Background-Free Volumetric Two-Photon Microscopy by Side-Lobes-Cancelled Bessel Beam

We have demonstrated the background-free volumetric two-photon microscopy by cancelling the side lobes of the fundamental 0th-order Bessel beam. The cancellation is based on the fact that the lateral ring patterns of the 3rd-order Bessel beam are well-matched with the side lobes of the 0th-order Bessel beam, so that the image obtained by the 3rd-order Bessel beam is capable to be subtracted as the background of the 0th-order-Bessel-beam-scanned image. We investigated the intensity of the imaging background introduced by the Bessel side lobes under different focusing conditions both in simulations and experiments. The performance of the side-lobes cancellation is showcased on a 50-μm-thick mouse brain slice, and the contrast ratio is enhanced by 2.7 dB for the neurons. This background cancellation technique provides a simple and versatile tool for enhancing the contrast ratio in the two-photon volumetric imaging, especially critical in high-NA cases when a better lateral resolution is pursued.