Difference between revisions of "Team:Slovenia/Hardware"

Line 81: Line 81:
 
<div align = "center">  
 
<div align = "center">  
 
<figure data-ref="1">
 
<figure data-ref="1">
<img class="ui large image" src="" >
+
<img class="ui large image" src=" " >
 
<figcaption><b>Different experiment configurations.</b><br/>
 
<figcaption><b>Different experiment configurations.</b><br/>
 
Testing homogeneity of pressure in each well with a plate immersed in the ultrasonic bath.
 
Testing homogeneity of pressure in each well with a plate immersed in the ultrasonic bath.
Line 99: Line 99:
 
<div style="float:left;">  
 
<div style="float:left;">  
 
<figure data-ref="1">
 
<figure data-ref="1">
<img class="ui medium image" src="" >
+
<img class="ui medium image" src=" " >
 
<figcaption>
 
<figcaption>
 
</figcaption>
 
</figcaption>
Line 108: Line 108:
 
<div style = "float:left;">  
 
<div style = "float:left;">  
 
<figure data-ref="2">
 
<figure data-ref="2">
<img class="ui medium image" src="">
+
<img class="ui medium image" src=" ">
 
<figcaption><b>Signal parameters.</b><br/>  
 
<figcaption><b>Signal parameters.</b><br/>  
 
Parameters that can be set for a typical stimulation signal.
 
Parameters that can be set for a typical stimulation signal.
Line 119: Line 119:
 
<div >  
 
<div >  
 
<figure data-ref="5">
 
<figure data-ref="5">
<img class="ui medium image" src="" >
+
<img class="ui medium image" src=" " >
 
<figcaption><b>Application interface.</b><br/>
 
<figcaption><b>Application interface.</b><br/>
 
Web based interface used for setting of the signal parameters.
 
Web based interface used for setting of the signal parameters.
Line 131: Line 131:
 
<div style="float:left;">  
 
<div style="float:left;">  
 
<figure data-ref="4">
 
<figure data-ref="4">
<img class="ui medium image" src="" >
+
<img class="ui medium image" src=" " >
 
<figcaption><b>Simulation scheme.</b><br/>
 
<figcaption><b>Simulation scheme.</b><br/>
 
Scheme used in LT Spice simulations.
 
Scheme used in LT Spice simulations.
Line 140: Line 140:
 
<div style = "float:left;">  
 
<div style = "float:left;">  
 
<figure data-ref="5">
 
<figure data-ref="5">
<img class="ui medium image" src="" >
+
<img class="ui medium image" src=" " >
 
<figcaption><b>Model of transducer.</b><br/>
 
<figcaption><b>Model of transducer.</b><br/>
 
Equivalent model of ultrasonic transducer based on a BVD model.
 
Equivalent model of ultrasonic transducer based on a BVD model.
Line 162: Line 162:
 
<div style="float:left;">  
 
<div style="float:left;">  
 
<figure data-ref="7">
 
<figure data-ref="7">
<img class="" >
+
<img class=" " >
 
<figcaption><b>Typical output signal without load.</b><br/>
 
<figcaption><b>Typical output signal without load.</b><br/>
 
Amplitude of acquired signal depends on the winding of transformer.
 
Amplitude of acquired signal depends on the winding of transformer.
Line 173: Line 173:
 
<div style="float:left;">  
 
<div style="float:left;">  
 
<figure data-ref="8">
 
<figure data-ref="8">
<img class="">
+
<img class=" ">
 
<figcaption><b>Ultrasound transducer V318-SU.</b><br/>
 
<figcaption><b>Ultrasound transducer V318-SU.</b><br/>
 
Handy waterproof case allowed us to use the transducer in most experiments.
 
Handy waterproof case allowed us to use the transducer in most experiments.
Line 183: Line 183:
 
<div style="float:left;">  
 
<div style="float:left;">  
 
<figure data-ref="9">
 
<figure data-ref="9">
<img class="ui medium image" src="" >
+
<img class="ui medium image" src=" " >
 
<figcaption><b>Electric power of ultrasonic transducer before and after compensation.</b><br/>
 
<figcaption><b>Electric power of ultrasonic transducer before and after compensation.</b><br/>
 
Electrical power with compensation was significantly increased with serial compensation.
 
Electrical power with compensation was significantly increased with serial compensation.
Line 204: Line 204:
 
<div style="float:left;">  
 
<div style="float:left;">  
 
<figure data-ref="11">
 
<figure data-ref="11">
<img class="ui medium image" src="" >
+
<img class="ui medium image" src=" " >
 
<figcaption><b>Hydrophone RP 31l.</b><br/>
 
<figcaption><b>Hydrophone RP 31l.</b><br/>
 
Calibrated hydrophone used to measure pressure.
 
