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For fluorophores commonly used in biological imaging, such as GFP, calculated 3-photon PSFs are usually slightly larger than calculated 2-photon PSFs.

3-photon excitation employs longer wavelengths than 2-photon excitation, resulting in a larger point spread function (PSF). This effect is partially offset by the cubic relationship between illumination intensity and excitation probability, the net result usually being a 3-photon PSF that's slightly larger than the 2-photon PSF. For 2- and 3-photon excitation of GFP at 910 nm and 1300 nm, the calculated full width half maximum (FWHM) of the central peak of the 3-photon PSF is ~15-20% greater in both transverse and axial dimensions than 2-photon excitation. Transverse FWHM for 3-photon excitation is very approximately ~0.33 lambda / NA.

Calculated PSFs for linear, 2- and 3-photon excitation at 490, 910 and 1300 nm. PSFs were calculated using vectorial diffraction theory (Richards & Wolf, 1959) for NA 0.9 and water immersion with no aberrations. (Code available on the Downloads page.)  Transverse FWHM for linear, 2- and 3-photon excitation 0.31, 0.41 and 0.49 µm. Axial FWHM 1.24, 1.66 and 1.95 µm.


3-photon PSFs measured from test slides and deep in cortical tissue are consistent with PSFs calculated with vectorial diffraction theory. Yildirim et al. (2019b) measured transverse and axial FWHM at 0.45 ± 0.05 μm and 1.9 ± 0.2 μm for 100 nm beads in agarose, similar to calculated values of 0.45 and 1.44 μm (1300 nm, NA 1.02). PSFs changed little when imaging into layers 5 and 6 of mouse visual cortex, indicating that ~1 mm of cortical grey matter adds only mild aberrations (lateral and axial FWHM of 0.5 and 2.5 μm; Yildirim et al., 2019b).

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