Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells.
J Vis Exp
; (165)2020 11 12.
Article
em En
| MEDLINE
| ID: mdl-33252108
ABSTRACT
Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Fluorescência
/
Membrana Celular
Tipo de estudo:
Guideline
Limite:
Animals
Idioma:
En
Revista:
J Vis Exp
Ano de publicação:
2020
Tipo de documento:
Article