Calibrated hydrophone used to measure pressure.
Line 215: Line 215:
 
<div style="float:left;">  
 
<div style="float:left;">  
 
<figure data-ref="12">
 
<figure data-ref="12">
<img class="ui medium image" src="" >
+
<img class="ui medium image" src=" " >
 
<figcaption><b>Voltage signal detected using the hydrophone.</b><br/>
 
<figcaption><b>Voltage signal detected using the hydrophone.</b><br/>
 
With calibration data, we can determine pressure which corresponds to acquired voltage amplitude.
 
With calibration data, we can determine pressure which corresponds to acquired voltage amplitude.
Line 226: Line 226:
 
<div style="float:left;">  
 
<div style="float:left;">  
 
<figure data-ref="13">
 
<figure data-ref="13">
<img class="ui medium image" src="" >
+
<img class="ui medium image" src=" " >
 
<figcaption><b>3D printer.</b><br/>
 
<figcaption><b>3D printer.</b><br/>
 
Printing a holder for the ultrasonic transducer that we used for a 6-well plate.
 
Printing a holder for the ultrasonic transducer that we used for a 6-well plate.
Line 235: Line 235:
 
<div style="float:left;">  
 
<div style="float:left;">  
 
<figure data-ref="14">
 
<figure data-ref="14">
<img class="ui medium image" src="" >
+
<img class="ui medium image" src=" " >
 
<figcaption><b>Ultrasound based experiments on the microscope.</b><br/>
 
<figcaption><b>Ultrasound based experiments on the microscope.</b><br/>
 
Ultrasonic transducer with a 3D printed holder in a 6-well plate.
 
Ultrasonic transducer with a 3D printed holder in a 6-well plate.

Revision as of 22:03, 15 October 2016

Hardware

  MODUSON - Generating ultrasonic power pulses for cell stimulation

Ultrasound is sound waves with frequencies above the upper audible limit of human hearing, which is approximately 20 kHz. The ultrasound (US) wave interacts with tissue and reflects back depending on the properties of the tissues such as velocity of sound in the tissue and its density, which can be modeled using wave equation (hyperlink to Žiga’s modeling[Requires formatting>]). Higher frequencies have better resolution (shorter wavelengths) but cannot penetrate as deep into the tissues. In diagnostic ultrasound frequencies above 1 MHz are used. For better tissue penetration frequencies of interest for our purposes are between 0.3 to 1 MHz yielding sufficient resolution and penetration at the same time Speed2001 [1]. Latest studies show that it is possible to use pulsed ultrasound for neurostimulation, ultrasound therapy such as treatment of pain and the repair of various tissues, however without clear knowledge which receptors or cell types we are targeting, and what is the mechanism of the ultrasound stimulation effects Vasquez2014 [2].

For the simple setup of the ultrasonic stimulation of cells we initially used ultrasonic baths (1) that are used to clean the laboratory equipment, small devices to clean jewelry or ultrasonic cell disruptors, which however offer little control over the intensity, frequency or pulse shapes and numbers of repetitions and are not appropriate to monitor activation of mechanosensors under the fluorescence microscope. However, one member of the team is a student of electrical engineering and this was the right challenge for him.

Different experiment configurations.
Testing homogeneity of pressure in each well with a plate immersed in the ultrasonic bath.

For the generation of specific shapes of ultrasound pulses researchers usually use a setup consisting of two signal generators (one for switching on and off the train of US pulses and the other one to produce the sinusoidal signal of suitable frequency). This signal is further fed to the amplifier and then to the ultrasonic transducer. Furthermore, an ultrasonic sensor is required to evaluate and control the magnitude of the ultrasound. Usually a hydrophone is used in combination with an amplifier and an oscilloscope. This setup is complex to establish and difficult to use. Therefore, the goal of a part of the iGEM 2016 group (student of electrical engineering) was to develop a device (named Moduson), which would be capable of providing appropriate signals required to perform specific ultrasonic experiments in a single apparatus and as such easy to use. The research and development of the device was performed in the Laboratory for Bioelectromagnetics/Faculty of Electrical Engineering/ University of Ljubljana under the advisor prof. Dejan Križaj and a company Noeto.

nbsp;The basic requirements for the device and realizations

Adaptability

The device should be designed to be as flexible as possible in order to be capable of delivering a wide range and different shapes of stimulation signals to stimulate cells under the microscope, cells in a petri dish, cells in a microplate immersed into a bath and to stimulate animals. In order to fulfill this requirement we selected a dedicated embedded measurement card Red Pitaya acting as an embedded computer. This embedded device is based on Linux system and can be completely customized to the user’s needs. Furthermore, it can be Wi-Fi controlled so the final application is based on modern programming tools such as JavaScript, C++, Html, etc. The final application is basically a web page, accessible with any computer capable of the Wi-Fi connection. This application gives complete control of all stimulation parameters and operation of the device. Another advantage of the platform used its capability of simple integration with Matlab through so-called SCPI commands, usually used in the instrumentation for easy control and data acquisition making the device perfect research and development tool. Simple example of SCPI commands in Matlab for pulsed bursts is shown below:

On the Figure 2 a typical sequence of a stimulation signal constructed of a set number of sine waves of defined frequency that is repeated for required number of times with selected repetition period is shown.

