0196-4763/81/0106-0377$00.

00/0
CYTOMETRY Val. 1,No. 6, 1981
Copyright 0by the Society for Analytical Cytology Printed in U.S.A.

ORIGINAL ARTICLES

Flow Cytometric Electronic Direct

Current Volume and Radiofrequency
Impedance Measurements of Single Cells
and Particles'
R. A. Hoffman, T. S. Johnson and W. B. Britt
Life Sciences Division, University of California, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (R.A.H.,
T.S.J., W.B.B.) and Ortho Diagnostic Systems, Westwood, Massachusetts 02090 (R.A.H.)
Received for publication December 2, 1980; accepted February 19, 1981

A flow-system instrument is described membrane produce rf-impedance clSanges

that uses direct current and radiofrequency
(rf) currents to detect simultaneously the
low and high frequency impedance changes
produced by biologic cells or particles sus-
pended in saline traversing through a 93-pm
diameter sensing orifice. For nonmembra-
nous particles and plastic microspheres both
the Coulter direct current volume and rf-
parameter signals are proportional to par-
ticle volume. Cells having an intact plasma
dependent additionally on the electrical
properties of the plasma membrane and in-
tracellular structures. Thus, biologically dif-
ferent cells having the same Coulter direct
current volume can be distinguished if they
differ in their electrical rf impedance prop-
erties.
Key terms: Flow cytometry, direct current
cell volume, radio-frequency impedance

One of the fist physical parameters measured using flow
cytometric methods was the electronic cell volume or Coulter
volume (3) which detects the change in direct current (dc)
electrical resistance of a small sensing orifice caused by entry
of a cell or other particle. Because of their highly insulating
plasma membrane capacitance becomes a relatively low
impedance, and the cell appears electrically as a conductive
particle with a complex ac impedance (5,15-17). Based on the
plasma membrane capacitance becomes a relatively low
impedance and the cell appears electrically as a conductive
particle with a complex ac impedance (5,15-17). Based on the
rationale that the rf impedance change due to entry of a cell
into a sensing oritice should give information about the elec-
trical characteristics of the cell membrane and/or cell interior
( 5 ) , we constructed a flow-system instrument that would
detect the rf impedance properties of single cells.
The present instrument extends the technique of electronic
cell volume measurement (3, 8) by applying both ac and dc
current through the sensing orifice, and it is an improved
' Performed under the auspices of the US Department of Energy,
with joint support from Grant 22585 from the National Cancer
Institute, Department of Health, Education, and Welfare.
377
version of an instrument previously described (5). To date,
other investigators have used ac techniques to detect large
organisms (2, 9) and rf sensing commercial instruments have
been developed by Toa Electronics, Ltd. (4) and Coulter
Electronics, Inc.*s3(21) for analysis of single cells or particles.
Our instrument uses phase-sensitive detection to measure rf
impedance properties of single cells and particles.
Materials and Methods
Description of Instrument Flow System:Figure 1 shows
the flow chamber, which is constructed from Polypenco
Q200.5 (The Polymer Corporation, Reading, PA) a cross-
linked polystyrene with low rf losses. It has: an entrance cap;
two body sections; a 93-pm diameter by 150-pm long cylindri-
cal sapphire orifice jewel (Richard H. Bird Co., Inc., Waltham,
MA); and an exit cap. The plastic pieces screw together. The
sample enters the flow chamber through tubing (90%platinum
plus 10%iridium) in the entrance cap, and a sheath flow (12)
constrains the sample to the center of the sensing zone. A
manifold is located in the entrance cap to distribute the sheath
Coulter WH, Hogg WR. US Patent No. 3,502,974, 1970
' Coulter WH, Ingram M, Ferro PV: U S Patent No. 3,836,849, 1974
378 HOFFMAN, JOHNSON AND BRITT

ENTRANCE CAP FLOW CHAMBER BODY EXIT CAP

PI WIRE

NUT

BOTTOM VIEW L 100 in I

OFENTRANCE CAP

FIG.1. Mechanical drawing of the flow chamber. The manifold in the entrance was made by drilling holes through the edge of the cap and
then sealing the edges with plugs (shown with narrow cross-hatching). Dimensions are in inches.

