WIXLIB

Figure 4 Equivalent electrical model of a cell. Re represents resistance of ECF. Ri represents resistance ofICF. Rm represents resistance of membrane. And Cm represents capacitance of membrane. (8)Based on the model introduced above, the impedance of a cell can be written as:Re 1 j CRi 1 Z Re Ri j C 1 j C Re Ri [4]At low frequencies, the electrical current is impeded by the membrane. So the ECF plays amajor role in conducting electrical current. In the extreme case, when a DC current is applied,the measured signal would be related only to ECF. This could also be demonstrated with theequation above. When the frequency 0, the expression for the impedance could be writtenas:Z Re .[5]At high frequencies, the electrical current passes through the membrane and the measurementis dependent on both ECF and ICF.2.2.2β dispersionThe dielectric properties of living tissues and cell suspensions have a dispersive behavior inresponse to electrical currents. This means their dielectric properties like permittivity andconductivity depends on frequency. According to the changes in permittivity and conductivity,Schwan defined three major dispersions: α, β and γ dispersion. A fourth dispersion widow, δdispersion, between β and γ dispersion has also been noticed. (10)

Figure 5 Frequency dependence of permittivity and conductivity of living tissue (11).β dispersion includes the frequency range from 1kHz to several Mhz. This dispersion isconsidered to be related to the behavior of membrane. The properties of cells are mainlyinfluenced by dielectric properties of membrane and the process of charging and dischargingthe membrane. Because of this reason, the measurement of electrical bioimpedance is usuallyperformed in this range. And for single frequency bioimpedance analysis, the frequency istypically 50kHz.2.3 Bioimpedance measurement system designThere are two methods to obtain the impedance. One is to inject an electrical current andmeasure the corresponding voltage. Another method is to apply voltage to the object of interestand measure the current. Both methods are used in bioimpedance measurement, and both havetheir advantages and limitations. Compared to the BIA device that applies voltage to the target,the current based BIA is able to manage currents within medical safety limits more easily.Besides, it suffers less noise due to spatial variation and produces better bioimpedancetomography results. However, current source gives poor performance at high frequency due toits decreased output impedance. Voltage sources applications work with higher bandwidth,however low dc SNR. (12) The current source based BIA is more commonly used in the EBImeasurement devices today. In this thesis work, the focus will be the impedance measurementsystem with a current source.2.3.1Electrodes and connectionBioimpedance measurement devices typically consist of two major functions: first inject analternating current to the biological material and then measure the potential difference fromtwo ends. Fulfilling these two functions requires two, three or four electrodes (13). Two‐electrode method and four‐electrode are introduced here. Three‐electrode method is not verycommon. One example using three‐electrode method can be found in (14).Two‐electrode methodThe two‐electrode method or bipolar method uses two electrodes to inject a known current tothe biological material and measures the voltage drop over this material from the sameelectrodes. See Figure 6. Apart from the impedance of biological material Zm, the impedances attwo electrode contacts Zc are also included in the measured impedance.6

Figure 6 Biopolar bioimpedance measurement system (a) and its equivalent electrical circuit (b).So the two‐electrode method only works when the impedance of the tested material is muchhigher than the impedance of electrode contacts at the BIA working frequency. If a BIS device isused, two‐electrode method is not an option because the impedance at electrode‐electrolyteinterface is susceptible to frequency variations. (15)Four‐electrode systemThe four‐electrode system is also known as tetrapolar system. It uses separate electrode pairs toinject current and measure voltage.Figure 7 Tetrapolar bioimpedance measurement system (a) and its equivalent electrical circuit (b).Assuming the voltmeter is ideal, the measurement result will involve only the impedance of thetested material. This makes the four‐electrode method most commonly used in today’sbioimpedance measurement.In practice, however, the impedance at the contact sites still has an impact on the measurementbecause of common voltage introduced by contacts’ impedance and common mode rejectionratio (CMRR) of the differential amplifier in the voltmeter. (15) (16)2.3.2Current generationAn alternating current source can be obtained by combining a voltage generator with a voltage‐to‐current convertor circuit. For a current source, high output impedance is desired in EBImeasurement, especially in BIS where the working frequency goes up to several Mhz. However,the output impedance of a current source drops as the frequency increases. This is caused bythe parasitic capacitances that come from many components in the circuit, for example betweenelectrodes. The output impedance of the current source should be at least 100 kOhm (17).Sinusoidal signal generationSinusoidal signal can be generated from either analog or digital method.

