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Multiphoton microscopy is a fluorescence imaging technique, and is considered an optimal technique for the imaging of living, intact biological tissues.
MPM is also referred to as two-photon excitation microscopy, two-photon laser scanning microscopy, or nonlinear excitation microscopy and is regarded as an alternative to confocal microscopy.
Fluorescence microscopy is a technique that employs fluorescent tagging to illuminate specific parts of a biological sample rather than using an external light source. The fluorescent tags are then “activated” using electromagnetic radiation, illuminating thick specimens like organs and proteins. This allows image detection and magnification to occur, ultimately revealing structure and function.
However, while it is a powerful technique, fluorescent imaging has drawbacks. Background fluorescent light can negatively affect the image when imaging relatively thick samples. Suppose you only want to observe something on the surface of a thick specimen. In that case, the background fluorescent light from the thickness of the material often drowns out the fluorescent light from the material’s surface.
Confocal microscopy is notorious for causing photobleaching and photodamage throughout the specimen. It collects the fluorescence signal only from the focal plane, which causes the sample to absorb the remaining excitation light, creating significant problems in live samples.
Multiphoton microscopy solves this problem by using two photons as the excitation light, rather than just one photon, and by eliminating the contamination of the fluorescence signal by the excitation light. The photons used in MPM also have longer wavelengths, which have lower energy and penetrate further into the sample. This results in high-resolution, in vivo, 3D images of living cells and biological tissue, and reduces photobleaching and photo-toxicity.
MPM has applications in several fields, from biology, physiology, embryology, neurobiology, neuroscience, and cancer research to tissue engineering and dermatology.
Just like wide-field or confocal microscopy, MPM is based on fluorophore excitation. This excitation results in light emission, which can then be observed and analyzed.
Typically, a fluorophore is used to dye the sample, which becomes excited when it absorbs a single photon of a specific wavelength. However, in the case of MPM, two or three photons of longer wavelengths are focused at the sample area that’s been dyed through an objective lens, resulting in a specific area of the sample fluorescing.
Unlike traditional methods, which employ one photon, the photons have to strike the molecule within femtoseconds of each other. When they strike the molecule simultaneously, they excite that sample’s electrons to a higher, less stable state. This energy difference between the normal state and excited state is equal to the sum of the energies of the two photons used to excite it.
Due to the need for precision, the focused laser used in MPM must function at very high pulse rates. These pulsed lasers are often called femtosecond lasers. The absorption of two photons during the fluorophore’s excitation, along with the tightly focused laser, dramatically increases the probability of fluorescent emission.
This effectively restricts excitation fluorescence to a tiny focal point in the sample, resulting in increased rejection of out-of-focus light. The fluorescence produced is collected by a highly-sensitive detector, such as a photomultiplier tube.
This localized excitation and fluorescence are one of the reasons that multi-photon imaging is such a powerful tool. In addition, the light source used is typically infrared or near-infrared, which reduces scattering, further increasing MPM’s capabilities to generate high-resolution images.Another benefit of using two-photon microscopy is that it allows optical sectioning at greater depths. It can do this due to the extreme localization of the lasers used, which allows for deep tissue penetration.
Three-photon excitation microscopy can be employed to reduce the out-of-focus fluorescent light further and decrease the amount of tissue scattering. Three-photon and two-photon excitation microscopy have very similar procedures.
The major difference is that the molecule must absorb three photons before it fluoresces rather than just two. This also means that the excitation wavelengths of the photons used in three-photon microscopy are much longer (about 1300 nanometers) than those used in two-photon excitation microscopy (about 910 nanometers).
Four-photon excitation is currently being looked into; however, no significant practical biological use has been found yet.
When fluorophores and fluorescence are used to image an object, phototoxicity and photobleaching become issues that need to be considered.
Second-harmonic imaging microscopy (SHIM) is a nonlinear optical technique that exploits the second-harmonic generation effect. This means that molecules are not excited, thus negating the issues of so phototoxicity or photobleaching.
SHIM can look at cells and living tissue’s structure by creating contrast by examining variations in a material’s ability to create second-harmonic light. Second-harmonic light occurs when two photons of the same frequency strike a material, combine, and generate a new photon of twice the energy of the initial photons. SHIM can be used to create 3D images of samples even if they are relatively thick.
Additionally, fluorophore tagging is often not required because organic structures produce second-harmonic generation signals naturally. This means that samples can be observed in an unaltered state.
Two-photon excitation microscopy is based on the reaction of two-photon absorption. This was first predicted in 1931 by Dr. Maria Goeppert-Mayer while at the University of Göttingen in her doctoral thesis.
It would, however, take thirty more years and the invention of the laser before her theory could be verified. There were three Nobel Prize winners on her doctoral committee: her advisor Max Born, James Franck, and Adolf Otto Reinhold Windaus.
In recognition of creating two-photon fluorescence light microscopy, the unit for a two-photon absorption cross-section is named after her, the Goeppert Mayer. She would continue her scientific career in the United States as a teacher at Sarah Lawrence College in the 1940s. While there, she joined the Manhattan project and worked with yet another Nobel Prize winner Harold Urey.
After the war ended, she found herself in Chicago as an associate professor in the Physics Department of the Institute for Nuclear Studies. She was recognized for her intellect and eventually was offered another position at the Argonne National Laboratory, to which she replied, “I don’t know anything about nuclear physics.”
During her time in Chicago, she penned her theory on the nuclear shell model. In it, she mathematically explained why specific configurations in an atom’s nuclei result in a stable structure. She would later be awarded the Nobel Prize in physics, becoming the second woman to do so after Marie Curie.
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