FIGURE 1.

Principle of DA-TPEF. When focusing a laser beam into thick tissue, the laser power becomes largely depleted by scattering before it attains the beam focus. TPEF background can then arise from out-of-focus ballistic excitation, particularly near the sample surface, or from “snakelike” scattered excitation near the beam focus, both of which can produce background fluorescence that is nonnegligible compared to the in-focus signal fluorescence (a). The introduction of extraneous aberrations in the illumination pupil leads to a spreading of the ballistic excitation profile that is more pronounced near the beam focus than away from the beam focus, thereby preferentially quenching the in-focus TPEF signal while leaving the out-of-focus TPEF background relatively unchanged (b). The subtraction of a TPEF image with extraneous aberrations (configuration b) from an image without extraneous aberrations (configuration a) then leads to enhanced out-of-focus TPEF background rejection (c).