Title: UltraWideband Electronics, Design Methods, Algorithms, and Systems for Dielectric Spectroscopy of Is
1Ultra-Wideband Electronics, Design Methods,
Algorithms, and Systems for Dielectric
Spectroscopy of Isolated B16 Tumor Cells in
Liquid Medium Erick N. MaxwellDefense of
Doctoral DissertationChairperson Venkat
Bhethanabotla, Ph.D. Major ProfessorThomas
Weller, Ph.D. Committee Kenneth A. Buckle,
Ph.D., P.E. Dennis K. Killinger, Ph.D.Geoffrey
Okogbaa, Ph.D.Arthur David Snider, Ph.D., P.E.
2OUTLINE
- Introduction
- Literature Review
- Research Question
- Approach and Methodology
- Contributions (4 of 5)
- Future Work and Direction of Research
- Conclusion
- Acknowledgements
3INTRODUCTIONBackground and Motivation
0200 / 0200
- Isolated tumor cells (ITCs) are individual tumor
cells - spread to the lymph nodes or general circulation,
which includes - Blood and
- bone marrow
- ITCs are believed to be a mechanism involved in
formation of tumors in distant sites - Significance of circulating ITCs in the blood has
not been established due to conflicting results - Reported on a connection between circulating
tumors in blood and the formation of hematogenous
metastasis - Keiichiro Uchikura and others,
- Reported that in 15 of sentinel lymph nodes in
which ITCs are detected, no metastasis is found - Hermanek, Hutter, Sobin
- Consequently, the independent prognostic
significance of circulating ITCs has not been
proven - The tumor-lymph node-metastasis (TNM)
classification system - The Most widely used means for classifying spread
of malignant tumors - Does not assign clinical significance to the
occurrence of ITCs
4INTRODUCTIONBackground and Motivation (Continued)
0100 / 0300
- The Pathology Associates of Lexington,
Pennsylvania stated the following - Regardless of the lack of any current consensus
as to the significance of isolated tumor
cells...excellence in surgical pathology practice
requires that a staging lymph node exam actually
be truly negative when diagnosed as negative. - Although a physician may consider the results
insignificant, it does not effect the reporting
that comes from the Pathologist - Detecting, quantifying, and characterizing ITCs
is an integral part of the metastatic work-up for
cancer patients - Surgical pathologist and cytologist have an
interest in characterizing ITCs - For its potential to provide data for cancer
staging, classification and treatment
5INTRODUCTIONResearch Question
0210 / 0510
- The Problem
- An optimal method for detecting single tumor
cells not established (Weaver, Krag, et. al.) - Two categories for available methods
- Morphologic methods i.e. HE-Staining and
IHC-Staining - Nonmorpologic methods i.e. Flow cytometry
- HE-staining is non-ideal because
- A single section evaluates less than 1 of the
lymph node - Not used exclusively for diagnosing disease
- Flow cytometry
- Has emerged as a useful application in clinical
pathology - Range in price from 50,000.00 to 500,000.00
- Paraskevas, et al used dielectric spectroscopy
(DS) for evaluating oil-based emulsions - high-sensitivity,
- low-cost tool
- This research applies frequency-and time-domain
dielectric spectroscopy to characterize and
quantify ITCs in medium - To what extent are different volumes of cancer
cells electrically distinctive? - What formalism should be applied for
distinguishing ITCs? - Does the literature support a hypothesis that
they are distinctive?
6LITERATURE REVIEWDielectric Spectroscopy
0320 / 0830
- What is dielectric spectroscopy (DS)?
- measures material properties over frequency by
capturing polarization effects - Dielectric Material
- A material is a dielectric if it
stores/dissipates energy with application/removal
of E-field - The term Permittivity describes this
interaction - Kramers-Kronig Relations
- Provides a set of mathematical properties that
connect the real and imaginary parts for any
complex analytic function - If an oscillatory force is applied to a physical
system - Force is applied in one direction during ½ cycle
and reversed - The system does not respond instantaneously to
the change in direction (or response function) - As frequency increases system has less time to
react before force changes direction - the response function diminishes with an increase
in frequency - Provides a fundamental description for dielectric
response of a material (real-dissipation) - Peter Debye
- Provided a formula to capture the Kramers-Kronig
system response for dielectric material - Called the system response dielectric relaxation
(or dielectric dispersion) - Called the delay between the application of an
external electric field and orientation of an
electric dipole moment a Phase delay - permittivity decreases from a static value at low
frequencies to a smaller value at high
frequencies - attributed occurrence of material relaxation to
dipole polarization
Dielectric mechanisms versus dispersion type.
