Maybe this is an excuse to show off these incredible images created with the Leica TCS SP8 MP, but why not take a look back at where this technology came from while we’re at it?
What we’re seeing here is a 3D image with optical sectioning using a multiphoton microscope. The phenomenon behind this technology is called two-photon absorption (TPA). Essentially, two photons are absorbed at the exact same time in order to excite a molecule from one state to a higher energy electronic state. Thinking back to high school chemistry, light is given off once that molecule decays back to a lower state.
Maria Goeppert-Mayer, the German-born American theoretical physicist, first predicted this process in her doctoral dissertation in 1931. It wasn’t until 30 years later, with the invention of the laser, that experimental verification for TPA was possible.
TPA was used as a spectroscopic tool until the 1980s. Once more developments in the field occurred, different applications were demonstrated. A few of them being photodynamic therapy, optical data storage, and imaging (obviously).
Watt W. Webb is best known for suggesting using TPA for imaging and microscopy. In 1990 he co-invented mulitphoton microscopy along with Winfried Denk and Jim Strickler. Earlier in his career, Webb pioneered techniques in fluorescent correlation spectroscopy (FCS). The combination of TPA and FCS resulted in high resolution, high signal-to-noise images.
One of the biggest advantages to multiphoton microscopes is the use of long wavelength, low energy excitation lasers. This is less damaging to live cells and introduces fewer toxic effects. This unique attribute is responsible for the in vivo microscopy you’re seeing in these images. Developments in medical endoscopy are being explored since the potential for in vivo, in situ real-time diagnostics is there.
This concludes our small lesson in multiphoton microscopes. Class dismissed.