Inducible fluorescent speckle microscopy

• Type: Article in International Scientific Journal
• Date: 2016

Authors

Pereira, AJ (Author) Other; Paulo Aguiar (Author) FMUP; Belsley, M (Author) Other; Helder Maiato (Author) FMUP

Journal

Title: Journal of Cell Biology

Vol. 212

Pages: 245-255

ISSN: 0021-9525

Publisher: Rockefeller University Press

Indexing

ISI Web of Knowledge - 2 Citations

Scopus - 2 Citations

Other information

Authenticus ID: 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 11