Two-photon excitation fluorescence microscopy with a high depth of field using an axicon

In conventional two-photon excitation fluorescence microscopy, the numerical aperture of the objective determines the lateral resolution and the depth of field. In some situations, as with functional imaging of dynamic events distributed in live biological tissue, an improved temporal resolution is needed; as a consequence, it is imperative to use optics with a high depth of field to simultaneously image objects at different axial positions. With a conventional microscope objective, increasing the depth of field is achieved at the expense of lateral resolution. To overcome this limitation, we have incorporated an axicon in a two-photon excitation fluorescence microscopy system; measurements have shown that an axicon provides a depth of field in excess of a millimeter, while the lateral resolution is maintained at the micrometer scale. Thus axicon-based two-photon microscopy has been shown to yield a high-resolution projection image of a sample with a single 2D scan of the laser beam while maintaining the improved tissue penetration typical of two-photon microscopy.