7LITERATURE REVIEWB16-F10 Cell Characterization
0300 / 1130
- Irimajiri, Hanai and Inouye demonstrated that
every shelled particle interface in a suspension
gives rise to a single Debye-type dispersion - Consider the structure of B16-F10 mice tumor
cells - are generally round in shape with a single cell
membrane and cytoplasm - Based on the microscopic structure it is
reasonable to anticipate molecular, atomic, and
sub-atomic dielectric mechanisms - Asami
- Characteristic polarization for heterogeneous
systems is interfacial polarization, which
demonstrates ß dispersion, - Asserted that the microscopic mechanisms may be
ignored because they are not dominant in a cell
suspension - Pauly and Schwan showed that for biological cells
the dispersions degenerate to a single dispersion
process - What this means is that a Cole-Cole dispersion
model is sufficient - Now Lets Consider What support is there for
relating permittivity to cell quantity? - Maxwell-Wagner
- formula for describing the dielectric behavior of
mixtures - provides a means to extract properties of
individual cells from measurement of the cell
suspension - cell suspension is a function of the permittivity
of the medium and cell as well as volume fraction
that cells occupy - Maxwell-Wagner formalism supports the possibility
for using the permittivity of a mixture to
estimate the volume fraction of cells in the
suspension!
8LITERATURE REVIEWB16-F10 Cell Characterization
(Continued)
0130 / 1300
- What measurement technique?
- There are many challenges with T/R methods
- T/R Fixtures
- Not suitable for holding liquid samples
- High fixture complexity and cost
- Frequency domain dielectric spectroscopy (FDDS)
- No ideal method for extracting permittivity
exists - Time domain dielectric spectroscopy (TDDS)
- Provides best resolution of structural properties
of cell - Simple, low-cost circuits for generating
sub-nanosecond tunable pulses unavailable
NIST-National Institute of Standards and
Technology
9RESEARCH QUESTION AND CONTRIBUTIONS
0100 / 1400
- Provide an answer to the question
- To what extent are different volumes of cancer
cells electrically distinctive? (by relating
dielectric properties to cell quantity) - Contribute
- Novel circuits for tunable pulse generation
(ultra-wideband generators) by proposing a new
approach to generator design, - A technique and circuits for shaping
sub-nanosecond UWB pulses, - A low-cost method for transmission-reflection
(T/R) measurements that is based on the
construction of a fixture from semi-rigid coaxial
transmission line with a Teflon-PolyTetraFlouroEth
ylene (PTFE) core, for measuring liquid
specimens, - A genetic algorithm (GA) approach for
permittivity extraction which circumvents the
problem of half-wavelength resonance in the
Nicholson-Ross-Weir (NRW) technique and extends
Oswalds approach, and - An investigation of the capacity for dielectric
spectroscopy to quantify isolated B16-F10 tumor
cells in McCoys liquid medium.
10APPROACH AND METHODOLOGYOverview
0100 / 1500
- UWB measurement of cancer cells in media
- circuits
- techniques
- models
11UWB SIGNAL GENERATIONOverview
0230 / 1730
- What is UWB?
- An intentional radiator that, at any point in
time, has a - Fractional bandwidth equal to or greater than
0.20 or - 10dB bandwidth equal to or greater than 500
MHz... - What has been done in low cost Generator Designs?
- Step Recovery Diodes (SRDs) usedAgilent, Lesha
et.al, - J. Han and C. Nguyen Microstrip Generators /
Tunable - What are the primary challenges?
- Theoretical framework not well defined
- Solid-state tunable generators have complex
topology - Loss in symmetry
- How we addressed these challenges?
- Conventionally
- SRDs in series configuration
- Generate impulse/Gaussian then tune pulse
- Change the philosophy for producing pulse
- New philosophy circumvents challenges
- Produce a step/square waveform using an SRD
- Provide a mechanism for tuning rise-time of the
step
UWB Signals
Complex Topology
Loss in symmetry
12UWB SIGNAL GENERATION Background
0200 / 1930
- A SRD is a p-n junction diode
- stores electric charge during forward conduction
- rapidly removes these charges during reverse
conduction - Time associated with transition can be less than
60 psec - called snap-off, charge-storage, and memory
varactor diode - Boff, Moll and Shen noted that this fast
transitioning edge occurs in a p-n junction diode
that is doped with linearly or exponentially
graded impurity - New philosophy requires square wave or step
- Model a SRD clipping circuit
- Peak of positive and negative going sinusoid is
clipped - Transforms a sinusoid to a fast edge square wave
13UWB SIGNAL GENERATION Results and Discussion
0130 / 2100
(C)
(E)
(A)
(D)
(B)
(F)
14UWB SIGNAL GENERATION Summary
0130 / 2230
- Contributions
- A new design approach
- Low complexity tunable UWB pulse generator
- Advantages
- Simple design,
- Positive- and negative-going pulses possible
- Disadvantages
- Lower voltages due to shunt diode
- Capacitive tuning complicates transmission line
approaches for differentiation - Capacitor is an integral part of RF/Microwave
differentiator - Must decouple the VERC and differentiator circuit
To circumvent disadvantages provide an
alternative method for derivation
15UWB PULSE SHAPINGIntroduction
0030 / 2300
16UWB PULSE SHAPINGOverview
0200 / 2500
- What is UWB pulse shaping?
