This research aims to develop line-scanning fluorescence imaging systems based on non-linear absorption with high speed and high spatial resolution, which have practical importance for fast 3D dynamics detection and large-scale imaging in biological applications. Conventional illumination and detection configuration in line-scanning systems constrains performance in large-volume imaging tasks. Typically, line-scanning adopts wide-field detectors like sCMOS and EMCCD in the detection arm and requires strict conjugation between the detector and focal plane; otherwise, the image quality will degrade out of the depth of field (DoF), which means the illumination region should be fixed under the objective lens and cannot probe sample randomly in 3D space. This can be alleviated by necessitating mechanical movements of samples or objective lenses for volumetric imaging but will increase acquisition time and system cost. Additionally, line illumination is highly asymmetric but lacks an effective strategy to modulate the intensity along the illumination line, which will cause strip artifacts in large-scale stitching scenarios.

To address these issues, two digital micromirror device (DMD)-based illumination methods, either in holography or projection mode, are presented, allowing flexible control over position and intensity modulation of the illumination line. In the first project, we present a multi-line generation method by combining DMD-based binary holography with a bucket detector, which can probe samples independent of DoF limitations. Specifically, a DMD generates a randomly distributed focus array on a plane (i.e., x-z plane) via binary holography. A galvanometric mirror then scans the focus array in a direction normal to the plane (i.e., y-axis) across the imaging volume. The emission is collected by a single-point detector, and the final volume image will be reconstructed using the Compressive Sensing (CS) algorithm. Experiments on standard samples and sliced mouse brain have been devised, and the built system achieves a volumetric imaging rate of 15 volumes/sec over 77 × 120 × 40 µm3 while the high-resolution optical cross-sectional images on selected region of interest (ROI) can reach up to 107 frames/sec.

In the second project, we present the method to modulate intensity along the illumination line using DMD projection based on the spatial and temporal focusing nature of femtosecond (fs) pulse laser. Being a dual-function device in our implementation, DMD functions as a dispersing element, i.e., a blazing grating and a binary programmable mask. By programming the number of “ON” pixels of DMD along the direction perpendicular to the axis of the cylindrical lens, the illumination intensity can be tuned pixel-wise, generating a flat illumination field or complex illumination field like Structured Light Illumination (SLI). In a corporation of a high-precision stage and strip-wise stitching, a large Field of View (FOV) over millimeter-scale imaging can be achieved. Verification tests are designed and conducted on a home-built line-scanning temporal focusing microscope. Artifact-free imaging over a millimeter scale is demonstrated by uniform intensity correction with a programmable binary mask and eliminates computational resources on post-processing. Moreover, the built system allows the generation of SLI patterns with arbitrary angles during a single scanning process, and a resolution enhancement of over 1.3 times, along with an improved signal-to-background ratio (SBR), has been demonstrated.

In summary, this work presents new methods of implementing line-scanning two-photon fluorescent microscopy to realize high-speed and high-resolution imaging for volumetric and large-scale imaging. The promising results obtained from point laser scanning and temporal focusing configuration have shown that technology may find significant applications in biophotonics and neuroscience.