A new expansion microscopy (ExM) technique from MIT makes it possible to use a conventional light microscope to generate high-resolution images at the nanoscale, by expanding specimens 20-fold before imaging them.
Historically, nanoscale structures in cells and tissues have been imaged with high-powered, expensive, superresolution microscopes. The new ExM protocol, which achieves 20-fold expansion in just one step, provides a simple, inexpensive method that can be used by most biology labs to perform imaging at a resolution of about 20 nm.
“What this new technique allows you to do is see things that you couldn’t normally see with standard microscopes,” professor Laura Kiessling said. “It drives down the cost of imaging because you can see nanoscale things without the need for a specialized facility.”
The original version of the ExM technique, developed by professor Edward Boyden and his team in 2015, expanded tissue about 4-fold and provided images with a resolution of around 70 nm. In 2017, Boyden’s lab modified the process to include a second expansion step, achieving an overall 20-fold expansion.
“We’ve developed several 20-fold expansion technologies in the past, but they require multiple expansion steps,” Boyden said. “If you could do that amount of expansion in a single step, that could simplify things quite a bit.”
The new method reaches the same level of performance possible with iterative expansion methods, but with the simplicity of a single-shot protocol.
To implement ExM, the researchers embed the tissue specimen in an absorbent polymer and added water, creating a hydrogel that expands the polymer and pulls the biomolecules in the specimen apart. For one-step, 20-fold expansion, the researchers use gel that is extremely absorbent and mechanically stable to ensure that the gel does not fall apart when the specimen is expanded by 20x.
To further stabilize the gel and enhance its reproducibility, the researchers remove oxygen from the polymer solution prior to gelation, preventing side reactions that could interfere with crosslinking. Unlike previous expansion gels that require another molecule to be added to form crosslinks between the polymer strands, the gel used for the single-shot, 20-fold ExM technique forms crosslinks spontaneously.
Once the gel is formed, select bonds in the proteins that hold the tissue together are broken and water is added to make the gel expand. After the gel expands, target proteins in the tissue can be labeled and imaged. The new technique supports post-expansion staining for brain tissue to facilitate biomolecular labeling.
“This approach may require more sample preparation compared to other superresolution techniques, but it’s much simpler when it comes to the actual imaging process, especially for 3D imaging,” researcher Tay Won Shin said.
In one round of expansion, the new ExM technique, which the team calls 20ExM, enabled the researchers to image hollow microtubule structures in cultured cells and synaptic nanocolumns in the mouse somatosensory cortex on a conventional confocal microscope. The team could also visualize mitochondria and the organization of individual nuclear pore complexes in the cells.
The new ExM technique could be used for a variety of experiments where high resolution and single-step simplicity are desired. The researchers are currently using the technique to image glycans — carbohydrates, found on the surface of a cell that help control how the cell interacts with its environment.
20ExM could also be used to image tumor cells, providing insight into how proteins are organized within these cells. In principle, the new ExM technique could be used to simplify or enhance the resolution of other expansion-based technologies, such as in situ RNA detection and sequencing and genome imaging.
The single-shot, 20-fold expansion microscopy method provides a robust, simple, affordable solution to nanoscale-resolution imaging of preserved cells and tissues using conventional microscopes. The researchers believe that any biology lab could use the technique at a low cost, because it relies on standard, off-the-shelf chemicals and equipment that most labs already have or can easily access.
“Our hope is that with this new technology, any conventional biology lab can use this protocol with their existing microscopes, allowing them to approach resolution that can only be achieved with very specialized and costly state-of-the-art microscopes,” researcher Shiwei Wang said.
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Historically, nanoscale structures in cells and tissues have been imaged with high-powered, expensive, superresolution microscopes. The new ExM protocol, which achieves 20-fold expansion in just one step, provides a simple, inexpensive method that can be used by most biology labs to perform imaging at a resolution of about 20 nm.