- Pulse formation (or pulse shaping)
- critical to the performance of a UWB system
- used to optimize spectrum for meeting FCC
emission spec - In communication, radar, and DS measurement
systems - What has been done in pulse shaping designs?
- GaAs MESFETs, non-linear transmission lines,
short-circuit stubs and resistive-reactive
circuits - waveform response to circuit reactance is
fundamental - reactive elements form a simple RC or RL network
- What are the primary challenges?
- RC time constant requires lt 20 pF cap for shaping
(50O) - Stray or parasitic capacitance can easily modify
waveform - How we addressed those challenges?
- Coupled-line coupler and Schottky detector
differentiators - Provides a means to isolate VERC and
differentiator - Mutual and junction capacitances are small enough
17WAVEFORM DIFERENTIATOR Background
0130 / 2630
(A)
(A)
(B)
(B)
(C)
18WAVEFORM DIFERENTIATOR Background (Continued)
0130 / 2800
(A)
(B)
(A)
(C)
(B)
19WAVEFORM DIFERENTIATORResults and Discussion
(Continued)
0100 / 2900
MCS3P Fabrication and Measurement
Multi-Port Circuit for Simultaneous Shaping of
Sub-nanosecond Pulses
20WAVEFORM DIFERENTIATORSummary
0050 / 2950
- Contributions
- Novel techniques and circuits for sub-nanosecond
pulse differentiation, including - Novel coupled-line coupler differentiator
- Novel Schottky detector differentiator
- Multi-port Circuit for Simultaneous Shaping of
Sub-nanosecond Pulses - Advantages
- Provides alternatives to sub-nanosecond pulse
shaping - Disadvantages
- Minor variations in fabricated design has a
notable effect on signal
21TEST FIXTRUE DESIGNIntroduction
0010 / 3000
22T/R TEST FIXTRUE DESIGNOverview
0250 / 3250
- Why T/R test fixture design?
- TEM mode of propagation
- Complex relative permittivity and permeability
- What has been done in T/R fixture designs?
- Applied in variety of dielectric measurement
systems including produce, biomaterials, and UWB - Constructed with an air core
- What are the primary challenges?
- Most suitable for measuring solids (NIST)
- Sample holder separate from fixture (must be
small) - Intricate and high cost!
- How we addressed those challenges?
- Construct a fixture without an air core
- Semi-rigid coaxial transmission line w/
Teflon-PTFE
23T/R TEST FIXTRUE DESIGNBackground
0200 / 3450
- Nicholson Ross Weir Technique
- provides a solution for extracting complex
relative permittivity and permeability - from measured S-parameters
- fixture step-discontinuity at PTFE-specimen
- characteristic impedance (Zw) for semi-rigid line
based on continuous interface - Somlo showed that a step capacitance (Cd) may
describes coaxial step in outer conductor
24T/R TEST FIXTRUE DESIGNResults and Discussion
0220 / 3710
Adjusting NRW Algorithm for step-discontinuity
Simulated
- A simple approach to correcting for the effects
of the coaxial step was developed. - This approach entailed
- Computing permittivity with and without a step
discontinuity over several simulated sample
values - Calculating an error for difference between
computed permittivity values, - Fitting an equation to the calculated error, and
- Correcting NRW permittivity
Measured
25T/R TEST FIXTRUE DESIGNSummary
0110 / 3820
- Contributions
- Low-cost, Low-complexity, T/R Fixture capable of
measuring liquids - Advantages
- (See Contribution)
- Disadvantages
- Sample preparation is difficult
- Fixture dimensions is critical in permittivity
measurement - Fixture is leaky
- NRW approach problematic
We need a new approach to permittivity
determination
Genetic Algorithm approach was developed to
circumvent problems
26PROPERTIES OF CANCER CELLS Introduction
0010 / 3830
27PROPERTIES OF CANCER CELLS Background
0150 / 4020
- Describe Tumor Cells
- B16-F10 tumor line was derived by
- Injecting B16 tumor cells into syngeneic C57BL/6
mice - Harvesting the secondary growth after two to
three weeks - Placing the tissue in a culture and
- Injecting into new syngeneic mice
- Note The above process was repeated ten times ?
