My name is Megan Michie, and I’m the first author of our paper entitled, Cyanine Conformational
Restraint in the Far-Red Range, which I worked on while I was a post bac at the NIH in Dr.
Martin Schnermann’s lab.
An important goal in optical imaging is to make tools that better visualize structures
in complex living environments. Fluorescence imaging experiments have been very useful
in this context.
Fluorescence-based experiments use molecules that are activated by exposure to a certain
wavelength of light. Once activated, the molecules, or fluorophores, emit light of different,
longer wavelengths that allow researchers to locate structures that are labeled with
a specific dye. These tools are important for a better understanding in both experimental
and clinical applications, such as fluorescence-guided surgery.
While these techniques work well for cells and tissues examined outside of the body,
visualizing them in living organisms is difficult because light from existing fluorophores can’t
pass through tissue. Most currently available fluorescent dyes work in the green and blue
range of the visible light spectrum.
Our lab has been focused on fluorophores that emit longer wavelengths of light in the far-red
and near-infrared range (longer than 650). We are interested in these longer wavelengths
because they are better able to penetrate through tissue, which is something everyone
has seen when you hold a flashlight to your hand and the red light passes through it.
However, most fluorophores that emit light in these wavelengths are not bright enough
for biological applications. Figuring out how to make molecules that are both bright
enough and emit long wavelengths of light has been a long-standing puzzle for chemists.
In fact most flurophores used today, including those used in the clinic, were often taken
from very different other areas, such as the dye industry, and, as a result, have significant
limitations for biological use.
Most of our work has been on a class of fluorescent probes called the cyanines, sometimes called
the “Cy” dyes, which have strong light-absorbing properties.
However, as fluorophores, they are not very bright, which limits their use for addressing
many important problems.
Our lab speculated that the reason cyanines are not very bright is because these molecules
rotate after they absorb light. We made the hypothesis that restricting the
molecule’s ability to rotate by attaching a rigid structure would make them brighter.
Using tricks from modern organic chemistry, we built a system of chemical rings and attached
them to the far-red cy5 fluorophores, which we call Conformationally Restrained Cy5 or
Cy5B for short.
There are two key steps in the synthetic approach used to make these molecules.
In the first step, a chemical reaction, called olefin metathesis, makes a precursor molecule.
This reaction was developed over the last 20 years to attach a key reactive functional
group that enables the cyclization step.
In the second step, this precursor molecule is exposed to strongly acidic conditions,
which drives the formation of four rings in a single step. This is an exciting chemical
reaction because it involves forming 5 bonds, including 2 carbon-oxygen bonds and 3 carbon-carbon
bonds, the latter of which are often hard to form.
With this novel synthesis complete, we were delighted to discover that the addition of
these new ring structures made the fluorophores over 400% brighter. In addition these molecules
also have a longer fluorescence lifetime, which will be useful for visualizing time
dependent changes in tissues and cells. Mechanistic studies show that these molecules are bright
because they can no longer rotate in this excited state, validating our original hypothesis.
In addition to being brighter, these probes have an improved ability to turn from on to
off, making their signal easier to distinguish from the background.
A little more specifically, these new dyes can be reduced through sodium borohydride,
and this gain of an electron turns them into a dark state. This dark state can be recovered
to the on state with UV light.
Surprisingly, these rings make this on and off process occur much more efficiently than
it does for cyanines lacking the rings. Our lab is currently carrying out mechanistic
studies to better understand these differences.
These new properties prove to be quite useful for super resolution light microscopy (SMLM)
imaging applications.
In SMLM, molecules in a tissue sample—such as tumor cell-surface markers—are fluorescently
labeled and turned on to a bright state one at a time, as is shown in this movie, allowing
researchers to count them and determine their location with nanometer precision. This approach
has been used to localize structures both inside and outside of cells, as is seen in
this example imaging actin with these new dyes.
Our lab is currently developing variants of these dyes that emit even longer, more deeply
penetrating near-infrared wavelengths, which is challenging because more rings must be
created. The challenge is worthwhile, however, as flurophores
than can penetrate tissue more deeply will be useful for clinical techniques such as
fluorescence guided surgery. In fluorescence guided surgery, these dyes can be used to
helo surgeons identify sensitive tissues to avoid, like nerves or the biiary tree, and
to help them identify tumors so they can successfully remove all of the cancerous cells. Ongoing
studies in our lab are working to synthesize molecules that are specifically designed for
these applications.
Going forward, these newly synthesized Cy5 far-red fluorescent probes are brighter and
more stable than the currently available counterparts, which means they could be used for imaging
live tissue over long timeframes. These promising properties point to several applications,
such as tracking tumor cells in live tissues, which could lead to a better understanding
of how cancer behaves in a living system.