Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

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This method can help answer key questions in mechanobiology, such as how biological samples respond to mechanical forces produced by low intensity ultrasound stimulation. The main advantage of this technique is that it allows an experimentalist to noninvasively apply mechanical forces to a sample and measure various biological perimeters in realtime. To begin, slowly drill a 12 millimeter hole at the bottom of a standard 35 millimeter culture dish using a vertical press drill.

Once drilled, remove pieces of plastic attached to the bottom of the dish using a blade, to create a smooth surface on the external side. Next, apply a thin layer of marine-grade epoxy or glue at the external bottom surface of the dish. On top of the adhesive, place a two-point-five-micron thick film of polyester, pressing firmly to make sure the adhesive spreads evenly between the film and the thick plastic surface.

Gently pull the film in a centrifugal manner with fingers, to create a flat surface. Once the adhesive has dried, briefly rinse the polyester bottom dish with 95%ethanol. Then, UV sterilize the dish by placing it under a strong 254 nanometer UV excitation source.

Adjust duration and intensity to deliver a UV dose of approximately 330 millijoules per square centimeter. Next, dilute a commercially available extracellular matrix protein mixture with the desired culture medium at a ratio of one to 100. Work on ice to prevent matrix polymerization, and quickly apply 100 microliters of the medium mixture onto the polyester film.

Place the lid back on the dish to maintain sterility. Once covered, incubate the matrix-coated, polyester bottom dishes in a cell culture, carbon dioxide-buffered incubator at 37 degrees Celsius for six to 12 hours. After incubation, aspirate the excess medium and directly seed the surface with cells at the desired density.

Place a water tank underneath the objective of an upright microscope. Then, using commercially available optomechanical components, position the sample holder between the objective and the transducer. Make sure that the moving parts and the actuators of the translation stages are either outside the tank or above the water line to avoid water damage.

Then, fill the tank with deionized and degassed water up to the horizontal plane of the sample holder, before utilizing the immersion transducer. Using noncorrosive, commercially available optomechanical components, make sure that the transducer in an oblique position with respect to the optical path. This will ensure that any reflected waves will be directed away from the sample.

Adjust the frequency of the function generator to the nominal peak frequency of the transducer. Then, create a sinusoidal voltage pulse of the desired duration and repetition frequency, using the burst mode of the function generator. Next, adjust peak-to-peak voltage to a desired value.

Make sure that the pulse duration is shorter than the elapsed time between two consecutive pulses. Connect the output of the function generator to the input of an oscilloscope. Use the oscilloscope to confirm that the waveform corresponds to the desired signal.

Then, connect the output of the function generator to the input of a power of a properly-sized RF amplifier. Using a hydrophone probe that operates with a frequency range and acoustic intensity compatible with that of the ultrasound transducer, carefully bring the probe's tip into focus within the objective's field of view at the position of the sample. Make sure that both probe and transducer are immersed in the water bath and perform a gross pre-alignment of the transducer by visually positioning its acoustic axis toward the hydrophone probe.

Make sure that the distance between the two correspond to the transducer's focal length. Do not bump the tip of the hydrophone with any physical object other than water, as this will alter its coating and affect the measurement. Next, connect the hydrophone output to one of the oscilloscope's signal input.

Then, connect the synchronization trigger from the function generator to another oscilloscope input. Visualize both signals simultaneously on the oscilloscope. Next, drive the transducer with few ultrasound cycles at a low duty cycle and low amplitude to avoid damaging the probe.

Check with the hydrophone's manufacturer safe operation conditions to avoid damaging the hydrophone tip. Adjust the S-division knob according to the travel time of the ultrasound from the transducer's surface to the hydrophone. Look for a hydrophone signal on the oscilloscope after the synchronization trigger.

Next, slowly actuate the transducer using a motorized or manual XYZ stage. Position the transducer in the position that correlates with the maximal hydrophone signal. With the beam now aligned, measure the peak-to-peak amplitude of the hydrophone output at the oscilloscope for various voltages driving the transducer.

Make sure not to exceed the pressure limit of the hydrophone. Replace the cell's culture medium with the desired imaging buffer containing five micromolar of a cell-permeant, calcium-sensitive dye, such as Fluo-4 AM.Then, incubate the culture dish in a carbon dioxide-buffered incubator at 37 degrees Celsius for one hour. Following incubation, carefully wash cells with the same buffer free of dye.

Then, place the dish in the sample holder. Excite the cells using blue light illumination at 490 nanometers. Adjust the excitation intensity and camera exposure to avoid excessive bleaching or pixel saturation.

Perform time-lapse imaging using an immersion objective with a long working distance for better image quality, and to reduce undesired reflections. Here, glioblastoma cells are shown on extracellular matrix-coated polyester film in standard culture medium. These cells have been incubated with a calcium-sensitive fluorescent reporter.

In this image, the red dots represent individual fluorescent cells, identified using an image processing software. Upon stimulation for 10 seconds with ultrasound, robust calcium elevations were visualized in many regions of interest. The number of activated regions of interest are shown here over time.

Following the 10-second activation with ultrasound, approximately 70%of the regions were found to be above user-defined threshold values. While attempting this procedure, it's important to remember to align the ultrasound beam before doing fluorescence measurement.

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Journal (Engineering)

Journal (Engineering)

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

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