Inducible fluorescent speckle microscopy

The Journal of Cell Biology
António J PereiraHelder Maiato

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.

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Citations

Jul 25, 2019·Molecular Biology of the Cell·Che-Hang YuDaniel Needleman

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Methods Mentioned

BETA
light microscopy
fluorescence imaging
fluorescence correlation spectroscopy
dissection

Software Mentioned

MATLAB
NIS
ISI
Elements
FSM

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