IOK IOK C IOK IOK

L ____-__ 1
SENSING ORIFICE
FIG.2. Schematic of the bridge/preamplifier circuit. Transformer TI(Model TM016-1, Mini-Circuits Laboratory, Brooklyn, NY) applies an
rf voltage across nodes C and f)of the bridge circuit. Buffer amplifiers U1 and U2 (LH0063C,National Semiconductor Corp., Santa Clara, CA)
provide the reference and signal voltages for the phase-sensitive detectors. The high-speed operational amplifier U3 (Model 148B, Intech, Inc.,
Santa Clara, CA) forms a current/voltage converter for the Coulter volume signal. The dc current supply and separate electrodes used to
deliver the dc current are not shown.

fluid through four outlets and equalize the flow. A platinum To prevent the accumulation of gas produced by electrolysis
tubing in the entrance cap is generally used as one of the dc when the dc current was applied, we oriented the flow cham-
current electrodes. The exit cap contains the other electrode ber so that the flow was upward. Experience with other flow
in the form of a platinum foil. The exit cap contains a long, chambers showed that a small capacitance change produced
small-diameter exit channel which provides a large electrical by fluid dripping off the exit tube produces large pulses in the
resistance to ground for electrical isolation. Each half of the ac detectors. This problem was solved by the large electrical
flow chamber body contains a ring-shaped platinum electrode resistance in the exit channel of the present design.
formed from 0.25-pm diameter platinum wire. These wire
electrodes apply the high-frequency electrical current and Electronics
sense the high frequency and low frequency impedance
changes across the sapphire orifice jewel (see legend for Fig. Description of Instrumentation Electronics:The sens-
2). The electrodes were placed as close as possible to the ing orifice forms one of the impedance legs in a balanced
orifice. bridge circuit (Fig. 2). The purpose of the circuit is to detect
 F L O W CYTOMETRIC DC CELL VOLUME AND R F I M P E D A N C E 379
amplifier U3. The 100-pf and 15-pF capacitors, in series with
I
' A
I

