Tissue heating, and the need to avoid thermal damage to the tissue, often limits imaging depth and speed during 3-photon excitation.
Damage to biological tissue can occur via non-linear and via linear absorption.
The damage threshold for non-linear absorption depends on the point spread function and pulse characteristics, including duration and shape, and may differ between microscopes. In one experiment, likely representative of many others, the damage threshold was ~2 nJ pulse energy with 40 fs pulses (Yildirim et al., 2019b). For a 1 MHz repetition rate, 2 nJ pulse energy corresponds to 2 mW of illumination at the focal plane. Only a minority of incident photons converge at the focus when imaging deep into biological tissue, necessitating >2 mW illumination at the tissue surface to drive significant 3-photon excitation in the focal plane. Illumination intensities of ~30 mW are commonly used to drive 3-photon excitation at 1300 nm ~800 µm below the surface of mouse brain without causing non-linear photodamage, even during prolonged imaging.
The dominant mechanism of linear absorption in many biological tissues is absorption by water molecules, resulting in tissue heating. Linear absorption is largely independent of point spread function and pulse characteristics and is likely similar across microscopes. Heating-related photodamage occurs with >250 mW of prolonged illumination at 800-1040 nm or >120 mW at 1320 nm (Podgorski & Ranganathan, 2016; Wang et al., 2020a). This ~2-fold difference in damage threshold is conststent with the ~2-fold difference in the extinction coefficient for absorption by water at 920 and 1320 nm (Curcio & Petty, 1951; Hale & Querry, 1973; Bertie & Lan, 1996). Likely heating, rather than non-linear photodamage or other factors, determines the maximum depth of 3-photon excitation in many biological imaging experiments.
A key advantage of 3- over 2-photon excitation is that 3-photon excitation permits deeper imaging into biological tissue.
With non-linear excitation, fluorescence is generated preferentially in the focal plane, but measurable fluorescence can also arise from outside the focal plane (Ying et al., 1999; Theer et al., 2003). When imaging deep in biological tissue, high illumination intensities are often necessary to generate sufficient intensity at the focus and these high intensities can drive 2-photon excitation of fluorophores above and below the focal plane (Theer & Denk, 2006; Takasaki et al., 2020). Out-of-focus fluorescence reduces image contrast and often limits the depth at which images can be formed with 2-photon excitation (Theer & Denk, 2006; Takasaki et al., 2020).
In tissue with a uniform spatial distribution of fluorophore molecules, the depth limit of 2-photon excitation occurs where in- and out-of-focus fluorescence are equal, at ~3 scattering length constants, 400-600 µm in cortical grey matter (Theer & Denk, 2006; Takasaki et al., 2020). When the focal plane is shallower than the depth limit, fluorescence is mostly from the focal plane. As the focus is shifted deeper into tissue, illumination energy must be increased to maintain intensity at the focus, which maintains constant excitation probability in the focus but drives greater out-of-focus fluorescence. Beyond the depth limit, most fluorescence originates from outside the focal plane. Since the depth limit is a function of out-of-focus fluorescence, the depth limit will be greater in tissues with few fluorophore molecules outside the focal plane.
3-photon excitation generates less out-of-focus fluorescence than 2-photon excitation, due to the steeper relationship between 3-photon excitation and illumination intensity (Takasaki et al., 2020). The result is an extended depth limit and deeper imaging (Horton et al., 2013; Kobat et al., 2009; Kobat et al., 2011; Ouzounov et al., 2017; Yildirim et al., 2019b; Takasaki et al., 2020). The depth limit of 3-photon excitation commonly results from tissue heating, not out-of-focus fluorescence. (Takasaki et al., 2020) and 3-photon imaging in mouse brain is possible to ≥1.4 mm below the tissue surface at 1300 nm (Takasaki et al., 2020) and ≥1.6 mm at 1700 nm (Kobat et al., 2009; Kobat et al., 2011).