(来源:MIT News)
Traditional imaging of cellular nanostructures often relies on expensive and high-performance super-resolution microscopy. As an alternative, researchers at MIT have developed a method to achieve imaging by expanding tissues, which allows them to achieve nanoscale resolution using a common light microscope.
In the latest version of the technology, researchers can scale their tissues up to 20x in one step. This simple and inexpensive method will make nanoscale imaging accessible to almost any biological laboratory.
"This really democratized imaging," said Laura Kiessling, Novartis Professor of Chemistry at MIT and a member of the Broad Institute at MIT and Harvard University, as well as the MIT Koch Cancer Institute. "In the past, if you wanted to see high-resolution details, you needed very expensive microscopes. Now, this new technology allows you to observe tiny structures that are not normally visible with a normal microscope. It dramatically reduces the cost of imaging because you don't need to rely on expensive professional equipment to see nanoscale details. ”
The resolution achieved with this technique is about 20 nanometers, which is enough for scientists to observe organelles and protein clusters within cells.
"The 20-fold expansion has reached the range of biomolecules. The building blocks of life are things at the nanoscale: biomolecules, genes, and gene products. said Edward Boyden, the Y. Eva Tan Professor of Neurotechnology at MIT. He is also a professor of bioengineering, media arts and sciences, and brain and cognitive science, a fellow of the Howard Hughes Medical Institute, and a member of the MIT McGovern Brain Institute and the Koch Cancer Institute.
Boyden 和 Kiessling 是这项新研究的资深作者,该研究今天发表在《Nature Methods》杂志上。 MIT 研究生 Shiwei Wang 和 Tay Won Shin(2023 年获得博士学位)是该论文的主要作者。
One-time expansion
Extended microscopy was first invented in Boyden's lab in 2015. This technique embeds tissue into an absorbent polymer and breaks down proteins that normally hold tissues tightly together. When water is added, the gel swells and separates the biomolecules from each other.
The initial version of the technology can expand the tissue by a factor of about four, obtaining an image with a resolution of about 70 nanometers. In 2017, Boyden's lab improved the process by adding a second scaling step to achieve an overall 20x scaling, resulting in higher resolution, but the process was also more complex.
"We've had several 20x scaling techniques in the past, but they all required multiple scaling steps." "Being able to scale to this extent in a single scale would greatly simplify the process." ”
With a 20x extension, researchers can reduce the resolution to about 20 nanometers using a common light microscope, enabling the visualization of cellular structures such as microtubules, mitochondria, and protein clusters.
In the new study, the research team tried to achieve a 20x scale with a single step. To do this, they needed to find a gel that was both highly absorbent and mechanically stable, ensuring that it would not break when it expanded 20-fold.
They used a gel made of N,N-dimethylacrylamide (DMAA) and sodium acrylate. This gel is different from the gels that require the addition of cross-linked molecules in traditional expansion techniques, and can form cross-links on their own with stronger mechanical properties. Previously, these gels had been used for extended microscopy, but only by a factor of about 10. The MIT team optimized the gel formulation and polymerization process to make it more robust and able to scale up to 20x.
To further enhance the stability and reproducibility of the gel, the researchers removed oxygen from the polymer solution prior to gel formation to prevent interference from the crosslinking reaction. This step is done by introducing nitrogen into the polymer solution, replacing most of the oxygen in the system.
After the gel is formed, the protein bonds inside the tissue are broken and water is added to swell. Once the expansion is complete, the protein of interest can be labeled and imaged.
"This method may require more sample preparation than other super-resolution techniques, but it is much simpler in the actual imaging process, especially when imaging in 3D." "We've documented the steps in detail in our paper to make it easy for readers to use." ”
Imaging of tiny structures
Using this technology, the researchers were able to image tiny structures within many brain cells, including those known as synaptic nanopillars. These protein clusters are arranged at the synapses of neurons in a specific way, allowing neurons to communicate by secreting neurotransmitters such as dopamine.
In the study of cancer cells, researchers have also observed microtubules – these hollow tubular structures that help cells maintain their form and play a key role in cell division. They also successfully imaged mitochondria, the cell's energy generators, and even saw the tissue structure of a single nuclear pore complex (a cluster of proteins that control access to the nucleus).
Wang is currently using this technique to image carbohydrates (sugar chains) on the cell surface, molecules that help cells interact with their environment. This method can also be used to image tumor cells, allowing scientists to look at the protein organization structures inside these cells in an unprecedented way.
The research team wanted it to be available at a lower cost to any biological lab, as the technique uses standard off-the-shelf chemicals and common equipment such as confocal microscopes and glove boxes.
"We hope that with this new technology, any biology lab will be able to use existing microscopes and apply this protocol to achieve a resolution that can only be achieved with very expensive and specialized top-of-the-line microscopes." Wang said.
The study was partially funded by the National Institutes of Health of United States, the MIT President's Graduate Fellowship, the United States National Science Foundation Graduate Fellowship, Open Philanthropy, Good Ventures, Howard Hughes Medical Institute, Lisa Yang, Ashar Aziz, and the European Research Council.
Original link:
https://news.mit.edu/2024/new-method-makes-high-resolution-imaging-more-accessible-1011