Free-moving-state microscopic imaging of cerebral oxygenation and hemodynamics with a photoacoustic fiberscope

Abstract

We report the development of a head-mounted photoacoustic fiberscope for cerebral imaging in a freely behaving mouse. The 4.5-gram imaging probe has a 9-µm lateral resolution and 0.2-Hz frame rate over a 1.2-mm wide area. The probe can continuously monitor cerebral oxygenation and hemodynamic responses at single-vessel resolution, showing significantly different cerebrovascular responses to external stimuli under anesthesia and in the freely moving state. For example, when subjected to high-concentration CO₂ respiration, enhanced oxygenation to compensate for hypercapnia can be visualized due to cerebral regulation in the freely moving state. Comparative studies exhibit significantly weakened compensation capabilities in obese rodents. This new imaging modality can be used for investigating both normal and pathological cerebrovascular functions and shows great promise for studying cerebral activity, disorders and their treatments.

Fig. 1. Photoacoustic fiberscope for cerebral imaging in the freely moving-state.

a A freely moving mouse wearing the imaging probe. b External and (c) internal structure of the imaging probe. d Stereoscopic and (e) side views of the laser beam scanning and ultrasound detection schemes. f Photoacoustic images showing the relative hemoglobin concentration (Hb) and (g) oxygen saturation (sO₂) in a selected region of the mouse cortex. h Photoacoustic signals after 558 and 532 nm excitation recorded in the artery and vein indicated in (g). The signals show different magnitude contrasts PA₅₃₂/PA₅₅₈ due to the different absorption spectra of oxy- and deoxygenated hemoglobin. Scale bar, 200 μm

Fig. 2. High-concentration CO₂ respiration induced cerebrovascular responses under anesthesia.

a–d Hb and sO₂ photoacoustic images in a normocapnia-hypercapnia cycle. Recorded variations in sO₂ (e) Hb (f) vessel diameter (g) and oxygen extration fraction (OEF) (h). n = 4. The data in (e–h) are shown as the mean ± s.e.m. The arrows in (e–h) show the transients corresponding to (a–d). The black lines indicate the period of the hypercapnia experiment. Scale bar, 200 μm

Fig. 3. Cerebral imaging in the awakening process over 30 min

a–d Hb and sO₂ photoacoustic images of the cortex during four selected periods. The motion trajectories over each period are also shown. The pseudo color scale of each trace line represents the time. e sO₂, (f) Hb, (g) vessel diameter, and (h) OEF changes in the arteries and veins. Scale bar: 200 μm. Dots in (e–h): raw data; solid curves in (e–h): lowpass filtered result

Fig. 4. Hypercapnia experiment in freely behaving mice.

a–e Hb and sO₂ photoacoustic images in freely moving mice in a hypercapnia-normocapnia cycle. f Motion trajectories recorded during the experiment. g sO₂, (h) Hb, (i) vessel diameter in the arteries and veins, and (j) Oxygen extraction fraction (OEF) change in the region of interest. The black lines in (g–j) represent the period of hypercapnia. The arrows represent the time transients corresponding to (a–e). n = 4. The data in (g–j) are shown as the mean ± s.e.m. Scale bar, 200 μm

Fig. 5. Hypercapnia experiment in freely behaving obese mice.

a–e Hb and sO₂ photoacoustic images in freely moving mice in a hypercapnia-normocapnia cycle. f Motion trajectories recorded during the experiment. g sO₂, (h) Hb, (i) vessel diameter in the arteries and veins, and (j) Oxygen extraction fraction (OEF) change in the region of interest. Comparison of changes in (k) sO₂ in the artery, (l) sO₂ in the vein, (m) Hb in the artery, and (n) Hb in the vein relative to the baseline during the experiment between healthy and obese mice. The black lines in (g–j) represent the period of hypercapnia. The arrows represent the time transients corresponding to (a–e). The data in (g–j) are shown as the mean ± s.e.m. Here, the P-values were determined by two-way ANOVA. *P < 0.05, **P < 0.01, ****P < 0.0001, ns not significant (healthy mice n = 4, obese mice n = 4). Scale bar, 200 μm