Three-photon Image-scanning-microscopy Enabling Deep Super-resolution Imaging

Laser scanning multiphoton microscopy allows imaging deep in samples, as the nonlinearity causes the excitation to be strongly confined in the axial direction, that is, multiphoton microscopy comes with intrinsic sectioning of the signal. However, the resolution in laser scanning microscopy is given by how tightly one can focus the beam, which is the so-called Abbe limit, given by the wavelength <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$\lambda$</tex>, divided by twice the numerical aperture NA of the focusing element (objective). Thus, compared to single photon excited microscopy, the two- and three-photon excited microscopy the resolution worsens by a factor <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$\sqrt{2}$</tex> or <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$\sqrt{3}$</tex> respectively. To overcome the Abbe resolution limit, several approaches have been developed for single photon excited microscopy [1]–[5]. Of those methods, structured illumination microscopy [2] and image scanning microscopy (ISM) [4] can be applied with virtually any fluorescent marker, which makes them the most widely applicable super-resolution methods. ISM is implemented with laser scanning confocal microscopy, and recently it was demonstrated to be applicable to two-photon microscopy [6], achieving deep imaging with a resolution better than what is achieved with confocal microscopy.