“What this new technique allows you to do is see things that you couldn’t normally see with standard microscopes,” professor Laura Kiessling said. “It drives down the cost of imaging because you can see nanoscale things without the need for a specialized facility.”
The original version of the ExM technique, developed by professor Edward Boyden and his team in 2015, expanded tissue about 4-fold and provided images with a resolution of around 70 nm. In 2017, Boyden’s lab modified the process to include a second expansion step, achieving an overall 20-fold expansion.
“We’ve developed several 20-fold expansion technologies in the past, but they require multiple expansion steps,” Boyden said. “If you could do that amount of expansion in a single step, that could simplify things quite a bit.”
The new method reaches the same level of performance possible with iterative expansion methods, but with the simplicity of a single-shot protocol.
To implement ExM, the researchers embed the tissue specimen in an absorbent polymer and added water, creating a hydrogel that expands the polymer and pulls the biomolecules in the specimen apart. For one-step, 20-fold expansion, the researchers use gel that is extremely absorbent and mechanically stable to ensure that the gel does not fall apart when the specimen is expanded by 20x.
To further stabilize the gel and enhance its reproducibility, the researchers remove oxygen from the polymer solution prior to gelation, preventing side reactions that could interfere with crosslinking. Unlike previous expansion gels that require another molecule to be added to form crosslinks between the polymer strands, the gel used for the single-shot, 20-fold ExM technique forms crosslinks spontaneously.
Once the gel is formed, select bonds in the proteins that hold the tissue together are broken and water is added to make the gel expand. After the gel expands, target proteins in the tissue can be labeled and imaged. The new technique supports post-expansion staining for brain tissue to facilitate biomolecular labeling.
“This approach may require more sample preparation compared to other superresolution techniques, but it’s much simpler when it comes to the actual imaging process, especially for 3D imaging,” researcher Tay Won Shin said.
In one round of expansion, the new ExM technique, which the team calls 20ExM, enabled the researchers to image hollow microtubule structures in cultured cells and synaptic nanocolumns in the mouse somatosensory cortex on a conventional confocal microscope. The team could also visualize mitochondria and the organization of individual nuclear pore complexes in the cells.
The new ExM technique could be used for a variety of experiments where high resolution and single-step simplicity are desired. The researchers are currently using the technique to image glycans — carbohydrates, found on the surface of a cell that help control how the cell interacts with its environment.
20ExM could also be used to image tumor cells, providing insight into how proteins are organized within these cells. In principle, the new ExM technique could be used to simplify or enhance the resolution of other expansion-based technologies, such as in situ RNA detection and sequencing and genome imaging.
The single-shot, 20-fold expansion microscopy method provides a robust, simple, affordable solution to nanoscale-resolution imaging of preserved cells and tissues using conventional microscopes. The researchers believe that any biology lab could use the technique at a low cost, because it relies on standard, off-the-shelf chemicals and equipment that most labs already have or can easily access.
“Our hope is that with this new technology, any conventional biology lab can use this protocol with their existing microscopes, allowing them to approach resolution that can only be achieved with very specialized and costly state-of-the-art microscopes,” researcher Shiwei Wang said.
Bio Photonics Research Award
Visit: biophotonicsresearch.com
Nominate Now: https://biophotonicsresearch.com/award-nomination/?ecategory=Awards&rcategory=Awardee
#MeatAnalysis #FluorescenceTech #FoodQuality #FoodSafety #SpectroscopyInFood #MeatAuthentication #RapidDetection #FoodScience #MeatFreshness #MolecularDetection #FoodIndustryInnovation #NonDestructiveTesting #FoodMonitoring #SpectroscopyApplications #QualityControl #AdvancedSpectroscopy #MeatSpoilageDetection #FoodIntegrity #SmartFoodTesting #RealTimeAnalysis #FoodAuthenticity #FoodSafetyInnovation #SpectroscopyResearch #NextGenFoodSafety #InnovativeFoodScience,
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