Superresolution has been a hot topic within the microscopy community since the 2014 Nobel Prize in chemistry was awarded. Breaking the diffraction limit is no small task. Despite efforts to simplify and perfect the process, it is well known that superresolution is difficult to perform in practice, and even more so with live samples. This is why it’s important to get the most out of live cell experiments by looking beyond methods and techniques. There are many common superresolution microscopy techniques offering various benefits. From localization techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), to structured illumination microscopy (SIM) and stimulated emission depletion (STED), choosing a 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 the experiment. If the cells die, you must be able to pinpoint the cause. And a major part of keeping the samples alive involves optimizing the system to best accommodate living samples. To keep cells happy, it’s necessary to minimize the amount of light they are exposed to. UV light is damaging to live samples and should be avoided whenever possible. A major part of keeping samples alive involves optimizing the system to best accommodate living samples. To keep cells happy, it's necessary to minimize the amount of light they are exposed to. Make sure you are also using fluorescent proteins with high quantum yields, which essentially measure how effectively a fluorophore converts light from one wavelength to another. If quantum yield is low, samples will be exposed to a lot of light but you won’t get much fluorescence in return. Short exposure times are preferred, and techniques to further shorten them, such as binning, should be investigated. Don’t go too far, however. A 1-ms exposure with 100% laser power is not always better for samples. Allow the cells time to recover between exposures when possible. Choose imaging software that is able to accommodate differing intervals for time-lapse experiments so you can ramp up exposure during critical periods without sacrificing cell health. Stay current with literature regarding superresolution (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 superresolution image. Cell health is the most important factor in live cell experiments, but there are other ways to improve the results from superresolution experiments. One metric for image quality is the ratio of intensities between the signal and the background. This can be quite literal. The biggest factors that hurt your signal ratio are the material your sample vessel is made of and your imaging medium. Always perform superresolution in coverslip, glass-bottom dishes as close to the objective as possible. Plastic is a poor choice for superresolution because it can autofluoresce, which is a huge issue for samples. Media components such as phenol red and FBS (fetal bovine serum) 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 when collecting photons through different cameras and detectors. Outside of the sample itself, environmental control is critical. Optimal temperature, humidity, and gas conditions must be maintained. These days it is easier than ever to accomplish this on a microscope, which also has the ability to change conditions 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 superresolution but also applies to all live-cell microscopy experiments. You’ll need to know whether the cells aren’t surviving because of a drug treatment or a toxic 405-nm laser. When performing experiments in the lab, further optimization is almost always needed inside and outside the microscope to keep samples happy and healthy. Meet the author Lauren Alvarenga is a product manager for research imaging in the Scientific Solutions Group at the Olympus Corp. of the Americas. She is currently responsible for imaging software, and inverted and superresolution microscopes. She has a Bachelor of Science degree in biomedical photographic communications from the Rochester Institute of Technology.