Fig 1.
(a) Side view of the Y scanning pair. The red and green lines show two parallel scanning positions. (b) The X scanning pair. X2 serves as an angle switcher for tilting the focus. (c) Control waveforms of the Stereo-scanner. (d) Switchable annular illumination at BFP. (e) Stereoscopically scanning with tilted Bessel beams. Scanning geometry with N×M scanning positions with each position excited twice from different angles (f) Data processing of interlaced raw images. From top row to bottom row: Interlaced raw data with odd and even lines for left (red) and right (cyan) views, respectively; Deinterlaced into two matrices; Rotated 90° and to create a stereo-pair. The acquired stereo-pair can be directly viewed with 3D glasses or processed by correspondence algorithm for depth recovering.
Fig 2.
Software control framework based on ScanImage.
(a) Modules modified (in red boxes) based on ScanImage workflow (from “Start” to “End”, see [22] for details). (b). List of all the modified files of ScanImage.
Fig 3.
Flowchart of depth-map reconstruction with feature-based method.
Fig 4.
Simulated and measured two-photon excitation PSF2p of Bessel beams with small tilted angle (≈2.5°).
(a) Numerical simulation. Scale bar 20μm. (b) Measured PSF2p. (c) PSF2p with Gaussian beam. Scale bar 1μm. (d). Lateral FWHM of PSF2p. (e) Axial intensity distribution along beam direction at different z position.
Fig 5.
Volumetric imaging and 3D reconstruction.
Stereo-pairs of (a-b) pollen grains and (c) fluorescent beads were captured in stereo-mode. (d) Sum projection of the stack along z-axis. (e) 5 selected images from an image stack consisting of 87 slices taken in standard two-photon mode. (f) 3D map of objects with their depth recovered from the stereo-pair in (c). Each object is labeled according to its depth error with a color map that spans from green to red. Blue spheres are unrecognized objects in (e), but are present in (d). (g) Histogram of the depth error with superposed normal distribution (red line). Scale bars in (a)-(e) are all 10μm.
Fig 6.
Feature-based correspondence algorithm and the recovered depth.
(a) 87 circular objects found in the left image (Fig 5c1). (b) The corresponding circular objects are found in the right image (Fig 5c2). (c) The corresponding circular objects are also found in the z-projection of the stack (Fig 5d) to determine the ground truth depths. (d) Comparing the recovered depths from the stereo-pair and the ground truth values from the image stack.
Fig 7.
Volumetric imaging of dynamic samples and 3D Reconstruction.
The fluorescent beads are moved at an average speed of about 1 μm/s by driving the sample stage. See S1 Movie for the dynamic tracking.