designation F10 - B16 line is desirable because
- Shows stable metastatic properties, even after
many tissue subcultures - American Type Culture Collection supplied the
primary B16-F10 tissue culture for this research - Harvesting tissue from mice was unnecessary
- Growing cells in culture mandatory to achieve
large counts for testing - Describe McCoys Medium
- Culturing environment for growing and testing
cells - Required 5-8 days in culture to achieve desired
cell volume (3 M cells/mL) - Cell growth/death continued even during testing
28PROPERTIES OF CANCER CELLS Results and Discussion
0230 / 4250
- Characterized B16 cell suspensions of 0, 1, 2 and
3 M cells/mL - Frequency domain system (32 samples, 8 per test
volume) - Time domain system (32 samples, 8 per test
volume) - Paired t-test was conducted to determine the
significance differences between these volumes - The Goal to disprove null hypothesis there is no
difference in the static permittivity - 90 confidence level
- If area under one-tail t-distribution was less
than 10 significance level ? Reject Null
Hypothesis
29PROPERTIES OF CANCER CELLS Results and
Discussion (Continued)
0200 / 4450
30PROPERTIES OF CANCER CELLS Summary
0040 / 4530
- Contributions
- A preliminary investigation of the capacity for
dielectric spectroscopy to quantify isolated
B16-F10 tumor cells in McCoys liquid medium - Advantages
- Despite cell contamination in frequency domain
data and - Secondary reflections in time-domain system
- ITCs can be characterized using dielectric
spectroscopy - dielectric spectroscopy relates to cell quantity
- Disadvantages
- Cell preparation difficult
- Dielectric spectroscopy requires an evenly
distributed population of cells - Advantage of UWB measurement system not evident
31Future Work and Direction
0310 / 4840
- Conduct FDDS testing again
- Verify cell contamination at 1 M cells/mL
- Goal of reducing standard uncertainty
- Solve problem with secondary reflection in TDDS
- Reflection due to reversed reflection from sample
interface - Delayed this reflection in subsequent experiment
- Increasing length of transmission line between
the Sinusoidal source and UWB Generator - Conduct TDDS testing again
- Standard uncertainty mainly due to uneven cell
distribution in population - Develop a method for ensuring consistent sampling
of population - Reduce standard uncertainty in the measurement
results - Test smaller volumes
- Different volumes of cancer cells were
electrically distinctive so long as the cell
volumes are large enough to overcome the sources
of uncertainty - Revisit hemocytometer for verifying cell volumes
32Conclusion
0120 / 5000
- Designed/Fabricated Novel UWB Pulse Shapers and
Generators - Establishing an approach for UWB generator design
- Modeling / Characterizing of SRD for simulation
- 3 Novel Circuits (2 Provisional patents)
- Schottky detector and
- coupled-line coupler differentiators
- MCS3P circuit
- VERC technique/Circuit
- Developed a T/R Fixture ( Provisional Patent)
- Introduced an enhanced Genetic Algorithm approach
to parametric extraction - C using Microsoft Visual C 2005 express
edition - Genetic Algorithm Library from MIT
- Preliminary testing of ITCs in medium
- Using low cost time and frequency domain
dielectric measurement system - Showed correlation between tumor cell quantity
and electric permittivity - Concluding Statement
- Tools of research are necessary for experiments
to assess the prognostic significance ITCs - DS has the potential to provide data unavailable
using conventional methods - It offers another modality from which information
can be assembled for increased prognostic value
for Cytologists and Pathologists
33Acknowledgements
0000 / 0000
- Special Thanks to
- Dr. Tom Weller (Advisor)
- Bernard Batson
- Dr. Tom Fare (Rosetta Inpharmatics)
- Dr. Mark Jaroszeski (Dept. of Chemical
Engineering) - Jose Rey (Dept. of Biomedical Engineering)
- Dr. Jeffrey Harrow (VA Medical Center)
- Committee Members
- Dr. Kenneth A. Buckle
- Dr. Dennis K. Killinger
- Dr. Geoffrey Okogbaa
- Dr. Arthur David Snider
- Chairperson
- Dr. Venkat Bhethanabotla
- This work was supported financially in part by
- Center for Wireless and Microwave Information
Systems - Harris Corporation
- Florida Education Fund (McKnight Fellow)
- National Science Foundation (USF-IGERT)
Thank You!