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Accepted Manuscript
TOOLS AND RESOURCES
Optimizing live-cell fluorescence imaging conditions to minimize phototoxicity
Alex Kiepas, Elena Voorand, Firas Mubaid, Peter M. Siegel, Claire M. Brown
Journal of Cell Science 2020 : jcs.242834 doi: 10.1242/jcs.242834 Published 27 January 2020
Alex Kiepas
Department of Physiology, McGill University, CanadaGoodman Cancer Research Centre, McGill University, Canada
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Elena Voorand
Goodman Cancer Research Centre, McGill University, CanadaDepartment of Biochemistry, McGill University, Canada
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Firas Mubaid
Department of Physiology, McGill University, Canada
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Peter M. Siegel
Goodman Cancer Research Centre, McGill University, CanadaDepartment of Biochemistry, McGill University, CanadaDepartment of Medicine, McGill University, CanadaDepartment of Anatomy & Cell Biology, McGill University, Canada
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  • ORCID record for Peter M. Siegel
Claire M. Brown
Department of Physiology, McGill University, CanadaAdvanced BioImaging Facility (ABIF), McGill University, CanadaDepartment of Anatomy & Cell Biology, McGill University, CanadaCell Information Systems, McGill University, CanadaCentre for Applied Mathematics in Bioscience and Medicine (CAMBAM), McGill University, Canada
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  • For correspondence: claire.brown@mcgill.ca
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Abstract

Fluorescence illumination can cause phototoxicity that negatively affects living samples. This study demonstrates that much of the phototoxicity and photobleaching experienced with live-cell fluorescence imaging occurs as a result of “illumination overhead” (IO). This occurs when a sample is illuminated but fluorescence emission is not being captured by the microscope camera. Several technological advancements have been developed, including fast-switching LED lamps and transistor-transistor logic (TTL) circuits, to diminish phototoxicity caused by IO. These advancements are not standard features on most microscopes and many biologists are unaware of their necessity for live-cell imaging. IO is particularly problematic when imaging rapid processes that require short exposure times. This study presents a workflow to optimize imaging conditions for measuring both slow and dynamic processes while minimizing phototoxicity on any standard microscope. The workflow includes a guide on how to (1) determine the maximum image exposure time for a dynamic process, (2) optimize excitation light intensity and (3) assess cell health with mitochondrial markers.

  • Received December 12, 2019.
  • Accepted January 9, 2020.
  • © 2020. Published by The Company of Biologists Ltd

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Keywords

  • Fluorescence microscopy
  • Phototoxicity
  • Cell migration
  • Mitochondrial dynamics
  • Microtubule dynamics
  • Adhesion dynamics

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Accepted Manuscript
TOOLS AND RESOURCES
Optimizing live-cell fluorescence imaging conditions to minimize phototoxicity
Alex Kiepas, Elena Voorand, Firas Mubaid, Peter M. Siegel, Claire M. Brown
Journal of Cell Science 2020 : jcs.242834 doi: 10.1242/jcs.242834 Published 27 January 2020
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Accepted Manuscript
TOOLS AND RESOURCES
Optimizing live-cell fluorescence imaging conditions to minimize phototoxicity
Alex Kiepas, Elena Voorand, Firas Mubaid, Peter M. Siegel, Claire M. Brown
Journal of Cell Science 2020 : jcs.242834 doi: 10.1242/jcs.242834 Published 27 January 2020

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