Author Affiliations
1Britton Chance Center for Biomedical Photonics and MoE Key Laboratory for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China2Current address: Department of Mechanical and Automation Engineering, Chinese University of Hong Kong, Shatin, Hong Kong3HUST-Suzhou Institute for Brainsmatics, JITRI Institute for Brainsmatics, Suzhou 215123, China4School of Biomedical Engineering, Hainan University, Haikou 570228, China5e-mail: huigong@mail.hust.edu.cn6e-mail: yuanj@hust.edu.cnshow less
Fig. 1. System configuration. The inset shows the enlarged view of the stepped mirror. The faint yellow plane represents the focal plane of O1. The positions of the stepped reflection surfaces were adjustable relative to the focal plane. Red lines and points represent two linear signals and their directions, respectively.
Fig. 2. Principle of stepped remote focusing and FOV-jointing. (a) Relative positions of the linear beams and the stepped mirror, as well as the geometric constraints of the distance between the linear beams and the step edge. Red, yellow, and pink points represent the linear beams perpendicular to the surface of the paper. (b) FOV-jointing module rearranges two parallel lines into a head-to-head line. The inset shows that the lateral interval between two linear signals is adjustable.
Fig. 3. Resolutions and Strehl ratio measurements in an axial range of ±150 μm. (a) and (b) Spatial resolutions of the two FOVs, respectively. Averaged pictures of beads are shown as corresponding insets at the top right corners. Pixel size, 0.244 μm in x and y as well as 1 μm in z. (c) FWHM results. (d) Measured Strehl ratio. Standard deviations of five beads are shown as error bars. (e) Position offsets of the remote focal planes of the objective O2 in FOV1 and FOV2 at different depths before and after correction.
Fig. 4. Imaging the edges of the knife-edge mirrors of the FOV-jointing module and the stepped mirror. (a) Edges of KM1, KM2, KM01, and KM02 are shown clearly on the camera when they are conjugated and in focus. Scale bar, 50 μm. (b) Enlarged view of white square in (a). (c) Imaging the same area of (b) by moving KM01 10 μm down. Scale bar, 5 μm.
Fig. 5. Optical sectioning ability of the system. (a) and (b) Defocusing responses of thin fluorescent sheet in the line confocal mode of the two FOVs. (c) Line confocal optical sectioning thickness with a line width of 1 pixel. (d) Line confocal and (e) LiMo-reconstructed images of the same layer of a pollen grain at different remote focal positions in the two FOVs. Scale bar, 10 μm. Right column shows normalized intensity profiles along corresponding colored lines in the images.
Fig. 6. Video-rate imaging of two-layer Brownian motion of 200 nm fluorescent beads aqueous solution. (a) Sandwich structure of the sample. (b) Steps to track motion trajectories of the beads. (c) and (d) Trajectories of Brownian motions of the beads in FOV1 and FOV2. (e) and (f) are enlarged views of the areas indicated by the white squares in (c) and (d), respectively.
Fig. 7. Simultaneous dual-plane functional imaging of a transgenic zebrafish brain in vivo. (a) and (b) MIPs of image sequences acquired in 240 s in two FOVs. Inset in (a) indicates that the two FOVs focus in optic tectum and hindbrain, respectively. White squares and colored circles represent the neurons with spontaneous activities. (c) and (d) Spontaneous fluctuations of neural cells marked by corresponding colored circles in (a) and (b), respectively. (e)–(g) Images of the same sample in TDI, LiMo, and wide-field modes. (h) Normalized intensity profiles along corresponding colored lines in (e)–(g).