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Photoactivatable Protein May Allow Single-Particle PALM Imaging of Live Cells

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Researchers at the Albert Einstein College of Medicine and the National Institute of Child Health and Human Development last week said they have developed a monomeric red-photoactivatable protein that matures more quickly and has better pH stability, faster photoactivation, higher photoactivation contrast, and better photostability than other monomeric red photoactivatable proteins.

They claim the lack of green fluorescence and single-molecule behavior make the protein, called PAmCherry1, a preferred tag for two-color diffraction-limited photoactivation imaging and for super-resolution techniques such as one- and two-color photoactivated localization microscopy, or PALM, according to the researchers.

Most notably, the absence of a green emission state could enable the researchers to use it in two-color live-cell and single-particle PALM, Vladislav Verkhusha, an associate professor in the department of anatomy and structural biology and the Gruss-Lipper Biophotonics Center at Albert Einstein, said this week.

The work was published online this week in Nature Methods.

Seeing Red

In their study, the researchers compared transferrin receptor-PAmCherry1 fusions with two other TfR fusions using other variants of mCherry, and showed that that PAmCherry1 had properties such as mean number of photons, mean molecular organization uncertainty values, and duration of molecular fluorescence that made it a more appropriate fluorescent probe for PALM imaging, the authors said.

The researchers also compared PAmCherry1 with tdEosFP, a more thoroughly characterized PALM probe, and found that they were comparable in fixed-cell PALM experiments.

To evaluate the use of PAmCherry as a marker in two-color PALM, the scientists made a photoactivatable green fluorescent protein-clathrin light chain chimera, and observed TfR-PAmCherry1 and PAGFP-CLC clusters in a total internal reflection microscopy image similar to those observed in one-color experiments, with molecules clustered throughout the plasma membrane.

“We use a pulse laser [with] a very small intensity, a very fast pulse for photoactivation [of these proteins],” said Verkhusha. The proteins are then imaged and are photobleached.

"While we image them, we are collecting photons, and our software will build a kind of Gaussian pattern of locations of those individual protein molecules,” said Verkhusha.

Then it repeats. “We apply a very short pulse of a low intensity laser to photoactivate other [fluorescent protein] molecules.” Again, the proteins are imaged and photobleached, and software again builds a small Gaussian distribution.

This process is repeated thousands of times, Verkhusha said.

He went on to say that, “When we add those Gaussian distributions to each other, we get a super-resolution image. This is how we resolved molecules with a precision of 20 nm.”

“The availability of PAmCherry1 will allow, maybe for the first time, the imaging of two proteins with super-resolution within the same living individual cell,” said Verkhusha. This was previously possible only in fixed cells using photoactivatable organic dyes.

“These two-color images [in the paper] are just stunning,” said Anne Marie Quinn, founder and CEO of Montana Molecular, a maker of fluorescent proteins for live-cell imaging. “The resolution is so beautiful, and is likely comparable to that seen in electron microscopy.”

In the current paper, all PALM imaging was done on fixed mammalian cells. “I guess the downside to the PALM method is that there is no temporal resolution,” said Quinn. “If you need to have temporal resolution, you would need to go to another technique such as stimulated emission depletion, or STED, microscopy.”

However, she went on to say that, “I think that PAmCherry1 will also be useful for live-cell applications, because it has such beautiful high contrast. I think it would be interesting for studying things such as cell migration or trafficking, where you really want to see movement over time.”

The authors only allude to this possible application in the paper, she said.

True Colors

In the greater context of the fluorescent probe market, PAmCherry1 is “one more variant of a fluorescent protein that has very nice properties for cell labeling,” said Quinn. The fact that PAmCherry1 is photoswitchable makes it useful for certain applications such as looking at cell migration or molecule trafficking.”

In comparison with other variants of mCherry, “you can definitely see in [the current Nature Methods paper] faster maturation time and nice pH properties,” Quinn said. “You see these every few months; new fluorescent proteins that have been tweaked to be just a little bit better.

“I think the real issue is signal-to-noise,” Quinn said. As fluorescent proteins are refined and improved, “I think that they will be a little better for all of these different applications. It will certainly help the market to have these fine-tuned tools to use,” she added.

“A whole spectrum of colors is now available, some of which are better than others for certain applications,” said Quinn. For instance, red and green are appropriate for co-localization studies, while yellow and blue are probably better colors for fluorescence resonance-energy transfer.

“In terms of high-content screening, I think people have tried a variety of colors,” Quinn said. “They are really just trying to optimize the signal-to-noise ratio.

“I think in terms of the [fluorescent probe] market, most people will tell you it has been slow to really take off,” she added. “I think there was really a lot of hope for using genetically encoded proteins in high-content screening.”

In the last few years, people have started to use the BacMam viral-expression system, which “has moved the high-content screening field forward quite significantly, because you can get that very stable expression without having to go through the trouble of creating stable cell lines,” said Quinn.

The next step for the Albert Einstein researchers will be to develop different colors of fluorescent protein probes. “Now we have PAGFP and PAmCherry. There is a need to develop PA blue, PA yellow, PA orange, and PA far-red,” said Verkhusha.

However, “We do not exactly know what will happen. It is not easy to develop these proteins, he said, adding that it took his lab “more than a year” to develop PAmCherry.

“The application of PAmCherry to live-cell PALM imaging is our next (and ongoing) step,” said Verkhusha.

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