Measurement of nanoscale three-dimensional diffusion in the interior of living cells by STED-FCS.
Nat Commun
; 8(1): 65, 2017 07 06.
Article
em En
| MEDLINE
| ID: mdl-28684735
ABSTRACT
The observation of molecular diffusion at different spatial scales, and in particular below the optical diffraction limit (<200 nm), can reveal details of the subcellular topology and its functional organization. Stimulated-emission depletion microscopy (STED) has been previously combined with fluorescence correlation spectroscopy (FCS) to investigate nanoscale diffusion (STED-FCS). However, stimulated-emission depletion fluorescence correlation spectroscopy has only been used successfully to reveal functional organization in two-dimensional space, such as the plasma membrane, while, an efficient implementation for measurements in three-dimensional space, such as the cellular interior, is still lacking. Here we integrate the STED-FCS method with two analytical approaches, the recent separation of photons by lifetime tuning and the fluorescence lifetime correlation spectroscopy, to simultaneously probe diffusion in three dimensions at different sub-diffraction scales. We demonstrate that this method efficiently provides measurement of the diffusion of EGFP at spatial scales tunable from the diffraction size down to â¼80 nm in the cytoplasm of living cells.The measurement of molecular diffusion at sub-diffraction scales has been achieved in 2D space using STED-FCS, but an implementation for 3D diffusion is lacking. Here the authors present an analytical approach to probe diffusion in 3D space using STED-FCS and measure the diffusion of EGFP at different spatial scales.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Fluorescência
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Proteínas de Fluorescência Verde
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Difusão
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Imagem Molecular
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Microscopia de Fluorescência
Idioma:
En
Revista:
Nat Commun
Ano de publicação:
2017
Tipo de documento:
Article