Published in

Rockefeller University Press, Journal of Cell Biology, 2(212), p. 245-255, 2016

DOI: 10.1083/jcb.201506128

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Inducible fluorescent speckle microscopy

Journal article published in 2016 by António J. Pereira, Paulo Aguiar, Michael Belsley ORCID, Helder Maiato
This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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Data provided by SHERPA/RoMEO

Abstract

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.