High-resolution fluorescence-guided transcranial ultrasound mapping in the live mouse brain

Abstract

Understanding the physiological impact of transcranial ultrasound in rodent brains may offer an important preclinical model for human scale magnetic resonance–guided focused ultrasound methods. However, precision tools for high-resolution transcranial ultrasound targeting and real-time in vivo tracking of its effects at the mouse brain scale are currently lacking. We report a versatile bidirectional hybrid fluorescence-ultrasound (FLUS) system incorporating a 0.35-mm precision spherical-phased array ultrasound emission with a fiberscope-based wide-field fluorescence imaging. We show how the marriage between cortex-wide functional imaging and targeted ultrasound delivery can be used to transcranially map previously undocumented localized fluorescence events caused by reversible thermal processes and perform high-speed large-scale recording of neural activity induced by focused ultrasound. FLUS thus naturally harnesses the extensive toolbox of fluorescent tags and ultrasound’s localized bioeffects toward visualizing and causally perturbing a plethora of normal and pathophysiological processes in the living murine brain.

Fig. 1.. Experimental setup and characterization.

(A) Schematic of the integrated wide-field fluorescence imaging and high-precision transcranial ultrasound system FLUS. (B) Fluorescence image of a mouse brain expressing GCaMP6f through the intact scalp and skull. A crosshair indicates the position of the array’s focus and the 6-mm circle the extent of the three-dimensional focus- steering range. The inset shows the extent of the field of view. (C) Skull insertion loss [mean, standard deviation (SD), and range] as a function of frequency measured using a hydrophone. (D) Normalized ultrasound pressure measured transcranially at 3 MHz. (E) Focal spot size at 3 MHz in free field (without the skull) and with the mouse skull in the ultrasound propagation path.

Fig. 2.. Wide-field recording of a mouse brain expressing GCaMP6f during sequential FUS delivery.

(A) Relative fluorescence change overlaid on the baseline fluorescence image shown on a gray scale. The arrowhead indicates the point of ultrasound delivery at the instant of minimum fluorescence. (B) Time trace of the relative change in fluorescence at the center of the ultrasound delivery region shown in (A). Details on the ultrasound delivery time shown on the inset. (C) RSC on two single FUS sonication. Addition of moderate spatial filtering (2 pixels wide) further improves the SNR. (D and E) Relative fluorescence change during ultrasound delivery at the targeted regions (red and green rings) averaged over 24 stimulation cycles. (F) Fluorescence time traces from (D) and (E) showing the average response of a single pixel at the ring’s center. (G) Averaged relative fluorescence change observed in E. coli colonies at the instant of minimum fluorescence overlaid on average fluorescence in gray scale. (H) Relative fluorescence change at the position of the red circle from (G). The emission was repeated for five cycles with a period of 2 s. (I) Relative fluorescence change as a function of the normalized ultrasound intensity.

Fig. 3.. Average fluorescence dip characterization with a thermal model.

(A) Change in fluorescence as a function of the temperature in a brain slice expressing GCaMP6f. An affine fit to four brain slices yields a fluorescence change of −1.9 ± 0.7%/°C. (B) Average fluorescence dip (n = 7, 140 stimulation cycles) at the instant of the minimum fluorescence. (C) Time traces of the average dip compared with the thermal model from the bioheat equation labeled following the rings on (B). The gray region depicts the instant of the FUS delivery, while a dashed vertical line indicates the time instant shown in (B). (D) Characterization of the spatial extent of the fluorescence dip compared against the thermal model. Dark and light green experimental curves correspond to cross sections of the same colors in (B). (E) Inferred relative temperature change for the model and the measured data. Gray region depicts the SEM, whereas the dashed line shows the identity map.

Fig. 4.. Functional connectivity (FC) before and after FUS delivery.

(A) Mouse brain schematic showing the regions included in the FC. Squares represent the seeds (7 × 7 pixels) while the orange circle depicts the FUS delivery spot. Average Pearson’s correlation (n = 3) map calculated before (B) and after 20 sonications using sequence 1 (C). The cross (B) shows the seed position whereas the orange circle encloses the FUS delivery point. The Fisher z-transformed FC is shown in (D to F) for the baseline, 20 FUS emissions at 3 MPa (three mice) (E) and 1 emission at 3.9 MPa (three mice). (G) Coronal mouse brain slice at the point of FUS delivery stained with DAPI, TUNEL assay, and Fluoro-Jade C. Sonications for a more conclusive histological analysis were performed on the right hemisphere (see Materials and Methods). (H) Difference after FUS delivery in standard units (z score) and masked for multiple-testing corrected P < 0.1 from a paired t test. Dark-green squares show P < 0.05. (I) Histology analysis on coronal slices after single sonication at 3.9 MPa stained with DAPI and c-Fos imaged with wide-field and confocal microscopy.

Fig. 5.. Wide-field recording of a CSD triggered by focused noninvasive ultrasound delivery.

(A to D) Relative fluorescence changes at different time points overlaid on baseline fluorescence shown in gray scale. (E) Time traces of the relative change in fluorescence at the points labeled by rings in (A). Ultrasound delivery point indicated by a crosshair. Light blue vertical lines at t = 0, 30, and 60 s. Represents the onset of consecutive ultrasound emission. Solid black vertical lines indicate the instants of the wide-field images shown in (A) to (D). (F) Mean probability of triggering a CSD as a function of the peak pressure and maximum temperature change. SD depicted by whiskers and the Mann-Whitney test. **P < 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. (G to I) Maximum intensity projection of the relative fluorescence change color-coded for time to 10% of the peak values for stimulation sequences 2 (G), 2 (H), and 3 (I). (J) Normalized fluorescence change with the colors represented by labels indicating the pulse duration and the I_SPPA. Lines above the main peak show the time delay 3 mm away from the stimulated spot (transparent curves).