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New technology developed by popular general researchers creates multicolor molecular movies

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Boston – Many techniques have been developed for the purpose of analyzing biological systems in operation at the molecular level. However, they are often hampered by the lack of visual details that distinguish the various molecules, or destroy cells in the process.

New technologies developed by researchers at Massachusetts General Hospital avoid these problems. This method, called cleavage-promoting fluorophore exchange (SAFE), uses a variety of immunofluorescent tags attached to molecules on living cells and combines unique fast but very mild chemical reactions to remove the tags. increase.

Dr. Jonathan Carlson, MD, co-author of the Center for Systems Biology at Massachusetts General Hospital, said: “By overlaying all the tagged molecules in different colors at the same time, we get live details and see that cells and tissues are moving, developing, and changing their functions.”

Details of the technique are published in Nature biotechnology..

The team’s goal was to develop a tool that would allow us to observe how cells interact and how tissues function. However, when viewed under a microscope, it is difficult to distinguish the molecules that layer the surface of the cell. Because they are essentially unexplained about visual markers.

Many forms of microscopy involve adding color to individual cells or molecules, but current techniques include a rich array of sequentially tagged molecules in samples that remain alive and intact. It lacks the ability to generate.

“This is a technique for seeing flags flying on the surface of cells,” said Hannes Mikula, who is now a member of the Institute of Applied Synthetic Chemistry in Vienna and previously completed a PhD, in collaboration with a new chemical tool. Carlson, who developed it, added. He is a researcher in the lab of Ralph Weissreder, co-author of the Massachusetts General Hospital.

“SAFE allows you to tag dozens of molecules in the same system, not just one or two, with a series of images collected in a short amount of time. In the meantime, a set of molecules and cells. Will survive. “

SAFE uses rapid bioorthogonal chemistry, a chemical reaction that can be performed in a system that does not destroy, interfere with, or degrade living cells or their natural functions, to add and remove immunofluorescent signals from the surface of labeled cells. To do.

Targets can be virtually any molecule on the surface of a cell, such as an important marker for distinguishing one cell type from another or classifying various types of immune cells. ..

In this process, two non-toxic reagents with nanomolar concentrations, customized tetrazine and Trance-Cyclooctene pair. It acts as a chemical scissors that quickly removes the immunofluorescent tag during imaging.

“It’s a much brighter and best tool when it comes to fast chemistry compatible with living systems,” explains Carlson, who explains that the chemicals are also mild, with only a small amount to find, connect, and interact with each other. He added that he didn’t need it.

The team demonstrated that when a fluorescent SAFE-labeled antibody was added, the molecule turned on rapidly and faded as quickly as the fluorescent tag was added and removed. “Chemical scissors allow us to quickly move from color to color, or from image to image, while cells are alive,” says Carlson.

“The secret here is not that you can erase the color, but that you can erase it with extraordinary speed, kindness, and efficiency in a way that the organization doesn’t even realize it’s happening. is.”

The team showed that over 99% of the color signal was removed in less than 30 seconds. SAFE overcomes the limitations of existing techniques that use harsh chemicals that destroy colors and molecules and cells in the process.

In this paper, researchers used SAFE to create a cinematic series of multicolor images showing the migration of immune cells in living mouse liver tissue.

They also create multiplexed 14-color images of live mouse bone marrow, tracking the maturation of bone marrow progenitor cells during neutrophil differentiation over 6 days, in the process “flagging” the cell surface. Was completely replaced.

“SAFE hopes to provide a wide range of useful new capabilities for visualizing complex biological systems over space and time,” says Carlson. “We envision that the dynamics of tumor cells and the immune system provide important new insights into both healthy physiology and disease, especially the internal workings of cancer, which are very interesting areas.”

Additional research authors include lead authors Jina Ko (now the University of Pennsylvania), Martin Wilkovitsch, Juhyun Oh, Rainer H Kohler, Evangelia Bolli, Mikael J Pittet, Claudio Vinegoni, and David B Sykes.

This work was partially supported by grants from the CSB Development Fund and the National Institutes of Health.

About Massachusetts General Hospital

Founded in 1811, Massachusetts General Hospital is Harvard Medical School’s first and largest teaching hospital. The Mass General Research Institute runs one of the largest hospital-based research programs in the country, with annual research activities of over $ 1 billion and more than 9,500 researchers working in more than 30 laboratories, centers and departments. It has been. In August 2021, Massachusetts General Hospital US News & World Report List of “America’s Best Hospitals”.