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.
Quantitative multi-image analysis (QMA) is the systematic extraction of new information and insight through the simultaneous analysis of multiple, related images. We present examples illustrating the potential for QMA to advance materials research in multi-image characterization, automatic feature identification, and discovery of novel processing-structure–property relationships. We conclude by discussing opportunities and challenges for continued advancement of QMA, including instrumentation development, uncertainty quantification, and automatic parsing of literature data.
- Award ID(s):
- 2004752
- NSF-PAR ID:
- 10375852
- Publisher / Repository:
- Cambridge University Press (CUP)
- Date Published:
- Journal Name:
- MRS Communications
- Volume:
- 12
- Issue:
- 6
- ISSN:
- 2159-6867
- Page Range / eLocation ID:
- p. 1030-1036
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Basic Protocol 1 : Preparation of live cells expressing protein constructsBasic Protocol 2 : Image acquisition and noise correctionBasic Protocol 3 : Drawing and segmenting regions of interestBasic Protocol 4 : Calculating the molecular brightness and concentration of individual image segmentsBasic Protocol 5 : Combining data subsets using a manual procedure (Optional)Alternate Protocol 1 : Combining data subsets using the advanced FIF spectrometry suite (Optional; alternative to Basic Protocol 5)Basic Protocol 6 : Performing meta‐analysis of brightness spectrogramsAlternate Protocol 2 : Performing meta‐analysis of brightness spectrograms (alternative to Basic Protocol 6)Basic Protocol 7 : Spot extraction and analysis using a manual procedure or by writing a program (Optional)Alternate Protocol 3 : Automated spot extraction and analysis (Optional; alternative to Protocol 7)Support Protocol : Monomeric brightness determination -
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