Although it is a well-known fact that the fluorescence process can cause phototoxicity to living cells
– it sometimes seems that this problem does not always get enough attention in planning biological experiments.
What is the best way to minimize the phototoxicity and still maintain images with high enough signal-to-noise? Should we minimize the exposure time and level up the illumination power or vice versa? Maybe find some middle ground for both features? Doesthe total light dose determine photobleaching and phototoxicity or the maximum light power that reaches the specimen?
New research led by Claire M. Brown at McGill University shows that although the total light dose determines the phototoxicity – long exposure times with lower light power seems to lower phototoxicity, but the reason behind this is illumination overhead.
What is illumination overhead? It is the time a sample is illuminated but fluorescence emission is not being captured by the microscope camera. The amount of illumination overhead vary depending on the hardware and software settings that are used, as can be seen in the following figure:
In the above figure the camera exposure time in the image acquisition software was set to 24ms but only LED illumination with TTL triggering illuminated the sample for 24 ms with no overhead. The total exposure time delivered with the USB cable connected to give the image acquisition software control of the LED (137 ms) was almost six times longer than the desired exposure time. Image acquisition with the mechanical shutter within the microscope stand was almost 10 times longer than the camera exposure time (230 ms).
If you are using a mechanical shutter, or even a LED light source that is controlled by the image acquisition software using a USB connection – you should try to capture with longer exposure times and less illumination power. This will reduce the relative contribution or the percentage of illumination overhead. For example – if you increase the exposure time from 24 ms to 240 ms and reduce the illumination power to 10% of the original power – you will get the same total illumination during the exposure time of 24 ms. The amount of light your sample will be exposed to will be more than 5 times higher in the short exposure option because of the illumination overhead when you are using a mechanical shutter.
Long exposure times may help in reducing phototoxicity, but this solution is not always possible for live cell imaging. You must consider the dynamic processes you want to image. When you want to image many fields, Z stacks and several channels – you will be even more limited in increasing the exposure time. For that reason, it is ideal to invest in devices such as TTL controlled LED light sources that can eliminate illumination overhead.
In Line-A Technologies we offer high speed TTL controlled LED light sources, such as the 7 channel Niji from Bluebox optics or the pE-300 Ultra from CoolLED. We also offer very sensitive cameras, such as the back illuminated sCMOS Sona from Andor that has 95% quantum efficiency. With such as sensitive camera you can reduce the illumination power and still detect the same number of photons without increasing the exposure time.
We also offer confocal devices that uses sensitive cameras as detectors instead of the much less efficient PMT’s used in standard scanning confocal microscopes. Among them are Andor’s Dragonfly, Confocal.nl’s RCM and Aurox’ laser-free Clarity.
Another solution we offer is the Livecyte from Phasefocus uses patented label-free Quantative Phase Imaging technology that allows you to identify, track and analyze cells without using fluorescence illumination at all.
Please contact me at Lior@Line-A.co.il for more information and advice on how you can upgrade your microscope to minimize phototoxicity and photobleaching.
*All images are from the article: 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 133: jcs242834 doi: 10.1242/jcs.242834 Published 21 February 2020