Concept, design, and characterization of tunable Bessel two-photon microscopy.

(A) Comparison of Gaussian (top) and Bessel (bottom) two-photon fluorescence microscopy (TPFM). Gaussian imaging requires sequential acquisition of 2D images at different axial positions, whereas Bessel imaging captures the entire volume in a single frame. (B) Fundamental trade-offs in Bessel beam imaging between spatial resolution, temporal resolution, and axial distinguishability, governed by the numerical aperture (NA), beam length, and side-ring confinement. (C) For targeted opto-stimulation, Bessel beam tuning must preserve a fixed axial beam center to maintain co-registration with stimulation or ablation beams. (D) Schematic of the tunable Bessel module (A1–A3, axicons; L1–L3, lenses; iris) and its integration with Gaussian-TPFM. A1 with L1 generates the Bessel beam; the iris adjusts ΔNA; A2–A3 spacing sets NA; L2–L3 form a 4f relay to the scanning galvo. (E) Zemax simulations showing pupil (left), sample (middle), and propagation (right) images for 0.8 NA with open iris (top), 0.4 NA with open iris (middle), and 0.4 NA with 10% iris opening (bottom). (F) Simulated versus experimental Bessel beam cross-section profiles at NA= 0.4 (top) and 0.8 (bottom). Experimental images were acquired using a camera conjugate to the objective’s sample plane. Scale bar: λ/n, where n is the refractive index of the immersion medium. (G) Theoretical and experimental results showing adjustment of the Bessel beam NA (blue), resolution (orange), and length (green) as a function of A2-A3 spacing. (H) Simulated versus experimental Bessel beam pupil profiles for ΔNA=0.005 (top) and 0.041 (bottom). Experimental images were acquired using a camera conjugate to the objective’s back focal plane. (I) Theoretical and experimental results showing adjustment of the Bessel beam ΔNA (blue), half total power radius (orange), and length (green).

Large-scale volumetric vascular imaging and neurovascular coupling.

(A) Left: 3D rendering of a 350×350×450-μm3 Gaussian stack showing dense cortical vasculature. Right: representative Bessel images with beam lengths of 17-μm (orange), 35-μm (magenta), and 140-μm (blue), illustrating the trade-off between information content and structural overlap. (B) tBessel scan combining lateral tiling (4×4) and focal jumps (3, color-coded), yielding 48 tiles covering a 2,500×2,500×450-μm3 cortical volume. Scale bar: 200-μm. (C) Combined millimeter-scale tBessel image from (B). White box: sub-capillary resolution. Green box: penetrating arterioles/venules (arrows). Right: vasoconstriction and vasodilation of the vessel marked in red. Scale bar: 200-μm. (D) Experimental setup for functional imaging of primary visual cortex (V1). Mice with cranial windows over V1 were presented moving gratings in eight directions. Five planes spanning a 260×260×400-μm3 volume were acquired at 2.5-Hz (5 trials per direction). Two neurons were analyzed for fluorescence dynamics, and vessels were outlined for resliced space–time plots of diameter changes. (E) Top: averaged calcium traces (green) from two neurons. Middle: raw and segmented resliced images from lines 1 and 2 in (D). Bottom: overlay of neuronal activity (green) and vessel diameter (magenta) showing strong correlation, with vascular changes lagging neuronal responses (arrows).

High-speed blood flow mapping in normal and ischemic stroke mice.

(A) Widefield and schematic view of mice brain vasculature. (B) Schematics (left) and images (right) comparing Gaussian (top; 2-μm depth of focus) and tBessel (bottom; 80-μm long) scanning. Gaussian imaging restricts measurements to vessels nearly parallel to the imaging plane, whereas tBessel enables blood flow measurement across a broad range of vessel orientations. Scale bars: 20-μm. (C) Workflow for blood flow measurement. Left: 3D Gaussian stack highlighting a vessel segment (blue). Bottom: real-time tBessel imaging showing moving blood cells. Right: kymographs, with blood cells appearing as dark streaks (arrows). Velocities are extracted from 2D tBessel streak slopes and corrected for vessel 3D length. (D) Mean intensity projection of a Gaussian stack (400×400×120-μm3, 40–160-μm below pia, depth color-coded) versus tBessel scans with NA = 0.4, 0.6, and 0.8. Insets: experimentally measured point spread functions (PSFs, scale bar: 1.5-μm) and optical transfer functions (OTFs). (E) Trade-off between spatial resolution and imaging speed: high resolution for capillaries versus low resolution for larger vessels. (F) Blood flow speed versus vessel diameter for 42 segments imaged with 0.4 NA tBessel at 58-Hz. (G) Frame scanning versus line scanning: restricting scans to vessel regions improves speed and accuracy for large vessels. (H) Blood flow speed versus vessel diameter measured with 1 kHz line scans. Red: arteries; blue: veins. (I) Schematics of stroke induction. (J) tBessel-TPFM hemodynamics of a 1,400×1,400×140-μm³ volume before stroke induction. Blood flow speeds (mm/s) measured by 1 kHz line scans; numbers indicate speed, arrows indicate direction (red: arterioles, blue: veins). Representative kymographs of three linecuts shown at right. Scale bar: 100-μm. (K) Same measurement after stroke induction. Scale bar: 100-μm. (L) Paired plot of blood flow speed before and after stroke induction (N = 17). Wilcoxon test, p = 1×10−5.

Video-rate volumetric functional imaging with targeted optogenetic stimulation.

(A) Experimental setup. A Gaussian beam (blue) delivers 3D localized optogenetic stimulation to a neuron, while a tBessel beam enables volumetric imaging symmetrically above and below the stimulation plane. (B) Schematics and axial PSFs of the stimulation Gaussian beam (blue), and short (yellow) and long (red) tBessel imaging beams, showing that the extended projection is always symmetric with respect to the Gaussian stimulation plane. The non-uniformity in intensity comes from input Gaussian beam. Scale bar: 10-μm. (C) Depth color-coded projection of volumetric functional imaging showing the stimulated neuron (neuron 1) and surrounding neurons; 16 neurons were selected for analysis. Scale bar: 10-μm. (D) Representative calcium transients from neurons numbered in (C). Light blue bars mark the timing of optogenetic stimulation pulses delivered to neuron 1. (E) Pearson correlation map of calcium dynamics among the neurons numbered in (C).

Rapid volumetric imaging of microglial responses to targeted ablation.

(A) Experimental setup: a Gaussian beam (green) delivers targeted ablation of a single microglial cell, while a tBessel beam (red) provides symmetric volumetric imaging above and below the ablation plane. (B) Mean intensity projection of a 200×200×120-μm3 Gaussian stack, depth color-coded. White box marks the targeted microglia. Scale bar: 20-μm. (C) Gaussian plane images of the targeted cell before (left, arrow) and after (right, arrow) ablation. Scale bar: 10-μm. (D) Representative 15 Hz volumetric tBessel data with a 120-μm-long beam (NA = 0.7). Raw (left), denoised (center), and deconvolved (right) images. Scale bar: 10-μm. (E) Time-lapse snapshots showing process extension toward the ablation site (arrowheads) at 1m15s, 4m0s, and 9m0s, revealing a two-wave response. Scale bar: 10-μm. (F) Zoomed views of two regions (green and blue boxes in (D)). Arrowheads indicate process extension toward the ablation site (top, green box) and retraction opposite the lesion (bottom, blue box) across the 10-min imaging period. Scale bar: 5-μm.