Fig. 1: Experimental apparatus for LIPUS treatment and two-photon imaging of microglia and vasculature following microelectrode implantation.
a To evaluate LIPUS stimulation power, a submersible hydrophone preamplifier was placed in degassed water (blue area) below the coverslip and PVA cone. The measurement of the spatial-peak temporal-average intensity (ISPTA) was conducted by setting the transducer’s voltage supply to 135 V. Axial and lateral ultrasound intensity plot from a representative trial (N = 1) used to characterize the transducer used for in vivo LIPUS experiments. Power was around 300 mW/cm2 at the center of the plane, 1 mm below the cover glass. Imaging planes covered a depth range from 0 to 0.3 mm below the cover glass. Attenuation of ultrasound power was assessed in both the X–Y plane (Top right figure) and the Y–Z plane (bottom right figure). Degree of attenuation was quantified by comparing the power at the various locations to the power at the center of the X–Y plane, specifically at the putative brain surface beneath the coverslip. b Representative trial (N = 1) of LIPUS induced tissue heating at two transducer operating intensities used for quantification of thermal effect from LIPUS stimulation. LIPUS was produced at 135 V (red) and 201 V (blue), a total of 15 min of LIPUS exposure, divided into three intervals of 5 min each, with 5 min of non-exposure between each sonication (red and blue periods). Driving the transducer at 135 V kept changes in brain temperature below 1 °C tested at 1.5 mm below the cortical surface. This driving voltage was used for subsequent experiments. c Illustration presents a timeline depicting the sequence of surgery, stimulation, and two-photon imaging. d Schematic representation of microelectrode implantation, sealing of craniotomy window, and LIPUS stimulation. Adapted from38. e A representative two-photon image of a Cx3CR1-GFP transgenic mouse following an I.P. injection of sulforhodamine 101 (SR101) showing microglia cells in green and cerebral blood vessels in magenta. Shaded blue region indicates the location of the implanted microelectrode. Scale bar = 100 µm.
