An Extreme Close Up: Making Nanoscale Visualization of Cell Structures Accessible

When we speak of nanometers, we are talking about one-billionths of a meter––one-hundred-thousandths the size of a single hair follicle. With these proportions in mind, it becomes even more fascinating to think that there are nanostructures in cells that range from five-to-ten nanometers in length. 

But despite their almost inconceivably small size, it is possible to see cell nanostructures using a method called correlative light/electron microscopy, or CLEM. However, although CLEM combines both fluorescence and electron microscopy to view nanostructures at high resolution and specificity, this method has a significant drawback: it requires specialized instruments and days of continuous data acquisition to obtain a single image. 

To circumvent these difficulties, researchers Ons M’Saad and Joerg Bewersdorf at the Yale Bewersdorf Lab have developed a new method—aptly named Pan-ExM—that produces similar results at a significantly faster pace, requiring only a standard light microscope.

In this new method, a sample can be enlarged twenty-fold in every direction through the use of two hydrogels: three-dimensional networks of hydrophilic polymers that, when introduced to water, swell in size whilst maintaining the structure of the sample. 

Pan-ExM achieves this by first embedding a sample into a hydrogel, which is initially expanded four-to-five-fold by adding water. The sample is then inserted into another hydrogel and treated with chemicals to help maintain its expanded state and prepare for further expansion . This composite hydrogel is embedded into a third hydrogel before being enlarged another four-to-five-fold. As a result of the two compounded expansions, the intracellular space is made wider twenty-fold in all directions—“almost an 8000-fold enlargement in volume,” M’Saad said. This makes an average distance of three nanometers between proteins become fifty nanometers after expansion, causing substantial molecular decrowding.  

Pan-ExM was also found to consistently resolve structures that were only thirty-to-one-hundred nanometers apart pre-expansion. When combined with a high-end confocal microscope, Pan-ExM can reach resolutions in the twenty-to-thirty nanometers range. “The size of molecules or proteins are around three-to-five nanometers… if you have this level of resolution in biological imaging, you’re good,” M’Saad explained. In addition, the sample preparation for the Pan-ExM process can be completed in just two days, with subsequent imaging taking less than a minute. 

Another key benefit of Pan-ExM is that it is not limited by label-size. In a process called labeling, a dye can be added to better visualize specific structures of a sample clearly.  “The size of dyes are actually pretty bulky—they are on the same order of size as the molecules themselves,” M’Saad said. As a result, the dye can cover up the nanostructures in the cell. However, due to the molecular decrowding by Pan-ExM, when labeled post-expansion, the dyes are unable to cover up any structures.

Pan-ExM has the potential to revolutionize light microscopic imaging of nanostructures as a whole. “These [traditional] techniques are extremely complicated, and people do it because there is no other option,” Bewersdorf added. “The method developed is doing ‘all that’ with just a light microscope.” Through its simpler and more efficacious approach, Pan-ExM provides an exciting new alternative for the expansion of nanostructures.