Aditya Vasan,Jeremy Orosco,Uri Magaram,Marc Duque,Connor Weiss,Yusuf Tufail,Sreekanth H Chalasani,James Friend
Abstract:
Ultrasound has been used to manipulate cells in both humans and animal models. While intramembrane cavitation and lipid clustering have been suggested as likely mechanisms, they lack experimental evidence. Here, high-speed digital holographic microscopy (kiloHertz order) is used to visualize the cellular membrane dynamics. It is shown that neuronal and fibroblast membranes deflect about 150 nm upon ultrasound stimulation. Next, a biomechanical model that predicts changes in membrane voltage after ultrasound exposure is developed. Finally, the model predictions are validated using whole-cell patch clamp electrophysiology on primary neurons. Collectively, it is shown that ultrasound stimulation directly defects the neuronal membrane leading to a change in membrane voltage and subsequent depolarization. The model is consistent with existing data and provides a mechanism for both ultrasound-evoked neurostimulation and sonogenetic control.
Figure: High-speed DHM imaging of membrane deflection. The deflection of the membrane under the influence of ultrasound was visualized using a) high-speed digital holographic microscopy (DHM). The DHM setup included a lithium niobate transducer driven by a signal generator and an amplifier at 6.72 MHz. The cells were mounted on a coverslip and placed in a custom perfusion chamber maintained at 37 °C. The DHM enables the b) quantitative reconstruction of phase images acquired by the high-speed camera at 40 000 frames s−1. Each recording began with 25 ms of no stimulus as a baseline, followed by a 50 ms ultrasound stimulus, and ended with a 25 ms baseline. c) The maximum deflection from the mean position was found to be 100–400 nm, with a median deflection of 214 nm for HEK293 cells and 160 nm for neurons (N = 30 for each cell type). Reconstructed phase profiles are shown for different cell types: d) HEK293 cells, e) neurons, and f) neuronal clusters. Displacement was measured as a function of distance along the green lines provided in the (d–f) contour plots and were g–i) plotted with (red line plot, max displacement during stimulus) and without (green plot, Baseline) ultrasound stimulus. A distance of “zero” in (g–i) is at the left end of the green line in (d) and (e) and at the bottom of the green line in (f). For the (green) baseline displacement, note the mean and 95% confidence intervals are provided. The maximum variation throughout all baseline responses was less than ±20 nm.
The Lyncée Tec Highspeed DHM® operates with a single wavelength and can be configured in either transmission or in reflection.
Its optical set up and laser source have been specifically optimized to operate with the highspeed camera FASTCAM NOVA by Photron. This system offers hologram recording rates up to 12’800 frames per second (fps) at megapixel resolution, 40’000 fps at a resolution of 512×512 pixels, and 116’000fps at 256×256 pixels.
A dedicated and intuitive software interface, fully integrated in our Koala acquisition and analysis software, enables to control the highspeed camera parameters (acquisition framerate, shutter), to handle the different camera trigger modes, to access optimally the camera internal memory buffer, to replay an acquired sequence, to select the relevant frames, and eventually to export the selected ones.
The Highspeed DHM® system enables novel research among others in the fields of energy harvesting, impact analysis, fluid interfaces, transients, stress analysis, evaporation, drying, absorption, desorption, and dissolution phenomena, etc.
