Puchner and Noireaux labs discover new photochemistry useful for live-cell single-molecule microscopy

Professors Elias Puchner and Vincent Noireaux, along with their research groups, have shown that a popular class of fluorescent dyes changes color under certain conditions, causing complications for its use in biological imaging. Their article, "Red Light Mediated Photoconversion of Silicon Rhodamines to Oxygen Rhodamines for Single-Molecule Microscopy," published in the Journal of the American Chemical Society, details the cause of this color change, how to prevent it, as well as how to utilize it for new imaging approaches. 

Single-molecule localization microscopy (SMLM) was a significant breakthrough for biophysicists trying to understand the physics of living cells. This technique can pinpoint a single biomolecule with ~20 nm precision, provided it is labeled with a fluorescent dye that can be turned "on" and "off" when exposed to UV light. Researchers have adopted a family of dyes called Silicon-Rhodamines, since they are very bright, stable, and readily penetrate live cells to label molecules of interest. While performing routine control experiments, researchers in the Puchner and Noireaux labs noticed the dye blue-shifting by over 100 nm, mimicking the fluorescent signal of another dye. This color change can create false correlations and can lead to inaccurate findings. 

“At first, I was convinced that what I was seeing was the result of a mistake I was making, since no one else had seen this before. Once I was sure I was looking at something real, I had to get the bottom of it. This work has many promising applications, but for me the most important thing was to get the word out so other researchers didn’t mistakenly publish results based on faulty data!” says Jacob Ritz, who recently transitioned from the Puchner lab to a HHMI microscopy specialist position at the University of Massachusetts, Amherst.

To get to the bottom of this surprising color change, they developed new characterization methods and found that the used Silicon Rhodamines react with oxygen from the environment to form Oxygen Rhodamines, causing the change in color. With an understanding of the phenomenon, they were then able to prevent artifacts and to leverage this new color change to obtain single-molecule images without the previously required UV-light, which is typically over 100 times more intense than what we get from the sun.

“Since we and the entire field are pushing to observe cellular processes in the context of living cells, it is very useful to have found this much milder and less invasive way of performing SMLM,” says Elias Puchner.

Since the color change of Silicon Rhodamines requires the presence of oxygen as shown in their study, the research group is very excited about the potential of this mechanism to be used as a molecular oxygen sensor. Being able to visualize oxygen concentrations in living cells opens up entire new research avenues.