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Promega, Leica to Develop Fluoroligands For Use with Superresolution Microscope

Promega and Leica Microsystems plan to develop fluorescent ligands for Promega’s HaloTag fusion proteins, which together could be used to label proteins within live cells at the sub-100 nm range, and visualize the proteins with Leica’s TCS stimulated emission depletion superresolution microscope.
Leica’s technology, also known as STED, has enabled Promega to show “that we can label our fusion proteins when the cells are still alive,” said Georgyi Los, the firm’s imaging group leader. However, the dye currently can only label the HaloTag when it is fused to proteins of interest on the cell surface.
Los said that Promega and Leica are trying to extend this approach of live-cell labeling to monitoring the translocation of the labeled proteins of interest in real time.
Leica has identified fluorescent dyes that are very suitable for STED microscopy, but the issue is how to attach those dyes to their proteins of interest. Leica scientists currently have to use antibodies to look at intracellular proteins, but their use requires fixing the cells. HaloTag technology potentially offers a way of looking at intracellular proteins in living cells.
“Today, we can only label proteins of interest on the surface of cells, but they are live cells, and we can monitor what happens in real time,” Los said in an interview with CBA News.
Promega scientists are already using the HaloTag for live-cell imaging using standard confocal microscopy and widefield microscopy. However, because STED microscopy is higher resolution than either confocal or widefield microscopy, “we hope the combination of superresolution STED microscopy and HaloTag labeling ability will help scientists to address new biological problems, and come to an even greater understanding of biological events,”  said Los.
The partners have not yet decided whether the STED-compatible ligands will be sold with the commercially available version of the STED microscope, or sold separately by Promega, “although either scenario is likely viable,” said Los. He added that the company would choose whichever commercialization strategy “would be better for our customers.”
However, he said “I think that we need to determine what exactly can be done using STED microscopy and HaloTag.”

“We hope the combination of superresolution STED microscopy and HaloTag labeling ability will help scientists to address new biological problems, and come to an even greater understanding of biological events.”

Promega has proteins ready in-house, and also has several models and ligands. “Now we have to combine these proteins with ligands in living cells, and this would be done by the lab at Leica that has a STED microscope,” said Los.
He said Promega scientists have already shown that they can label the HaloTag fusion proteins expressed on the surface of mammalian cells using ligands appropriate for STED microscopy.
“Our next goal is to demonstrate that we can monitor the movement of these proteins in live cells in real time. That is exactly what the Leica people will be doing,” Los said.
Promega is sending Leica all of the necessary materials for this type of experimentation. Leica investigators have cultured cells that they will transfect with the DNA encoding the Halotag proteins. After that, they will label proteins with the ligands provided by Promega, and apply their STED microscope.
At least one of Leica’s customers is using the HaloTag technology in combination with its STED microscope. “We have signed almost a three-way collaborative agreement with Leica and Max Planck,” Los said. He did not elaborate.
STED microscopy was invented in the laboratory of Stefan Hell at Max Planck. Promega decided to set up a collaboration with Wolfgang Fischle’s laboratory at Max Planck where they had STED microscopes, and Promega provided them with some pieces of HaloTag technology such as ligands and DNA encoding HaloTag proteins. Promega’s collaborations with Leica and Max Planck are moving forward in parallel.
“We are still in the process of setting [the experiments] up,” said Fischle, the research group leader at the laboratory of chromatin biochemistry at Max Planck. He explained that the fluorescent dye that can be used for STED microscopy is very hydrophobic when put onto living cells.
“It seems to be sticking, it does not penetrate the membrane, and it seems to aggregate,” Fischle told CBA News this week.
Fischle’s group at Max Planck is currently making some progress in its attempt to inject the dye into the cells to deliver it. “When you want to do dynamics and look at living cells, you have to have a dye that is cell permeable, or that can be delivered into the cell. So we are currently trying to figure that problem out,” he said.
It is not a new problem. “I was told by Georgyi Los when we started that they encountered this sort of problem with this particular ligand, but we have the technology available to do injection experiments and then do microscopy,” Fischle said. “It looks promising, but we are not there yet. We still need to adjust the injection protocol so that imaging can be done without harming the cells.”
Fischle said that ultimately, he is confident that he and his team can make that happen. “We have two different dyes that are in a similar spectral range, it is just that one dye is a lot brighter than the other.”
Fischle and his colleagues have started their work with the brighter dye because they claim it works better for STED microscopy, but Fischle said the brighter dye behaves “oddly.” The Max Planck researchers have tried the other dye, and it can be delivered, but “we have not done imaging experiments yet because it is dim.”
“We are closely collaborating with the microscopists and those developing the microscopes, and if we cannot use the best dye in terms of brightness and stability, then we will move on to the other dye, and the microscopes will have to be adjusted,” said Fischle.
STED microscopy is a “fantastic” technology for getting deep inside cellular structures, said Fischle. However, it currently comes with the drawback of not being able to be used with all kinds of dyes. Once that hurdle is overcome, “I am sure that STED microscopy will result in some really interesting new insights,” he said. In terms of overall cell imaging, live-cell imaging at a very high resolution has just been started, with papers coming out six months ago or so.
“I believe there will be big applications for STED microscopy,” Fischle said. He also said that he feels if one finds a way to link up live-cell imaging to high-resolution technology for microscopy, it will be very interesting to basic scientists. “I do not know how fast it will be adopted by industry; but for probing intercellular structures, it will be the way to go.”  

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