Bessel beam side lobe suppression via non-degenerate two-photon excitation

Bessel beams are commonly used in two-photon microscopy to extend the depth of field and thereby achieve functional volumetric imaging of the living brain. In practice, this approach suffers from background signals and limited lateral resolution due to the Bessel beam’s strong side lobes. We introduce and demonstrate a new approach to side lobe suppression based on non-degenerate two-photon excitation, in which dual wavelength illumination produces an imaging point-spread function that is the product of the two coaxial Bessel beams. This technique can reduce the main side lobe intensity of a Bessel beam by 50% or more. We illustrate the approach conceptually with an analytical paraxial model and use detailed physical simulation to show that the approach is effective in the presence of the symmetry-breaking aberrations that amplify side lobes in high NA systems. We experimentally demonstrated the technique using a refractive axicon and the pump and tunable beams of a femtosecond laser. This work establishes non-degenerate two-photon excitation as a practical and broadly applicable strategy for improving point spread-function quality in high-resolution volumetric microscopy.