IMPEDANCE SPECTROSCOPY FLOW CYTOMETRY:
PARAMETERS FOR LABEL-FREE CELL DIFFERENTIATION
Karen Cheung, Shady Gawad and Philippe Renaud
Microsystems Laboratory, STI-LMIS
Swiss Federal Institute of Technology Lausanne (EPFL), CH-1015 Lausanne, Switzerland
Abstract
This work presents label-free separation of cells in an on-chip flow cytometer based on
impedance spectroscopy. The amplitude, opacity, and phase information can be used for
discrimination depending on the cell types. Red blood cells, ghosts, and fixed cells are
differentiated based on differences in their membrane and cytoplasm dielectric properties. Among
other applications, detecting changes in cell conductivity and membrane capacitance will allow the
indentification of early-stage apoptosis or cancer.
Keywords: impedance spectroscopy, label-free, separation, flow cytometry
1. Introduction
As a non-invasive technique, dielectric spectroscopy is suitable for the characterization of
living biological cells. Impedance measurements over a wide frequency range give information on
cell size, membrane capacitance, cytoplasm conductivity, and cytoplasm permittivity as a function
of frequency [1]. This information can be used to distinguish cell populations without the need for
fluorescent, magnetic, or other cell markers.
The impedance spectroscopy flow cytometer used here permits rapid dielectric
characterization of a cell population with a simple microfluidic channel [2]. As cells flow
individually through the detection area of the microfluidic channel, they are measured at two
discrete frequencies which are applied simultaneously. The reference frequency is kept low and
constant, while the second frequency is swept from 300 kHz to 20 MHz. The amplitude and phase
information from the individual cells are averaged to give an impedance spectrum for the entire
population.
2. Theory
Biological cells are polarized in an AC electric field. At low frequencies, the cell is essentially
nonconducting due to the plasma membrane, which does not allow the passage of current. The low
frequency conductivity is the impedance parameter detected in a Coulter volume measurement, and
particles of the same volume but different dielectric properties will not be differentiated solely with
measurements at low frequencies. At intermediate frequencies, this membrane polarization
decreases. This phenomenon is known as the beta dispersion, or dielectric relaxation.
Measurements in the intermediate range of the β-dispersion give information about plasma
membrane properties. At high frequencies, the plasma membranes are minimally polarized, and
measurements here give information about the dielectric properties of the cell interior. The ratio of
the impedance amplitude at a high frequency to a low frequency, also known as opacity, can be
used as a measure of the difference in particle resistivity at the two frequencies [3]. Relative phase
is defined as the difference of phase to a reference signal. The amplitude, opacity, and/or phase can
be used to discriminate between different cell populations; the key parameter(s) will depend on the
cells under consideration.
3. Experimental
Three cell models were used: red blood cells (RBCs), ghosts, and RBCs fixed in
glutaraldehyde. Fresh blood was collected from healthy human donors in heparinized tubes. After
centrifugation, the buffy coat and plasma proteins were removed. The RBCs were washed and
suspended in phosphate-buffered saline (PBS).
RBC ghost preparation: RBCs were suspended in a hypotonic solution which lyses the cells and
releases the cell contents. The membranes were washed and resuspended in PBS to reseal the
membrane sacks. The resulting ghosts are clear membranes filled with saline solution.
RBCs fixed in glutaraldehyde: RBCs were suspended in various concentrations of
glutaraldehyde (0.02, 0.2, 0.5, 2.0, 5.0%) in PBS and incubated for 1 hour at room temperature.
Glutaraldehyde fixation cross-links protein amine groups in the lipid membrane. The fixed cells
were washed and resuspended in PBS.
4. Results and discussion
Since RBCs, RBC ghosts, and fixed RBCs are all similar in size, they can not be differentiated
using impedance signal amplitude (Fig. 1). The opacity of RBCs above 1 MHz increases with
increasing glutaraldehyde concentration (Fig. 2). With opacity, RBCs and ghosts follow the same
trend and cannot be discriminated (Fig. 3). In contrast, the fixed cells appear more opaque than
normal cells and are differentiated at high frequencies. In the low frequency range, where the
measurement only gives information about cell size, the opacity spectra for normal RBCs and fixed
RBCs overlap. As the measurement passes the dielectric relaxation, the spectra for normal RBCs
and fixed RBCs diverge. The relative phase above 10 MHz indicates that the ghosts, which are
filled with the buffer solution, are more conductive on the interior than RBCs (Fig. 4).
Increased opacity in the high frequency range can be attributed to a decrease in cytoplasm
conductivity. Both a decrease of ion mobility and the fixation of proteins within the cytoplasm can
contribute to decreased cytoplasm conductivity. Red blood cells fixed using increasing
concentrations of glutaraldehyde show increasing opacity, possibly indicating a higher degree of
crosslinking between cell membrane and the cytoskeleton. A change in the membrane properties
will shift the characteristic frequency of the beta dispersion and change the opacity spectrum.
While normal RBCs are modeled as thin membranes surrounding a conductive interior, crosslinking of cell proteins using glutaraldehyde could have bonded the cytoplasmic membrane proteins
more tightly to the membrane. Since capacitance scales inversely with distance of charge
separation, merging the thin membrane with a thicker layer of cytoplasmic proteins effectively
increases the membrane thickness and decreases membrane capacitance.
5. Conclusions
Measurements from several frequency regions give information about the dielectric properties
of the membrane and cytoplasm since cell membranes exclude low-frequency currents and pass
high-frequency currents. Red blood cells, ghosts, and fixed cells are differentiated based on
differences in their membrane and cytoplasm dielectric properties. This label-free method
implemented in a microfabricated flow cytometer is a powerful tool for the characterization of cells.
Future work will aim to distinguish more subtle differences between cell types.
Acknowledgements
This work was funded by the Swiss Innovation Promotion Agency (CTI project 5803.1) and
LEISTER Microsystems. Microfabrication was done in the Center of MicroNanoTechnology
(CMI).
References
[ 1]
K Asami, T Yonezawa, H Wakamatsu, N Koyanagi, Bioelectrochemistry and Bioenergetics, 40
(1996) 141-145.
[ 2]
S Gawad, L Schild, Ph Renaud, Lab on a Chip, 1 (2001) 76-82.
[ 3]
RA Hoffman, TS Johnson, WB Britt, Cytometry, 1:6 (1981) 377-384.
Figure 1. Amplitude measurements of RBCs,
fixed RBCs, and ghosts show no differentiation
based on signal amplitude since the cells are
similar in size. Amplitude measurements are
taken at a single frequency. The low frequency
conductivity is the impedance parameter detected
in a Coulter volume measurement.
Figure 2. Opacity measurements of RBCs and
fixed RBCs. Opacity is defined as the ratio of
signal amplitude at a given frequency to a low
frequency (602 kHz) amplitude. Glutaraldehyde
fixation crosslinks the cytoskeletal network and
decreases cytoplasm conductivity. The high
frequency opacity increases with increasing
glutaraldehyde concentration.
Figure 3. Opacity measurements of RBCs, ghosts,
and fixed RBCs. RBCs are not discriminated
from ghosts by opacity, while fixed cells and
RBCs are clearly differentiated in the high
frequency range of opacity.
Figure 4. Relative phase measurements of RBCs,
ghosts and fixed RBCs. Relative phase is defined
as the difference of phase to a reference signal
(602 kHz). RBCs and fixed RBCs are well
differentiated from ghosts at high frequency by
the phase information.
Each point represents measurements from ~ 800 cells.