Signal parameters.
Parameters that can be set for a typical stimulation signal.

Simple graphical user interface

Users of the device do not need to be computer experts and do not need to have knowledge on the signal generators, amplifiers and oscilloscopes. Therefore, the motivation was to design and develop a simple user-friendly graphical interface with all the relevant parameters that need to be set in order to run the experiments from the Web page. To accomplish this, dedicated software was written that enables users to design and run the experiment and evaluate the results. Figure 3 presents a developed user interface. On the right side (US burst settings -> STIMULATION PULSE) we can set parameters, such as the amplitude of the signal, frequency and number of sine waves. Below these settings, repetition parameters of the signal (PULSE REPETITION) such as the number of pulse repetitions and its frequency can be set. When all parameters are set, the signal can be previewed by clicking the SHOW BURST button. The pulse sequence is initiated with the START button. The acquired signal from hydrophone is seen on the main plot. At the top of the interface, there are options to export the image of a graph and csv data of the acquired signal. Chosen values of parameters can also be saved for the next use of the device.

Application interface.
Web based interface used for setting of the signal parameters.

Signal amplifier

Once a suitable signal is generated, it needs to be amplified from a signal level (amplitude of 2 V) to several hundred volts or even above 1000 V, which poses a serious engineering challenge. For this purpose, we first designed a circuitry on a conceptual level and simulated it with LT Spice (4). In the simulations an electric model of a transducer was considered as a combination of capacitors, inductors and resistors wired in parallel (5). This model enables simulation of ultrasonic transducers with several resonant frequencies. Simulations show that our device can reach the power at transducer around 92W, which is close to the measured 86W in the real system.

Simulation scheme.
Scheme used in LT Spice simulations.
Model of transducer.
Equivalent model of ultrasonic transducer based on a BVD model.

We built the circuitry with real elements on a prototype board where several additional alterations were required, as the real elements do not have ideal characteristics as the ones included in the simulation. Another challenge was the design of a suitable transformer to increase the voltage amplitude of the signal. The transformer is also required to operate as an impedance transformer. Lowering impedance of the ultrasonic transducer enables higher current levels and consequently higher power values.

Moduson.
Constructed device with a simple interface; an ON/OFF button, a BNC output for the transducer and a button to trigger stimulation pulses. The pulses can also be triggered directly through the Web interface.

Evaluation of the developed device

Functionality of the Moduson was first tested without any connected load. Figure 7 presents a typical output measured with an oscilloscope for a signal of frequency 310 kHz.

Typical output signal without load.
Amplitude of acquired signal depends on the winding of transformer.

Developed device was tested with several transducers with resonance frequencies ranging from 300 kHz up to 1 MHz. Most experiments were performed with an unfocused transducer Olympus V318-SU (8) with a waterproof case, allowing it to be used in in vitro experiments.

Ultrasound transducer V318-SU.
Handy waterproof case allowed us to use the transducer in most experiments.

Figure 9 presents measured electric power using the transducer V318-SU. After partial compensation of reactive part, the measured real power reached 86 W. This result exceeded the set requirements.

Electric power of ultrasonic transducer before and after compensation.
Electrical power with compensation was significantly increased with serial compensation.

With compensation, voltage was considerably increased at contacts of transducer and reached around 900Vpp as shown in Figure 10.

Voltage at input of a transducer V318-SU.
Transducer requires relative high voltage to operate properly.

We measured the emitted ultrasonic pressure indirectly with a hydrophone RP 31l shown in Figure 11. This hydrophone has a sensitivity of 50mV/100kPa at a frequency of 310kHz. It can be connected directly to the oscilloscope input to detect the pressure of the ultrasonic waves. Pressure measured 4 mm from the transducer reached 7 kPa which gives the intensity of 33 W/cm2.

Hydrophone RP 31l.
Calibrated hydrophone used to measure pressure.

Example of an output signal measured by a hydrophone is presented in the Figure 12.

Voltage signal detected using the hydrophone.
With calibration data, we can determine pressure which corresponds to acquired voltage amplitude.

Set-up for experiments with ultrasonic pulses

After we successfully tested the Moduson device, we aimed to design measurement set-up for stimulation of cells cultivated in plastic 6-well plates, which allows insertion of the ultrasound transducer. In order to ensure repeatable conditions in every experiment the position of a transducer was fixed with a suitable holder. Several models of holders were designed for different experimental configurations and a 3D-printer was used to fabricate a dedicated holder (13). This allowed positioning of the transducer at the fixed height above the bottom of a well, which was crucial due to the series of maximal and minimal intensity of generated pressure.

3D printer.
Printing a holder for the ultrasonic transducer that we used for a 6-well plate.
Ultrasound based experiments on the microscope.
Ultrasonic transducer with a 3D printed holder in a 6-well plate.