200 - 0 dc
COULTER VOLUME inductors Lo and L3, respectively, prevent shunting of the low-
frequency Coulter volume signal (bandwith approximately
0.1-100 kHz). The rf voltage is applied to the bridge through
a broadband step-up transformer. The 0.02 pF capacitor at
[r the rf source input blocks low-frequency noise that could
w
m degrade the dc Coulter volume signal. The 10 kC2 metal film
E resistors are connected in series to produce 20 kC2 resistance
3
with a minimum shunting capacitance. Inductor LOforms a
100
_I tuned circuit with the orifice capacitance Co, and inductor L
Ld
z and a 50 kC2 cermet potentiometer are used to balance the
z bridge. The input capacitance of UZis tuned out by inductor
a
I L3.
0
The circuits of Figure 2 are housed in an aluminum box,
and U1 and U2 are connected to a separate detector module
with 50 C2 cables. A two-way power splitter (Model ZSC-2*-,
C Mini-Circuits Laboratory, Brooklyn, NY) is generally used to
I 2 3 4
divide the output signal from U2 between a monitoring oscil-
VOLUME(p3) x 10 loscope and the detector module. The output of U3 is further
FIG.3. Plot of the mean peak channel number of the dc-resistance amplified by an amplifier specifically designed for dc Coulter
(Coulter volume, and OD, 4.5 MHz rf impedance (0)parameters for volume signals (19). Stray capacitances, which are not shown
9.55, 12.50, 15.61 and 18.52 pm plastic microsphere reference particle in Figure 2, are largely responsible for determining the values
diameters. Inset, oscilloscope traces of the ( a )dc and ( b )demodulated
of inductors L, LOand Ls. As a point of reference the inductors
rf signals obtained from 10 pm dia. plastic microspheres. Vertical scale
is 2V/div.; time base 10 psec/div. and the duration of the undershoot used to construct a circuit which operated at 4.5 MHz were L
represents about 25 psec for dc and 250 psec for the rf signal. = 38-85 pH, LO= 65-125 pH, L3 = 65-125 pH and Lq = 330
pH. A commercial signal generator (Ferris Model 22-D) pro-
vides the rf voltage applied to the bridge. The voltage from
simultaneously the dc Coulter volume signal (3) and the signal the signal generator is amplified by one or two stages of a
due to the change in ac orifice impedance and to provide these simple, low-noise power amplifier.
signals as separate outputs. The output of the bridge circuit Instrumentation Operation: Preamplifier signals were
due to the passage of a cell through the orifice is an asynchro- demodulated by a reference signal in phase with the voltage
nous pulse modulation which is buffered by preamplifier U2 applied to the bridge circuit (i.e., O", rf detector). The bridge
and demodulated by a phase sensitive detector not shown in impedances were adjusted to balance simultaneously the
Figure 2. The reference voltage for phase detection is sent out bridge and provide the maximum demodulated pulse ampli-
by an identical preamplifier UI. The basic bridge circuit and tude for homogeneous test particles, ie.,plastic microspheres.
phase-sensitive detector have been described previously (5) These 0" phase-detected rf signals were used as one parameter
and the theoretical analysis for the phase-sensitive detection to a dual-parameter data acquisition system (14). The other
used is described in the Appendix. parameter was from the dc Coulter volume signal. Mathe-
The balanced bridge circuit is necessary to reduce the matical analysis of the bridge circuit indicated that under the
steady signal applied to the detectors and preamplifier and to conditions described above the demodulated signal was de-
reduce the noise introduced by the rf source. This noise pendent almost entirely on the resistance rather than capaci-
reduction can be appreciated by noting that a very good tance change in orifice impedance. Experimental evidence to
commercial signal generator typically will have a noise level support this fact was the linear relationship between the dc
in a 100 kHz bandwidth greater than -100 dB or 0.001% of Coulter volume signal and rf impedance signal for nonmem-
the output, while the percentage change in voltage a t node A brane bound particles including plastic microspheres, gas bub-
due to a red blood cell traversing a 93-pm diameter orifice is bles and ethanol-fixed cells. For practical purposes we refer to
only about 0.001%. Thus, detection of small cells requires a the rf signal as a measure of the ac resistance change in the
quieter rf source than is generally available. In practice, it has sensing orifice, keeping in mind that in some cases the rf signal
been possible to balance the bridge sufficiently to make the may include a small effect due to cell capacitance.
noise from the rf source negligible and to null out the funda- The dc current through the orifice was 0.2-0.3 mA, and the
mental, but not the harmonics, of the voltage applied by the rf voltage applied across nodes C and D of the bridge circuit
rf source. was approximately 20 Vrms (5 Vrms input to the bridge/
For the dc Coulter volume signal, dc current is supplied preamplifier module). The amplitude of the reference voltage
