Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging.
Proc Natl Acad Sci U S A
; 111(48): 17164-9, 2014 Dec 02.
Article
em En
| MEDLINE
| ID: mdl-25404337
ABSTRACT
Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Algoritmos
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Processamento de Imagem Assistida por Computador
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Imageamento Tridimensional
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Microscopia de Fluorescência
/
Modelos Teóricos
Tipo de estudo:
Incidence_studies
/
Prognostic_studies
/
Risk_factors_studies
Limite:
Humans
Idioma:
En
Revista:
Proc Natl Acad Sci U S A
Ano de publicação:
2014
Tipo de documento:
Article