Inducible fluorescent speckle microscopy

• Tipo: Artigo em Revista Científica Internacional
• Data: 2016

Autores

Pereira, AJ (Autor) Outra; Paulo Aguiar (Autor) FMUP; Belsley, M (Autor) Outra; Helder Maiato (Autor) FMUP

Revista

Título: Journal of Cell Biology

Vol. 212

Páginas: 245-255

ISSN: 0021-9525

Editora: Rockefeller University Press

Indexação

ISI Web of Knowledge - 2 Citações

Scopus - 2 Citações

Outras Informações

ID Authenticus: P-00K-5M9

DOI: 10.1083/jcb.201506128

Abstract (EN): The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration.

Idioma: Inglês

Tipo (Avaliação Docente): Científica

Nº de páginas: 11