Simultaneous head-mounted imaging of neural and hemodynamic activities at high spatiotemporal resolution in freely behaving mice

Abstract

Exploring the relationship between neural activity and cerebral hemoglobin-oxygenation responses in freely behaving mice can advance our understanding of neurovascular coupling. Head-mounted microscopes enable neuroimaging in freely behaving mice; however, investigating neurovascular dynamics remains challenging because of a lack of hemodynamic information, especially blood oxygenation, or insufficient resolution. Here, we report a head-mounted microscope for neurovascular imaging that enables the simultaneous recording of neuronal burst firing and multiparametric hemodynamics such as vascular oxygen saturation at high spatiotemporal resolution. The 1.7-gram lightweight microscope integrates confocal fluorescence and photoacoustic microscopy, allowing recordings at 0.78 hertz with 1.5-micrometer lateral resolution across a field of view of 400 micrometers by 400 micrometers. We identified cell type-specific neurovascular responses to hypoxic challenges, observed active regulation of arterioles during sensory stimuli, and detected abnormal oxygen depletion and vasodilation preceding burst neuronal discharges in epileptic disorders. This technique provides valuable insights into neurovascular coupling and holds potential for studying the pathology of neurological brain diseases.

Fig. 1.. Head-mounted dual-modal imaging platform.

(A) Schematic of simultaneous imaging of vascular oxygen metabolism and neural Ca²⁺ dynamics in freely moving mice. FC, fiber collimator. (B) Spectral curves. Left axis: Absorption spectra of HbO₂ and HbR. Right axis: Fluorescence excitation and emission spectra of the GCaMP6s calcium indicator. The middle rectangular shading represents the bandwidth of the fluorescence filter (ET 525/50 m). The colored vertical dashed lines indicate the wavelengths (488, 558, and 570 nm) used for dual-modal imaging. a.u., arbitrary units; Norm., normalized. (C) Design of the miniature microscope. Left: Transparent three-dimensional (3D) view showing the internal path, light path (green), and mechanical assembly of the head-mounted probe; the inset shows the acoustic detection scheme using a miniaturized UT. Right: The solid model of the probe. Obj, objective. Scale bar, 5 mm. (D) Photograph of a freely moving mouse wearing the head-mounted dual-modal microscope. Scale bar, 1 cm. (E) FOV measurement by imaging a grid array with 100-μm grid spacing. Scale bar, 50 μm. (F) Spatial resolution test using a 1951 USAF resolution test target. Scale bar, 40 μm. (G) Cross-sectional intensity profile along the green line in (F); the spacing between the lines in element 6 (group 7) is approximately 2.2 μm.

Fig. 2.. Neurovascular response to hypoxia.

(A) Schematic of the hypoxia test paradigm. Pure N₂ and air gases were used to create normoxic and hypoxic environments. (B) Synchronized acquisition of dual-modal images during normoxia (top), hypoxia (middle), and recovery (bottom) states. Columns from left to right: Ca²⁺ fluorescence images, photoacoustic images of HbT and sO₂, and merged images. Each image represents a single moment under different states. Red and blue circles mark typical excitatory and inhibitory neurons. Scale bar, 50 μm. (C) Neuronal and hemodynamic responses to oxygen changes. (a) Average Ca²⁺ dynamic change from all neuronal traces; (b) overall hemoglobin (Hb) concentration changes within the FOV, with fractional changes in HbT, HbO₂, and HbR concentrations plotted separately; (c) overall blood sO₂ change; (d) vessel diameter change indicated by the blue arrow in (B); results are presented as means ± SEM. (D) Statistical comparisons of sO₂ variations in normoxia, hypoxia, and recovery states (n = 6 trials over three mice, two-sided paired t test, **P < 0.01). The data are presented as means ± SEM. (E) Statistical comparisons of vessel diameter changes measured in the normoxia, hypoxia, and recovery states (n = 6 trials over three mice, two-sided paired t test, **P < 0.01). The data are presented as means ± SEM. (F) Heatmap of the calcium transients from all neurons. Scale bar, 40 s. (G) Example traces of calcium transients from excitatory (left) and inhibitory (right) neurons, classified by correlation with overall blood oxygen change in the FOV. (H) Neuronal population classification based on hypoxia response: excitatory (left), inhibitory (middle), and composite (right). Numbers 1 to 10 indicate neuronal locations in (G). (I) Interneuron correlation coefficient matrix for neurons in (G). (J) Distribution of pairwise Pearson correlation coefficients of all extracted neurons.