through the electrodes on the entrance and exit caps of the for the phase-sensitive detector was approximately 0.3 Vrms.
flow chamber. A set of batteries in series with a large resistor The bridge was sufficiently balanced so that the steady voltage
(not shown in Fig. 2) provides the dc current. Connections to applied to the bridge-preamplifier was less than 200 mV peak-
the flow chamber are made through suitable rf chokes (not peak. The total flow rate (sheath plus sample) was 3 ml/min,
shown in Fig. 2) to isolate the dc supply from the rf circuitry. while the sample flow rate was 0.6 ml/min or less and the
R F choke Lq provides low-pass fdtering for the dc Coulter velocity of particles traversing the orifice was on the order of
volume signal, which is amplified by the transimpedance 5 m/s.
380 HOFFMAN, JOHNSON AND BRITT
Calibration Microspheres and Biologic Cells
Plastic Microspheres:The resolution and linearity of the
instrument were tested using uniform polystyrene micro-
spheres (Coulter Electronics, Inc., Hialeah, FL). Microspheres
of 9.55, 12.50, 15.61 and 19.52 pm diameter were used corre-
sponding to volumes of 456.1, 1022.7, 1991.6 and 3894.4 pm3,
respectively.
Biologic Cells:Asynchronous Chinese hamster cells (V79)
were used in these experiments. Stock cultures were main-
tained in exponential growth as monolayers in plastic tissue
culture flasks (Falcon, Oxnard, CA) at 37% C in a humidified
5% co2/95%air atmosphere using Gibco BME (G-133, Grand
Island Biological Co., Grand Island, NY) nutrient medium
supplemented with 50 U/ml potassium penicillin, 50 pg/ml
streptomycin sulfate and 10%fetal calf serum (Flow Labora-
tories, McLean, VA). Exponentially growing cells were rinsed
with Earle's salts solution, then dissociated using 0.25%trypsin
(DIFCO, 0152-17, Detroit, MI) in phosphate-buffered saline
solution (Mallinckrodt Na2HP04=7H20no. 7914; KH? PO4 no.
7100 and NaC12). Chinese hamster cells (line CHO) were
maintained free of contamination with Mycoplasma in F-10
medium (Schwartz/Mann, Ham modified, cat. no. 7712-21)
supplemented with antibiotics (100 U/ml of penicillin G, 100
pg/ml of streptomycin sulfate) and 10%calf and 5%fetal calf
sera. Cultures of exponentially growing cells were grown in
spinner flasks and analyzed as saline washed cells.
Several Murine cell types were analyzed including em-
bryonal carcinoma cells (line ECC); differentiated teratocar-
cinoma cells (lines PYS and SE); Chinese hamster lung (line
V279-171b); and mechanically dissociated cells from solid
flank Colon-26 tumors propagated in BALB/c mice. Freshly
harvested samples of Murine and chicken erythrocytes and
lymphocytes were also analyzed. To test for the effects of
fixation on the dc resistance and ac impedance properties of
cells, representative samples of 70% ethanol-fixed and 2%
buffered glutaraldehyde-fixed CHO cells and human eryth-
rocytes were compared with unfned cells. To assess the effects
of physical and chemical agent-induced damage on the dc
resistance and ac impedance properties of mammalian cells
we compared control V79 cells versus X-ray exposed at 400
rad, (colony assay survival fraction = 0.35); actinomycin-D
exposed 0.3 pg/ml, 30 min (colony assay survival fraction =
0.85) and combined x-ray plus actinomycin treated cells (col-
ony assay survival fraction = 0.31).
Results and Discussion
Plastic Microsphere Instrument Calibration: Typical
output pulses from the 0" 4.5 MHz rf detector and dc Coulter
volume detector obtained from our instrument are shown in
the inset of Figure 3. The oscilloscope traces represent the
superposition of several hundred pulses due to 10-pm diameter
plastic spheres traversing the 93-pm diameter orifice. These
particles caused a resistance change of approximately 10 a in
the sensing orifice and produced a modulation of about 0.01%
on the voltage at node A (Fig. 2) in the bridge circuit. The
main amplifier (19) for the Coulter volume signal contains
active baseline restoration circuitry that minimizes the dma-
tion of the undershoot on the pulse. Our 0" rf amplifier does
not contain baseline restoration, but this was not a problem
since the counting rates used were typically a few hundred
cells/sec. The coefficient of variation for the Coulter volume
pulse-height distribution was typically 2% for the microsphere
data. The 0" rf parameter usually has a slightly smaller
coefficient of variation than the Coulter volume parameter,
and coefficients of variation of 1.6% have been obtained for
t,he 0" rf distributions of 12.50-pm plastic spheres. The mean
channel numbers of the pulse-height spectra for the Coulter
volume and 0" rf parameters are plotted versus the volume of
the plastic spheres in Figure 3, illustrating that both param-
eters were directly proportional to particle volume.
dc Resistivity and rf Impedance Measurements of
Biologic Cells:Electronic dc resistance sensing ( i.e., Coulter
volume, reference 3) based on detection of dc electrical resist-
ance changes in a small sensing orifice due to the passage of
a cell or particle has been widely used for characterizing cell
populations. Only limited data are available for rf impedance
