A brief history of 3-photon excitation: early studies

The first experiments with 3-photon excitation were in the 1960s and 1970s, with solid samples that absorb in the near-IR and emit fluorescence in the UV-blue spectrum (Singh & Bradley, 1964; Catelano & Cingolani, 1979). In the subsequent 2 decades, several groups characterized 3-photon absorption by liquid benzene and by various organic molecules in solution, again with near-IR excitation and UV emission (Davey et al., 1995; He et al., 1995; Gryczynski et al., 1995a; Gryczynski et al., 1996a; Gryczynski et al., 1996b; Grubb et al., 1984; Cable & Albrecht, 1986; Shreve & Albrecht, 1991).

 

Two advances occurred in the 1990s: measurement of the 3-photon excitation cross-sections of several fluorophore molecules used in biological imaging, including DAPI, fura-2 and indo-1 (Gryczynski et al., 1995b; Xu et al., 1996); and the construction of the first laser-scanning 3-photon microscopes, used to acquire images of polystyrene beads doped with an organic fluorophore (Hell et al., 1996) and fixed C. elegans embryos stained with DAPI, fluorescein and Texas red (Wokosin et al., 1996).

1500 and 1700 nm 3-photon excitation

Early studies of 3-photon excitation were performed mostly with Nd:YAG and Ti:sapphire lasers. Limited to <~1.1 µm and with broad (>100 fs) pulses, these lasers systems were rarely capable of efficient 3-photon excitation of fluorophores commonly used for biological imaging, such as green and red fluorescent proteins. More widespread adoption of 3-photon excitation for biological imaging awaited laser systems with briefer pulses and greater peak power at 1-2 µm.

3-photon excitation in the 1-2 µm range was first achieved with an optical parametric oscillator (OPO), which converts incoming illumination into two output beams of lower frequency. Converting a 1064 nm pump into 1500 nm light with an OPO, Norris et al. (2012) demonstrated 3-photon autofluorescence imaging in plant cells.

<100 fs pulses and high peak power was achieved at ~1700 nm in 2013. Horton et al. (2013) employed a 1550 nm fiber laser and soliton-shifted the output wavelength to 1675 nm with a photonic crystal rod, enabling excitation of Texas red and the red fluorescent protein DsRed tdimer2(12) >1 mm deep in the mouse brain. A similar source was employed to excite mCherry and jRCaMP1b in Drosophila (Tao et al., 2017).​

Commercial laser systems

Biological imaging is dominated by green fluorescent proteins that are 3-photon excitable at 1300 nm. A different technology was required for 1300 nm illumination and the solution was the optical parametric amplifier (OPA). Most commercially-available 3-photon laser sources are two-box systems that combine a pump laser at ~1040 nm and an OPA that's tunable across the 1-2 µm range, suitable for 3-photon excitation of fluorophores at ~1300 nm or 1700 nm.

 

Pump-OPA systems are the laser sources in most current 3-photon experiments and their commercial availability has permitted the widespread adoption of 3-photon excitation for imaging into biological tissues.