Original news release was issued by the Colorado State University, written by A. J. Manning.
Two-photon excitation microscopy was first observed in 1961. Although it was first theorized about back in 1930s, only the invention of laser made it possible. Then it had to wait another thirty years, when in the 1991 it was perfected as living tissue imaging method. Until recently, there have not been major improvements on this method within the field of optical microscopy. Now, an innovative technique was used by researchers from Colorado State University. To better understand this, one has to dive into the recent developments surrounding microscopes.
The last major breakthrough happened in 2014, when the Nobel Prize in Chemistry was awarded to a team responsible for super-resolved fluorescence microscopy. This was an important change as it went past the standing limits of light diffraction – a key measure in microscopy. Diffraction of a wave as a phenomenon can be observed the easiest with a sound coming through, let’s say, a door. With light, it is a bit different, as we do not normally perceive it. We see a shadow and assume the light did not diffract. But it does diffract, as we now know that light also behaves as a wave.
In the field of microscopy, the humanly-imperceivable levels of diffraction can destroy the whole image. That is why the scientists are trying hard to limit it as much as possible. Optical microscope could reach the unprecedented level of resolution using fluorescence instead of more traditional reflection or absorption, as shown by the winners of the 2014 Nobel Prize in Chemistry. The CSU team is using this technology where a specific wavelength is emitted on the object, which either has the needed properties (otherwise a dye is added) and the light returned has a much longer wavelength, limiting the diffraction.
The other method used at CSU is called second-harmonic generation. This means the two photons are destroyed, creating a single photon with a higher frequency. The aforementioned methods are often used with one another, but team from CSU pushed the limits a bit further, making their custom microscope capable of using the two methods simultaneously for creation of a single image. The result is an unprecedented resolution achieved by an optical microscope.
Besides the obvious benefit of higher resolution, the microscope still allows for an imaging on whole living organisms, there is no need for glass slides as with most super-resolution techniques. “If we can do this below the surface of a biological sample such as live tissue, that is the utility of this,” Randy Bartels, professor in the Department of Electrical and Computer Engineering, said. “We can beat the diffraction limit of a canonical two-photon microscope.” The far reaching implication of the CSU’s microscope project a bright future, in which doctors could obtain more biological information with a less complicated procedure.