measurements of mammalian cells using flow techniques3 (5,
6, 10, 20-22). These data indicate that intact cells appear
electrically as conductive particles having impedance proper-
ties which are related to size and in general corresponds to
cellular buoyant density3 (10, 21). In the present study we
measured the flow-through dual-parameter dc resistance and
O", 4.5 MHz rf impedance properties of single mammalian
cells sampled from suspension cultures or dissociated from
monolayer and spheroid populations. Figure 4 schematically
illustrates the general pattern we observed: ( a ) that the rf
impedance for intact mammalian cells is smaller than the rf
impedance for plastic microspheres having the same dc vol-
ume; and ( b ) the contour patterns observed tended to be
dependent on cell-type and proliferative state. This is due to
- .
0
...
....
z
-I
..
W
z
z
a .* V79-171b
I
0
v
W
0
z
a
a
w
a
z
*
L
N
r
I
10
ti
0 -
0
:'
COULTER dcVOLUME (CHANNEL No.)
FIG. 4. Schematic illustration of the typical dual-parameter dc
Coulter volume and Oo, 4.5 MHz rf impedance contour pattern ob-
served for plastic microspheres and intact and viable mammaiian cell
populations, e.g., V79-171b and CHO cells. The contour pattern
appears to be dependent on cell-type, proliferation state and state of
differentiation.
 10970320, 1981, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cyto.990010605, Wiley Online Library on [07/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
FLOW CYTOMETRIC DC CELL VOLUME A N D RF IMPEDANCE 381
the fact that the intracellular conductivity is much less than populations, whereas cell-cycle related cell volume and rf
the plasma membrane conductivity and cell size generally impedance changes were readily detectable within 1-2 hr after
increases with cell-cycle age. Theoretically, the rf impedance moderate x-ray and low dose actinomycin-D exposures. Figure
properties of single cells in saline suspension should be sensi- 7 shows typical examples of control and treated dual-param-
tive to internal cell structure (5, 7, 15-17) and we obtained eter contour data obtained for samples taken at 6 hr postex-
evidence for asynchronously exponentially growing V79 cells posure, representing: moderate x-ray dose exposure; low dose
that this may indeed be valid. For example, we detected two drug exposure and combined x-ray and drug exposure. Note,
apparently different subpopulations, one having a higher rf
impedance (thought to be mainly G2-cells)and one having a
lower rf impedance (considered to represent mostly mitotic
cells) cells having G2M Coulter dc volumes as shown in Figure
5. One explanation for this observation would be that while ;50
the cell volumes of Gz and dividing cells are quite similar, u
40
interphase cells (in this case Ga-cells) have an intact nuclear 3
U
membrane and usually have higher buoyant densities than 30
dividing cells (e.g., 1, 11, 23). Other available flow-through LL
data for mammalian cells have suggested that rf impedance '= 20
N
sensing is useful for discriminating between biologically dif- 10
ferent cells having similar cell volumes3 (5, 6, 10, 21). M
In a separate series of experiments, we tested the effects of 9
trypsin versus pepsin treatment; and ethanol versus glutaral- 0
dehyde-fixation on the dual-parameter dc resistance/O", 4.5
mHz rf impedance contour patterns of CHO cells. Figure 6
shows contour data obtained for CHO control cells ( 6 b ) com-
pared to trypsin-treated cells (612) which underwent little
change and pepsin-treated cells (6d) which displayed an up-
ward shift of contour lines toward the plastic microsphere
reference line (Fig. 6 a ) . Glutaraldehyde fixation altered the
electrical resistivity properties of CHO cells (Fig. 6 e ) ,partic-
ularly for G2M cells (Fig. 6 e ) , while ethanol-fixation totally 10 20 30 10 20 30 10 20 30
destroyed the rf impedance information by shifting all the
COULTER dc VOLUME
events onto the plastic microsphere reference line (Fig. 6 f ) .
To assess the efficacy of using dual-parameterdc resistance/ FIG. 6 . Contour plot patterns for fresh and treated CHO cells.
Single cells in suspension culture were treated by adding various
O", 4.5 MHz rf impedance measurements to detect rapidly substances to the suspension. The solid line in the upper left panel
physical and chemical cellular damage, we compared the is a calibration provided by plastic microspheres ( a ) .Trypsin-treated
contour patterns of control versus x-ray and actinomycin-D ( c ) and glutaraldehyde-fixed ( e ) cells are not grossly different from
exposed V79 cells. Control and treated cells were sampled the fresh cells ( b ) .Some pepsin-treated cells ( d ) produced events
near the solid line which is suggestive of membrane damage. Ethanol-
from 0 to 24 hr after exposure in three duplicate experiments. fixed cells ( f ) all lie along the solid line which correlates with electron-
Iso-cell lines (where each line represents a constant cell num- microscopic evidence that ethanol fixation completely destroys the
ber) were characteristic of experimental growing control V79 plasma membrane.

