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Cell Health and Other Ways to Improve the Results from Super-Resolution Experiments.

Super-resolution has been a hot topic within the microscopy community since the 2014 Nobel Prize in Chemistry. Breaking the diffraction limit is no small task--and in spite of efforts to simplify and perfect the process, it is well known that super-resolution is difficult to perform in practice, even moreso with live samples. This is why it's important to look beyond methods and techniques to get the most out of your live cell experiments.

There are many common super-resolution microscopy techniques offering various benefits. From "localization microscopy" like PALM and STORM to Structured Illumination (SIM) and Stimulated emission depletion microscopy (STED), choosing your method is only half the battle. The most important thing to remember when working with live cells is that they must remain alive and happy at the end of your experiment. If your cells die, you must be able to pinpoint the cause. And a major part of keeping your samples alive involves optimizing your system to best accommodate living samples.

To keep cells happy, we must minimize the amount of light they are exposed to. Just like you, UV light is damaging to live samples and should be avoided whenever possible.

You also want to make sure that you are using fluorescent proteins with high quantum yields, which is essentially a measure of how effectively a fluorophore converts light from one wavelength to another. If this is low, you will expose your sample to a lot of light but won't get much in return.

Short exposure times are preferred, and techniques to further shorten them such as binning should be investigated. Don't go too far, though--a 1 ms exposure with 100 percent laser power is not always better for your samples.

You also want to allow your cells some time to recover between exposures when you can--your imaging software should be able to accommodate differing intervals for time lapse experiments so you can ramp up exposure during critical periods without sacrificing cell health.

It is important to stay current with literature regarding super-resolution, as processing algorithms are constantly improving to extract more out of samples without additional exposure to light.

SIM processing, for example, can be applied to specific confocal images to achieve similar levels of resolution with fewer frames. Even localization microscopy is evolving with high-density processing algorithms that reduce the number of frames needed to form a super-resolution image.

Cell health is the most important factor in live cell experiments, but there are other ways to improve the results from your super-resolution experiments.

One metric for the quality of your images is the ratio between the signal and that which surrounds it. This can be quite literal. One of the biggest factors that hurts your signal ratio is what your sample is sitting in. You should always perform super-resolution in coverslip, glass bottom dishes, as close to the objective as possible. Plastic is a bad choice for super-resolution as it can autofluoresce.

Autofluorescence is a huge issue for samples. Media components like phenol red and FBS can also be subject to this. Often, separate imaging media are stored without these items for use during time-lapse microscopy experiments.

You can also improve your signal ratios by increasing your efficiency collecting photons through different cameras and detectors.

Outside of the microscope itself, environmental control is critical. Optimal temperature, humidity and gas conditions must be maintained, and these days it is easier than ever to accomplish this on a microscope and be able to change on the fly as needed.

The health of your sample is the most important aspect of live cell imaging. Period. This is especially true for super-resolution but applies to all live cell microscopy experiments. You'll need to know if your cells aren't surviving because of a drug treatment or a toxic 405 laser.

When you are performing experiments in the lab, further optimization is almost always needed inside and outside the microscope to keep your samples happy and healthy.

By Lauren Alvarenga, Product Manager for Research Imaging, Scientific Solutions Group, Olympus Corp. of the Americas

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Title Annotation:Last Word
Author:Alvarenga, Lauren
Publication:Laboratory Equipment
Date:Apr 1, 2019
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