Fig. 3.. Neurovascular response to sensory stimulus.

(A) Schematic showing the foot shock setup and the electrical stimulus signal setting. The monophasic electric pulse trains had a 3-mA pulse amplitude and 0.25-ms pulse duration at a frequency of 5 Hz. (B) Synchronized acquisition of dual-modal images before (top) and during (bottom) electrical stimulation. Columns from left to right: Ca²⁺ fluorescence (FL) image, photoacoustic image of HbT concentration, photoacoustic image of sO₂, and dual-modal merged image. Scale bar, 50 μm. (C) Example trace of calcium transients from 10 neurons. The vertical red shading indicates the electrical stimulus applied. The scale bar applies to all individual traces. (D) Heatmap of calcium transients from all extracted neurons. The color bar indicates the z-score normalized ΔF/F value. Scale bar, 30 s. (E) Mouse movement trajectories during four electrical foot shock rounds; the colors denote the moving speeds. The red and purple arrowheads mark the moments at which the electric shock begins and ends, respectively. Scale bar, 5 cm. (F) Average Ca²⁺ dynamic change from 10 example neuronal traces in (C); the line and shading denote means ± SEM. (G to I) Hemodynamic responses of arterioles and venules [indicated by the red and blue arrows in (B), respectively] to electrical foot shock. The changes in sO₂, HbT, and vessel diameter are shown in the figures. The data are presented as means ± SEM. Dia., diameter. (J) Segmentation of neurons and blood vessels. (K) Adjacency matrices between neuronal dynamics and hemodynamic responses. The sequence numbers of representative neurons and vascular segments, including arteriole, venule, and capillary, are labeled in (J), respectively.

Fig. 4.. Neurovascular response to acute epileptic seizures.

(A) Schematic showing the timing of imaging and PTZ-induced acute epileptic seizures in mice. (B) Left: Images of Ca²⁺ dynamics and sO₂ at baseline (top) and during seizures (bottom). Right: Representative overlay of photoacoustic (PA) and fluorescence (FL) images at baseline. Scale bars, 50 μm. (C) Ca²⁺ dynamic images from single frames at the indicated time points during seizure wave propagation. Scale bar, 50 μm. (D) Average (Avg.) Ca²⁺ dynamic change from all frames of FL images acquired during seizures. The FL intensity is z-score normalized. (E) Heatmap of calcium transients from all neurons. The entire epoch can be divided into three periods: preictal, ictal, and postictal, separated by the red dashed lines. Scale bar, 100 s. (F) SO₂ changes in blood vessels [indicated by the white arrow in (B)] at baseline versus the seizure state. (G) Changes in blood vessel diameters [indicated by the white arrow in (B)] at baseline versus the seizure state. Two sets of data with the same duration (0 to 256 s) were used for comparison. (H) Example traces of calcium transients during baseline (left) and seizure (right). Time for baseline data, 0 to 256 s; time for seizure data, 128 to 384 s. (I) Interneuron correlation coefficient matrices of Ca²⁺ dynamics from the baseline (left) and seizure (right) states. (J) Statistical comparison of average cross-correlation coefficients in the baseline and seizure states (n = 3 mice, two-sided paired t test, *P < 0.05). The data are presented as means ± SEM. (K) Average Ca²⁺ response magnitude in the preictal and ictal states (n = 3 mice, two-sided paired t test, **P < 0.01). The data are presented as means ± SEM. (L) Mouse movement trajectories in the baseline and seizure states; the colors denote the moving speeds. Scale bars, 5 cm.