Better Resolution in Microscopy

By Jonathon Marioneaux

For centuries, light microscopes have helped biologists understand the inner workings of the cell by using the unique properties light and how it bends when traversing through different mediums.  The first light microscope led to the discovery of cells and ushered in a new era of biology: microbiology. Since that time, new technological advances have helped push the boundaries of cell anatomy to ever smaller structures and better resolution of those structures.  Several types of microscopes include the scanning electron microscope, transmission electron microscope, florescent microscope, bright field microscope, laser capture micro-dissection microscope, multifocal plane microscope, and x-ray microscope; each one with its unique capabilities.

Traditional optical or light microscopes take advantage of a light source to illuminate a specimen from behind allowing the viewer to see the object. However, the greatest limitation is the resolution of a microscope itself.  The resolution is the ability to differentiate between two objects in a viewing plane and is directly dependent on the wavelength used for viewing.  For example, a light microscope can only view objects to a resolution of 1250 times (theoretical limit 0.250 micrometers) whereas the scanning electron microscope has a limit of 1 nanometer.  As mentioned, this difference is based on the type of wavelength used for viewing, and in the optical microscope this is directly dependent on the type of light used; ultraviolet light achieves a better resolution than infrared.  Another problem with light microscopes is the material must be transparent to the wavelength, the best medium is dependent on the sample, however, oil or water are the most common.

Fei Chen and colleagues have shown that light microscopes can now achieve a higher resolution then previously thought due to a new technique called Expansion Microscopy.  This technique takes advantage of polyelectrolyte gels hydrolyzing in the presence of water homogenized throughout a specimen.  In their work, the researchers showed that a fixed permeabilized brain tissue can be infused with polyelectrolyte gel triggered by an ammonium persulfate accelerator, tetramethylethylenediamine accelerator and digested with tissue-polymer composite protease and can swell 4.5 times without distortion. As the polyelectrolyte gel is mostly water optical microscopes can resolve cellular structures.  The researchers showed that structures such as microtubules, dendrites, and mossy fiber boutons in the dentate were observable in high resolution.

To further increase the resolution of the new technique, the researchers attached fluorescently labeled proteins to the gel matrix and infused the cell before hydrolyzing it.  As the radical hydrolyzing occurred the florescent tags were bound in the matrix and allowed specific regions of the cell to be analyzed in more detail and higher resolution then without the tags.  The tags were created using a specific oligonucleotide sequence on a protein corresponding to an antibody which was then bound to either green florescent protein or yellow florescent protein.  The florescent antibodies also allow for macrostructures to be seen in more refined structural detail then previously possible.

This new technique is still in its infancy and will take advantage of further advancements in both the physical expansion of polyelectrolyte gel and diffraction-limited microscopes.  However, this new technology will allow light microscopes to see smaller cellular structures in finer detail then previously possible in fixed cell microscopy.  Microscope technology continues to advance far beyond the simple refraction lens used to discover the cell.

Image Credit: Erwin94


Chen, F., Tillberg, P., & Boyden, E. (2015). Expansion Microscopy. Science, 347(6221), 543-548.

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