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.
Highlights
Live cell imaging has become common practice across the physical, life and health sciences
The laser power of a 473 nm laser on the confocal laser scanning microscope (CLSM) was held constant at 40% and the bleaching decay rate was measured during continuous imaging of a 100 × 100 pixel (8.7 μm × 8.7 μm) region of interest (ROI) of the sample (Fig. 2A)
Most cells are never exposed to light during their lifetime and it is well known that high levels of light can induce toxic effects on living systems
Summary
Live cell imaging has become common practice across the physical, life and health sciences. Most microscope mounting media for fixed samples contain oxygen scavengers such as anti-fade agents to reduce photo-bleaching of fluorescent probes This is not an ideal solution for imaging of living mammalian cells or tissues that require an oxygen rich environment. Continuous and pulsed confocal laser light on the nanosecond and microsecond time scales was applied to fixed and live samples to investigate the effects of light pulsing on photo-bleaching of fluorescent proteins and photo-toxicity to live cells. Our results suggest that the photo-bleaching and photo-toxicity depend on excited triplet state molecular destruction and the generation of oxygen radicals as the photo-bleaching is negated with the application of an oxygen scavenger This is a novel but straightforward technique that researchers can immediately implement in the CLSM software without any need for hardware upgrades. It should be used to reduce photo-damage to fixed samples and photo-toxicity to live samples
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