z
t Ir
Y

z
0 L - X
COULTER d c VOLUME
FIG.5. Dual-parameter oscilloscope display of Coulter dc volume and 0", 4.5 MHz rf impedance data obtained for asynchronous, exponential
growing V79 monolayer cells analyzed as intact, viable cells. Isometric display. Note the two different rf impedance subpopulations in the G&
cell volume region.
382 HOFFMAN, JOHNSON AND BRITT

I l l I l l sufficiently conductive that the cellular dc and rf conductivity

PLASTIC are equivalent and the cell appears electrically homogeneous.
Events from such nonmembrane-bound particles lie on the
line defined by plastic microspheres. Events from cells with
slightly leaky membranes will lie intermediate, between the
plastic microsphere reference line and intact cells such as we
observed for the x-ray and drug-damaged cells illustrated in
Figure 7 . As a measure of the deviation from homogeneous
plastic microsphere particles we determined the relative opac-
ity, for various control and treated cells, where the term

L 50
1 XRT-5h
/
4 ACT-0-5h 4 X-ACT-0-5h
relative opacity is defined as follows:
rf channel (cells)/
dc channel(cells)
40 relative opacity =
2 rf channel (plastic spheres)/
dc channel(p1astic spheres)
tr? 30
*- 2 0 - opacity (cells)

-b
0
10
opacity (plastic spheres)
(The ease with which a radiofrequency current will go through
z a particle or cell suspended in an electrolyte, e.g., saline, has
been termed “electrical transparency” analogous to light
transmission; the impedance to the radiofrequency current of
a particle or cell has been termed “opacity.”2* Limited pub-
lished data obtained from the Coulter opacity instrument

Table 1
Mean relative opacitiesashof Cells
Relative Opacity at 4.5
Cell Type
MHz
10 2 0 30 10 20 3 0 10 20 30 Untreated
Mouse lymphocyte 0.82
COULTER d c V O L U M E Human lymphocyte 0.75
FIG. 7. Effects of radiation and chemical-agent cellular damage on Chick erythrocyte 0.73
dual parameter dc volume/rf impedance properties of exponential Human erythrocyte 0.70
growing V79 cells. Reference plastic microspheres are shown in the Human monocyte 0.68
upper left panel. The contour patterns for cells analyzed at the same Chinese hamster (V279-171b) 0.65
instrument settings are shown for control cells; 400 rad x-ray exposed Colon-26 tumor 0.64
cells; 0.3 pg/ml actinomycin-D exposed cells; and more severely Chinese hamster (CHO line) 0.60
damaged 400 rad x-ray plus actinomycin-D exposed cells. The cells Teratocarcinoma-parietal yolk sac 0.63
were analyzed as unfixed cells sampled at 5 and 6 hr after treatment. Teratocarcinoma-undifferentiated 0.52

