title: Inducible fluorescent speckle microscopy.
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.
authors.affiliation: Chromosome Instability and Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal Instituto de Investigação e Inovação em Saúde - i3S, Universidade do Porto, 4200-135 Porto, Portugal apereira@ibmc.up.pt.
authors.forename: António J
authors.initials: AJ
authors.lastname: Pereira
authors.affiliation: Instituto de Investigação e Inovação em Saúde - i3S, Universidade do Porto, 4200-135 Porto, Portugal Instituto de Engenharia Biomédica, Universidade do Porto, 4200-135 Porto, Portugal.
authors.forename: Paulo
authors.initials: P
authors.lastname: Aguiar
authors.affiliation: Center of Physics, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors.forename: Michael
authors.initials: M
authors.lastname: Belsley
authors.affiliation: Chromosome Instability and Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal Instituto de Investigação e Inovação em Saúde - i3S, Universidade do Porto, 4200-135 Porto, Portugal Cell Division Unit, Department of Experimental Biology, Faculty of Medicine, Universidade do Porto, 4200-319 Porto, Portugal.
authors.forename: Helder
authors.initials: H
authors.lastname: Maiato
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D004330:Drosophila; D006801:Humans; D021621:Imaging, Three-Dimensional; D008677:Metaphase; D008856:Microscopy, Fluorescence / Q000379:methods*; D008870:Microtubules; D008941:Spindle Apparatus