Investigating Diffusion Dynamics and Interactions with Scanning Fluorescence Correlation Spectroscopy (sFCS).
Methods Mol Biol
; 2654: 61-89, 2023.
Article
em En
| MEDLINE
| ID: mdl-37106176
ABSTRACT
Activation of immune cells and formation of immunological synapses (IS) rely critically on the reorganization of the plasma membrane. These highly orchestrated processes are driven by diffusion and oligomerization dynamics, as well as by single molecule interactions. While slow macro- and meso-scale changes in organization can be observed with conventional imaging, fast nano-scale dynamics are often missed with traditional approaches, but resolving them is, nonetheless, essential to understand the underlying biological mechanisms at play. Here, we describe the use of scanning fluorescence correlation spectroscopy (sFCS) and scanning fluorescence cross-correlation spectroscopy (sFCCS) to study reorganization and changes in molecular diffusion dynamics and interactions during IS formation and in other biological settings. We focus on the practical aspects of the measurements including calibration and alignment of the optical setup, present a comprehensive protocol to perform the measurements, and provide data analysis pipelines and strategies. Finally, we show an exemplary application of the technology to studying Lck diffusion during T-cell signaling.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Simulação de Dinâmica Molecular
Tipo de estudo:
Guideline
Idioma:
En
Revista:
Methods Mol Biol
Ano de publicação:
2023
Tipo de documento:
Article