Non-degenerate two-photon absorption

The near-simultaneous injection of different-energy photons into a specimen poses advantages over the traditional method of same-energy degenerate two photon excitation. These advantages can be explained by the enhanced VSL and, thus, larger absorption cross-section.

Because of the longer VSL, there is a higher likelihood of promotion of an electron to an excited singlet state by the second photon, compared to degenerate two-photon excitation. Instead of increased Rayleigh or Raman scattering taking place from the virtual state, more electrons in a given excitation plane are likely to promote to the excited state, followed by larger rates of emission to the ground state. This larger amount of emission events translates to higher fluorescence intensity in a sample at a given spot, increasing signal-to-noise ratio and decreasing the effects of out-of-focus excitation.^[9]

Even though same-energy two-photon excitation microscopy provides a better depth of penetration than confocal microscopy, it is still confined to ~1mm depths, which is approximately the transport mean free path of biological tissue.^[8] Due to the increased VSL, absorption cross-section, and thus fluorescent intensity, ND-TPA provides a larger depth of penetration than degenerate two-photon microscopy, allowing for fluorescent emission deeper in a sample. At every depth location in a sample, ND-TPA provides brighter fluorescence than traditional two-photon absorption, thus allowing for visualizable fluorescence at depths impossible for traditional two-photon microscopy. Due to the high scattering nature of higher energy photons, and the ability of beam fluxes to be combined multiplicatively,^[8] beam fluxes can simply be tuned so that lower-energy photons are administered at a higher fluence rate, thus accounting for the loss in higher-energy photons at larger depths within a sample.

The combination of two photons of different wavelengths allows for a larger absorption cross-section, thereby accommodating for larger ranges of excitation than degenerate two-photon microscopy. Traditional degenerate two-photon microscopy is confined to photons with energies that, when doubled, account for the energy difference between ground and excited electron states, however, in biological tissue, this confines degenerate two-photon excitation to the near-infrared wavelength optical window, due to enhanced depth of penetration and energy requirements. With ND-TPA, virtually any wavelength may be used, so long as the second photon accounts for the remaining energy difference between the virtual state and the excited singlet state.^[10] This combined two-photon excitation has been demonstrated for fluorophores requiring equivalent one-photon excitation wavelengths of 266nm ^[11] and 1013nm.^[8]

When combined with degenerate two-photon absorption in microscopy settings, ND-TPA can provide better spatial resolution and axial sectioning. Because of the requirement for laser beams to be approximately synchronized in ND-TPA, a desynchronization event can "turn off" entire fluorophores, while the degenerately-excited fluorophores remain "on".^[12] This allows for the overlay of degenerate and non-degenerate two-photon microscopy images to pinpoint locations of specific structures like genes or sub cellular components. The use of further optical or reconstructive additions, like a shaded ring filter or beam-shaping techniques, enables further resolution optimization.^[13]

To implement non-degenerate two photon excitation microscopy, two photon pulses of differing energies must be synchronized to interact with a specimen at the sample plane near-simultaneously. Due to the enhanced absorption cross section and VSL, more time is possible for excitation to occur, and thus perfect synchronization is unnecessary. However, close synchronization of pulses, within ~10ns is preferred. For this and other logistical reasons, a single Ti:Sapphire femtosecond laser is used to create a single laser pulse train. After passing through a half wave plate to rotate the plane of polarization, the laser beam passes through a polarization beam splitter and is separated into two beams. One beam passes into an optical parametric oscillator (OPO), which splits the incoming high frequency beam into lower frequency components, and the resulting beam is of longer wavelength and lower energy than the incoming beam; this beam also passes through an automated defocuser. The second beam is redirected through a delay line, with mirrors optimized to near-perfectly synchronize the higher energy laser pulse with the lower energy output of the OPO. Both beams pass through half wave plates once again before meeting at a dichroic mirror which allows preselected low wavelength laser beams to pass through, while high wavelength beams are reflected orthogonally to meet and mix with the lower-wavelength beam. This mixed beam, consisting of two different-wavelength beams, passes through another dichroic mirror before being focused by an objective onto the specimen. The resulting incoherent fluorescence is partially redirected through the objective and reflected off the second dichroic mirror into another dichroic mirror, which again reflects the beam into a band-pass filter before it passes into a photomultiplier tube (PMT). This signal is then imaged.