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
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
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
Cell Health and Other Ways
to Improve the Results from
By Lauren Alvarenga, Product Manager for Research Imaging, Scientific Solutions Group,
Olympus Corp. of the Americas firstname.lastname@example.org
Editor’s Note: We invite you to submit your personal
commentaries to Last Word on topics that impact your
work and affect the overall industry. Please send your
commentaries to MaryBeth DiDonna, Editor,
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
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