Background-Free Volumetric Two-Photon Microscopy by Side-Lobes-Cancelled Bessel Beam
Hongsen He, Yu‐Xuan Ren, Ryan K. Y. Chan, W. L. So, Albert Hiu Ka Fok, Cora Sau Wan Lai, Kevin K. Tsia, Kenneth K. Y. Wong
Abstract
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.