title: Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity.
abstract: It is important to determine the most effective method of delivering light onto a specimen for minimal light induced damage. Assays are presented to measure photo-bleaching of fluorophores and photo-toxicity to living cells under different illumination conditions. Turning the light off during part of the experimental time reduced photo-bleaching in a manner proportional to the time of light exposure. The rate of photo-bleaching of EGFP was reduced by 9-fold with light pulsing on the micro-second scale. Similarly, in living cells, rapid line scanning resulted in reduced cell stress as measured by mitochondrial potential, rapid cell protrusion and reduced cell retraction. This was achieved on a commercial confocal laser scanning microscope, without any compromise in image quality, by using rapid laser scan settings and line averaging. Therefore this technique can be implemented broadly without any software or hardware upgrades. Researchers can use the rapid line scanning option to immediately improve image quality on fixed samples, reduce photo-bleaching for large high resolution 3D datasets and improve cell health in live cell experiments. The assays developed here can be applied to other microscopy platforms to measure and optimize light delivery for minimal sample damage and photo-toxicity.
authors.affiliation: Department of Physiology, McGill University, Montreal, QC, Canada.
authors.forename: Colton
authors.initials: C
authors.lastname: Boudreau
authors.affiliation: Department of Physiology, McGill University, Montreal, QC, Canada.|Advanced BioImaging Facility (ABIF), McGill University, 3649 Prom. Sir William Osler, Bellini Building Rm137, Montreal, QC, Canada, H3G 0B1.
authors.forename: Tse-Luen Erika
authors.initials: TL
authors.lastname: Wee
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada.
authors.forename: Yan-Rung Silvia
authors.initials: YR
authors.lastname: Duh
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada.
authors.forename: Melissa P
authors.initials: MP
authors.lastname: Couto
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada.
authors.forename: Kimya H
authors.initials: KH
authors.lastname: Ardakani
authors.affiliation: Department of Physiology, McGill University, Montreal, QC, Canada.|Advanced BioImaging Facility (ABIF), McGill University, 3649 Prom. Sir William Osler, Bellini Building Rm137, Montreal, QC, Canada, H3G 0B1.
authors.forename: Claire M
authors.initials: CM
authors.lastname: Brown
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D008027:Light / Q000009:adverse effects*; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D038761:Photobleaching / Q000528:radiation effects*; D011829:Radiation Dosage