Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy
Jerome J. Lacroix, Alper D. Ozkan
Journal of Visualized Experiments 2019 · 10.3791/58781
Abstract
By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.
Abstract via europepmc.
Exposures
Exposure 1: LIPUS-induced calcium imaging in cultured glioblastoma cells (representative example)
Target: other — “human glioblastoma cells (A-172) grown on a polyester-bottom culture dish”
Device: not reported
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 4,000 | ✓✓✓ |
| Pulse duration (ms) | 0.1 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 100 | ✓✓✓ |
| Duty cycle (%) | 1pulse duration × PRF gives 1% | ✓✓✓ |
| Sonication duration (s) | 10 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | 88 | ✓✓✓⚑ |
| Free-field Ispta (W/cm²) | 0.877 | ✓✓✓ |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable |
LIPUS was applied by manually driving a 4 MHz transducer with a pulse waveform of 158 V peak-to-peak amplitude, 0.1 ms pulse duration and 10 ms pulse repetition frequency (i.e. 1% duty cycle). Images were acquired at a speed of 1 frame per second by a standard CCD camera and LIPUS was applied for 10 sec between frames 20 and 30; the waveform consisted of 100 usec pulses containing 400 cycles at 4 MHz and repeated every 10 ms for 10 sec.
Flags from extraction
exposures[0].device.family— The protocol's Table of Materials lists several possible transducer suppliers (Olympus, Benthowave, Precision Acoustics, Ultrasonic-S-lab) but the specific device used for the representative 4 MHz experiment is not identified by manufacturer or model.exposures[0].free_field.isppa_w_cm2— Intensities were obtained from hydrophone calibration curves measured in the same degassed water tank used to hold the sample (no skull or intervening tissue), so classified as free-field per the bath convention; the paper does not use the term 'free field' explicitly.direction_of_effect— This is a general bioeffect (calcium transient) demonstration in a non-neuronal cell line, not a neuromodulation experiment with an excitatory/inhibitory neural readout; 'excitatory' is used loosely to reflect the increase in the calcium signal.