One example of analog method is to use a sinusoidal oscillator that converts DC power to aperiodic waveform. This method requires complexity in circuits and high accuracy in some ofthe electrical components. And it lacks flexibility compared to digital methods.Generating waveforms with digital modules is called direct digital synthesis (DDS). The blockdiagram below shows the idea of a simple DDS system (Figure 8) (18). DAC takes values from alook‐up table where the address counter points. As the counter steps through the look‐up tablerepeatedly, a periodical wave is obtained from the DAC output. The low‐pass filter (LPF) afterthe DAC filters out the high‐frequency harmonics and makes the curve gisterDACLPFFigure 8 Block diagram of a simple DDS system.VCCSMany studies have been conducted on the VCCS design to achieve as large output impedanceat high frequencies as possible (17) (19). Some typical VCCS configurations include Howlandcircuit, load‐in‐the‐loop circuit, etc. More detailed descriptions can be found in Section 3.3.1.2.3.3 Impedance analyzingOne of the most popular methods for analyzing the impedance of biological material is thequadrature demodulation (QD) method (20). The basic idea is introduced here.Consider i t I cos( 0t ) , by adding the phase shift caused by the impedance Z, theexpression of voltage can be written as:v(t ) Z i(t ) I Z cos( 0t ) I Z cos( 0t ) cos I Z sin( 0t ) sin , [6]where the two items are in phase and in quadrature with the injected current. Then theimpedance Z can be expressed as:Z R jX Z cos j Z sin [7]In the upper branch, the voltage is multiplied by cos( 0t ) , so the signal before low‐pass filter is:v1 (t ) v(t ) cos( 0t ) I Z cos( 0t ) cos( 0t )[8]1 cos(2 0t )sin(2 0t ) I Z (cos sin )22The high frequency components are filtered out by the low‐pass filter, so the output containsonly1III Z cos which can be written as R . Similarly, the output of lower branch is X2228

Figure 9 Quadrature demodulation method.Other impedance analyzing methods include measuring the amplitude and phase using Gain‐phase detector (21), bridge method (22), etc.2.4 Communication protocolCommunication between devices requires a common language that could make dataidentifiable. Communication protocols are such rules that define the formats of data and maycontain other information like the speed of transmission, the order of data bits, direction ofoperation, etc.2.4.1I 2CInter‐integrated circuit (I2C) is a multi‐master and multi‐slave protocol. It uses two bidirectionalsignal lines. One is the serial data line (SDA) for data transmission, and the other is the serialclock line (SCL) for two‐wire interface clock frequency.Figure 10 I2C connection with one master and three slaves (23)Data transmitted between master and slave device always begins with a START bit and endswith a STOP bit. After the master device starts data transmission, the master device reaches toeach slave device with the slave’s unique address. If the address matches, the slave will respondto the master with an AKCNOWLEDGE signal.2.4.2SPISerial peripheral interface (SPI) is a four‐wire full‐duplex interface allowing a single master tocommunicate with one or multiple slaves. Master In Slave Out (MISO) is used for sending datafrom slave to master. Master Out Slave In (MOSI) is used for sending data from master to slavedevice. Serial Clock (SCK) is configured by the master device and is used to synchronize datatransmission. Slave Select (SS) is used to select the slave device that the master wants tocommunicate with. MOSI, MISO and SCLK are common to all slave devices, while SS is specificfor each slave. The slave device is enabled only when its SS pin is low. See Figure 11.

Figure 11 SPI communication with single master and single slave (24)2.4.3I2C vs. SPIBoth I2C and SPI are commonly used in electronic devices. However, they have some importantdifferences that make them suitable for different purposes. Understanding the differencebetween I2C and SPI helps to select a proper interface for certain project. Two aspects arediscussed below:Wiring: I2C is a two‐wire interface, while SPI typically requires 3 N signal lines for N slavedevices.Speed: I2C is slow compared to SPI. It transfers data at a speed of 100 kbps in standard mode, 400kbps in fast mode, 1Mbps in fast mode plus and 3.4 Mbps in high‐speed mode (23). But high‐speed mode is not always feasible because of limitations on the master side. SPI has no specificworking mode or any defined limit. The throughput rate varies from device to device, and cango higher than 10 Mbps.2.5 Arduino UnoAruidno Uno (Figure 12) is the latest revision of the basic Arduino USB board. It is based on themicrocontroller ATmega328. The system clock is 16 Mhz. The board has 14 digital input/outputpins and 6 analog inputs. Some of these pins have special functions for communicating withother devices or microcontrollers. (25)For SPI communication using SPI library:Table 2 SPI pins on Arduino UnoPin 10Pin 11Pin 12Pin 13Slave selection (SS)Master out slave in (MOSI)Master in slave out (MISO)Serial clock (SCK)For I2C communication using Wire libray:Table 3 I2C pins on Arduino UnoPin A4Pin A5Serial data (SDA)Serial clock (SCL)Arduino Uno could be programmed with Arduino software by simply connecting the board tocomputer via USB port. Arduino boards come with an integrated development environment(IDE) which is an open‐source software based on Processing, avr‐gcc, and other open sourcesoftware (26). The Arduino programs can be written in C/C . Arduino itself has its ownstandard library and some other libraries that have made some transforms on C/C language.10

A typical Arduino program has two major functions: setup(), which runs in the beginning of aprogram and initializes settings; and loop(), which is called repeatedly to fulfill desired tasks.Figure 12 Arduino Uno front (25).

