178 / 2024-06-14 21:14:01

Volumetric two-photon microscopy with a novel Bessel beam

Bessel beam,Two-photon microscopy,volumetric imaging,light field manipulation,zebrafish

We demonstrate a volumetric two-photon microscopy (TPM) using the novel Bessel beam as illumination. In free space, the self-reconstruction and non-diffraction characteristics of the Bessel beam can provide incomparable advantages of the Gaussian beam, which can obtain greater depth of field for imaging and further improve imaging depth and speed. Under the same NA condition, the axial length of the traditional Bessel beam is about 4 times that of the Gaussian beam. To further extend the axial length of the conventional Bessel beam, we engineer a novel Bessel beam through advanced light field manipulation techniques. Direct mapping of the imaging trajectory shows that the novel Bessel beam extends the imaging range around 1.5~2 times longer than a conventional Bessel beam, while providing a better lateral resolution. The volumetric specimen is mapped layer by layer under Gaussian mode, while the three-dimensional structure is projected to a single two-dimensional image under Bessel mode, leading to a significantly increased acquisition speed. The performance of this advanced TPM system was comprehensively assessed using a phantom model of agarose gel embedded with fluorescent beads and living transgenic zebrafish. Our results conclusively show that the novel Bessel beam can obtain more imaging information than the conventional Bessel beam under the same imaging condition. This innovation has the potential to transform optical imaging, particularly for applications demanding high-resolution, rapid, and deep-tissue visualization.