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Optimizing live-cell fluorescence imaging conditions to minimize phototoxicity
Alex Kiepas1,2, Elena Voorand2,3, Firas Mubaid4
1Department of Physiology, McGill University, Montreal, Canada, H3G 1Y6 alex.kiepas@mail.mcgill.ca claire.brown@mcgill.ca.
Journal of Cell Science
|January 29, 2020
Summary
Minimize phototoxicity in live-cell fluorescence imaging by addressing illumination overhead (IO). This study offers a workflow to optimize imaging conditions, reducing light damage and preserving cell health on standard microscopes.
Area of Science:
- Microscopy
- Cell Biology
- Biophysics
Background:
- Fluorescence illumination can induce phototoxicity and photobleaching in living biological samples.
- Phototoxicity is often exacerbated by 'illumination overhead' (IO), where the sample is illuminated without simultaneous image acquisition.
- Existing solutions for IO are not widely adopted or standard on many microscopes.
Purpose of the Study:
- To identify and quantify the impact of illumination overhead (IO) on phototoxicity and photobleaching in live-cell fluorescence imaging.
- To develop and present a practical workflow for optimizing live-cell imaging conditions to minimize phototoxicity on standard microscopy equipment.
- To provide guidance for biologists on managing light exposure during the imaging of both dynamic and slow biological processes.
Main Methods:
- Quantified phototoxicity and photobleaching attributed to illumination overhead (IO) during fluorescence microscopy.
- Developed a workflow incorporating strategies to determine optimal exposure times for dynamic processes.
- Included methods for optimizing excitation light intensity and assessing cellular health using mitochondrial markers.
Main Results:
- Demonstrated that illumination overhead (IO) significantly contributes to phototoxicity and photobleaching in live-cell imaging.
- Validated a workflow that effectively minimizes phototoxicity across various imaging scenarios on standard microscopes.
- Established protocols for assessing cell health post-imaging, crucial for reliable experimental outcomes.
Conclusions:
- Illumination overhead (IO) is a major, often overlooked, source of phototoxicity in live-cell fluorescence microscopy.
- The presented workflow provides a practical solution for researchers to mitigate phototoxicity and improve cell viability during live-cell imaging.
- Implementing this workflow on standard microscopes enhances the reliability of data obtained from dynamic biological processes.
Keywords:
Adhesion dynamicsCell migrationFluorescence microscopyMicrotubule dynamicsMitochondrial dynamicsPhototoxicity