3 METHODS3.1 System ImplementationA simple impedance measurement system was implemented in this thesis. Arduino Uno wasused as a microcontroller. It wrote to a DAC and generated a sine waveform. A low‐pass filterwas connected to the DAC output to smooth the signal and a high‐pass filter was connectedafter the low‐pass filter. The high‐pass filter was used to remove the DC component from DACoutput, since the output range of DAC used in the project was 0‐5V (27). The voltage sourcewould then be transferred to a current source after VCCS. A DC offset was added to the voltagesignal over the RC model to fit the analog input range of ADC. The ADC measured voltagesover the RC model and a reference resistor. Voltage data was then sent to Arduino Uno. Finally,samples were sent to Matlab and impedance was calculated and displayed in Matlab. The blockdiagram of major function blocks in the system is shown in Figure 13 below.DC power supplyDACVVCCSI2R1C modelVADCMicrocontroller(Arduino Uno)MatlabSamplesFigure 13 Simplified block diagram of the impedance measurement system.3.2 Source generation3.2.1Generating waveforms from look‐up tableA sinusoidal voltage signal was generated from DAC using the DDS method. To generate thiswaveform, a sine wave look‐up table is needed. The look‐up table stores both amplitude andphase information of a sine (or cosine) wave. When reading from the look‐up tablecontinuously, the index of the amplitude value increases and so does the phase of the outputwaveform. To achieve n‐bit resolution, a sinusoidal cycle is divided into 2n phase points (Figure14). If these points are read by DAC successively without skipping points, the frequency of theoutput waveform obtained will be:f out 1 fin2n[9]

Figure 14 A sinusoidal cycle is divided into 2n points.The frequency is changeable by changing the input clock frequency, rewriting the look‐up tableor skipping points.In our project, the sine wave look‐up table was stored in the flash memory of Arduino. Toachieve the highest frequency, 32 points were taken for one sinusoidal cycle.3.2.2MCP4725MCP4725 (27) is a single channel 12‐bit DAC supporting I2C interface. This chip is chosenbecause it is easy to use. It has three modes: standard mode supports a baud rate of 100 kbps;fast mode supports a baud rate of 400 kbps; high‐speed mode supports 3.4 Mbps. Themaximum SCL clock frequency supported by ATmega328 on the Arduino Uno board is 400kHz (28). So it is only possible to use standard and fast mode with Arduino Uno.Fast mode communication was used in the thesis to get as high speed as possible with availablehardware components. Commands that need to be written in the microcontroller are shown inFigure 15.The output voltage is given by:VOUT VREF Dn4096[10]whereequals to the single power supplyandis the input code. In our case,is3.3 V from Arduino Uno board. The output signal ranges from 0 to 3.3 V. And the smallestvoltage difference that MCP4725 can produce is 0.8 mV.Arduino Uno supports two‐wire interface communication with the wire library. Commoncommands include: Wire.begin() to initiate the wire library, Wire.beginTransimmission(address) tobegin transmission to the I2C device with the given address, Wire.endTransmission() to end thetransmission and so forth. Detailed description and instruction for the library can be seen in (29).14

Figure 15 Fast mode write command. (27)3.3 VCCS3.3.1VCCS configurationsLoad‐in‐the‐loopA single operational amplifier (Op‐Amp) can work as a simple and effective VCCS. In a circuitlike Figure 16, if the Op‐Amp is ideal, the electrical current flowing through the load will beequal to that in R1. The output current can be written as:I load V1R1[11]which is independent of the load value.Figure 16 Simple load‐in‐the‐loop circuit.

Seoane et al (30) proposed an enhanced version based on simple load‐in‐the‐loop configuration.A current conveyor was added to the first stage so that the output current would beindependent of the value of resistor R3 that is connected to the inverting input of the secondstage. This resistor can be changed to get higher output impedance (Figure 17).Figure 17 A single Op‐Amp VCCS circuit driven by current conveyor. (30)Figure 18 shows a simplified equivalent schematic of AD844. The current flowing between theinput nodes gets replicated and flows in resistor Rt. In the method proposed by Seoane et al,this current was connected to the next stage via AD844 Pin 5. The amplitude of current inFigure 17 can be calculated approximately by:I V1R1 RIN[12]where the typical value of RIN is 50 Ohm (31)Figure 18 Equivalent circuit of AD844. (31)Enhanced Howland circuitThe enhanced Howland circuit has an advantage over other current source configurations: itneeds only one Op‐Amp and five resistors to work. The circuit is comparably simple. Theoutput current is given by:Vin R3R4 R2[13]R3 R4 R5 R2R1[14]IL The resistance follows the ratio:16

Figure 19 Enhanced Howland

to create a voltage source with DAC and Arduino Uno; to collect electrical signal with ADC and Arduino Uno; to collect samples from Arduino Uno in Matlab; to calculate and display the impedance in Matlab; 1.3 Outline This thesis is divided into eight chapters.