How to Overcome Photobleaching and Phototoxicity in Live Cell Imaging

Challenge Background

One of the most significant challenges in live imaging is to overcome photobleaching and phototoxicity. Excessive light exposure can cause cellular damage which may in turn lead to errors with experimental data and result in incorrect conclusions being drawn. All samples subjected to live imaging experiments will be affected by the light used, from single cells through to thick organisms. The underlying processes such as intracellular dynamics are also affected by excessive light, and other processes such as mitosis or vesicle trafficking (among others) are well known to be severely affected by the imaging light.

Photobleaching is the process by which, upon irradiation, a fluorescence emitting molecule is chemically altered in such a way that will be irreversibly unable to fluoresce. Photobleaching is caused by high-intensity illumination and/or by prolonged light exposure. The cellular processes related to photobleaching are also implicated in phototoxicity.

Phototoxicity is the process by which upon illumination, with high laser power or for prolonged periods, the imaged organisms/cells are damaged. Phototoxicity can cause cellular membrane blebbing, vacuole formation and even cell death. Both phototoxicity and photodamage can obviously negatively influence cell survival and data quality.

Technology Solution

A camera-based confocal system can be a better solution for live imaging without compromising cell viability due to photobleaching or phototoxicity. New developments in multi-point scanning confocal technology result in the ability to perform ultrafast live imaging with very low light exposure. New detectors with back-illuminated sensors and high QE, can capture even dimmer fluorophore emission. Ultra-fast camera-controlled shutters will result in precise control of exposure times, reducing the sample illumination to the minimum. The use of longer wavelengths (NIR) for live-cell imaging applications will reduce the energy that hits the sample resulting in increased cellular viability.

Andor Solutions for live-cell imaging Studies

Andor strongly recommends the Dragonfly confocal equipped with either the iXon Ultra 888 back-illuminated EMCCD, or the ZL41 Cell sCMOS cameras. The microdisk on the dragonfly allows the simultaneous scan of thousands of microbeams, resulting in decreased photobleaching and phototoxicity. The EMCCD and sCMOS detectors from Andor´s cameras used in the dragonfly give a 3 to 5 times increase in sensitivity when compared to point scanning confocal detectors; there is simply no need to image with high laser power when imaging with the Dragonfly.

Andor´s Borealis patented perfect illumination delivery system, allows the use of extended spectral range (from 400-800 nm) giving researchers the benefits of a broader choice in fluorescent probes, and the ability to use probes within the NIR range. Post-acquisition of the best quality images with Andor´s equipment, data analysis and visualisation tools are required. Imaris provides a full package of analysis solutions adapted to all the fields of life sciences including 3D analysis, visualisation, segmentation and rendering tools.

Key Requirement	Live imaging solution: Dragonfly and Andor High QE cameras
Decrease light intensities on samples while acquiring good quality data	The dual microdisk configuration allows the scan of thousands of microbeams simultaneously minimizing loss of light. The combination of the dual spinning disk with high QE detectors makes for a system that can run at high speed while maintaining highly efficient light capture. Result 1 - Increased signal-to-noise (SNR). Result 2 - Decrease excitation intensities and exposure times. Result 3 - Decrease photobleaching and phototoxicity.
Reduce exposure times, to minimise phototoxicity and photobleaching	Active blanking allows laser illumination to be precisely synchronised to camera exposure. Result 1 - Reduce sample exposure to radiation to a minimum. Result 2 - Reduce photobleaching and phototoxicity.
Image NIR wavelengths, using lower energy radiation for image acquisition	Dragonfly uses a multimode optical fiber system: Borealis. The fiber system, coupled to Dragonfly optics, allows an exceptionally broad excitation and detection ranges of 400–800 and 425–850 nm respectively. Result - NIR excitation can be used for live imaging, diminishing high energetic radiation on samples and increasing cell viability.
Detect weak signals while maintaining good quality data	EMCCD and sCMOS detectors (cameras) used with the Dragonfly are 3 to 5 times more sensitive than point scanning confocal detectors, with up to 95% QE. Result 1 - Low laser powers can be used for imaging. Result 2 - Shorter exposure times for acquisition. Result 3 - Increased sample viability.
Visualize and analyze data	Imaris provides a full package of analysis solutions adapted to all the fields of life sciences. Imaris for tracking provides tools for automatically analyzing moving objects, create a lineage tree plot and generate quantitative information from your live imaging data. Result 1 - High-quality data acquired with Dragonfly and Andor's cameras can be easily quantified, analyzed and 3D rendered. Result 2 - High-quality movies can be created.