligomerization (i.e., association) state of said molecules. We have recently developed a new FFS‐based method, termed Two‐Dimensional Fluorescence Intensity Fluctuation (2D FIF) spectrometry, which provides quantitative information on the size and stability of protein oligomers as a function of receptor concentration. This article describes protocols for employing FIF spectrometry to quantify the oligomerization of a membrane protein of interest, with specific instructions regarding cell preparation, image acquisition, and analysis of images given in detail. Application of the FIF Spectrometry Suite, a software package designed for applying FIF analysis on fluorescence images, is emphasized in the protocol. Also discussed in detail is the identification, removal, and/or analysis of inhomogeneous regions of the membrane that appear as bright spots. The 2D FIF approach is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest. © 2022 Wiley Periodicals LLC.
The dynamics of the cellular actomyosin cytoskeleton are crucial to many aspects of cellular function. Here, we describe techniques that employ active micropost array detectors (AMPADs) to measure cytoskeletal rheology and mechanical force fluctuations. The AMPADS are arrays of flexible poly(dimethylsiloxane) (PDMS) microposts with magnetic nanowires embedded in a subset of microposts to enable actuation of those posts via an externally applied magnetic field. Techniques are described to track the magnetic microposts’ motion with nanometer precision at up to 100 video frames per second to measure the local cellular rheology at well‐defined positions. Application of these high‐precision tracking techniques to the full array of microposts in contact with a cell also enables mapping of the cytoskeletal mechanical fluctuation dynamics with high spatial and temporal resolution. This article describes (1) the fabrication of magnetic micropost arrays, (2) measurement protocols for both local rheology and cytoskeletal force fluctuation mapping, and (3) special‐purpose software routines to reduce and analyze these data. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.
- PAR ID:
- 10381140
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 2
- Issue:
- 5
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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This article was corrected on 27 July 2022. See the end of the full text for details.
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