Treated
Chinese hamster (CHO line)
the x-ray-treated cells displayed what we interpreted as a Gz- ethanol-fixed 1.0
block in addition to cellular hypertrophy and damage to S- Human erythrocyte-glutaralde-
phase and G2M cells (13, 18). Only minimal perturbations hyde-fixed 0.95
were evident for the low dose drug-treated cells during the 24- Chinese hamster (CHO line) glu-
hr sample period. Those cells exposed to both radiation and taraldehyde-fixed 0.63
Human erythroc yte-lysed (ghosts) 0.36
drug showed more severe damage by 6 hr after treatment, as Chick erythrocyte-lysed (ghosts) 0.34
evidenced by abnormally high conductivity cells located on
Relative opacity
the contour plots intermediate between the intact cells and rf channel (cells)/
the nonconducting plastic microspheres (Fig. 7, bottom right). -
- dc channel (cells)
These results are encouraging since they suggest that dual rf channel (plastic spheres)/
parameter dc electronic cell volume/O”, 4.5 MHz rf impedance dc channel (plastic spheres)
sensing of unfixed, unstained cells may be useful for rapid - opacity (cells)
detection of radiation and chemical agent-induced cellular -
opacity (plastic spheres)
damage.
It is apparent from both theoretical considerations3 (8) and buncertainty within each run was 256, whereas the day-to-day
variability for relative opacity for biological cells was about 5%.For
our experimental data that as the plasma membrane becomes a given sample, the relative opacity showed up to a 10-20% difference
electrically leaky ( ie., conductive) the dc Coulter volume for cells having narrow Coulter volume (e.g., a 10-15 channels wide
signal decreases. At some point the plasma membrane will be window).

FLOW CYTOMETRIC DC CELL VOLUME A N D RF IMPEDANCE
have described opacity as the ac resistance divided by dc
resistance (10,21).We use the term relative opacity as defined
above since there have been no complete Coulter opacity
instrument descriptive studies published to date that would
allow a critical comparison of opacity and the Los Alamos
instrument measured relative opacity data.) Thus the relative
opacity can be used as a measure of the difference in particle
resistivity at high and low frequency electrical current. Ho-
mogeneous particles and plastic microspheres have a relative
opacity value of 1.0, whereas mammalian cells with intact
plasma membranes will have values less than 1.0. Table 1
summarizes the measured relative opacity values for various
cell types analyzed at O”, 4.5 MHz rf in the present study and
examples of values for fixed cells. In general, we observed
relative opacity values for lymphocytes having high nuclear/
cytoplasmic ratios to be 0.75-0.82. Erythrocytes were usually
around 0.7; cultured differentiated mammalian cells ranged
from about 0.63 to 0.68; and undifferentiated cells appear to
have lower values than differentiated cells (Table 1,reference
6). Ethanol-fixed cells were located essentially on the plastic
microsphere reference line, and hence had relative opacity
value = 1.0;glutaraldehyde-fixed cells showed scattered values
from 0.60 to 0.95. The uncertainty for the mean relative
opacity data for a given run was 2%, while the day-to-day
variability was about 5%. Therefore, within the variability of
the measured values it can be concluded that for the cells
studied (Table 1):( a )intact cells have relative opacity values
ranging from about 0.5 to greater than 0.8, depending on cell
type; ( b ) blood cells tended to have higher values than cul-
tured somatic or tumor cells; and ( c ) the relative opacities of
gluteraldehyde-fixed cells were nearly the same as unfixed
cells, whereas ethanol-fixation destroyed the cellular rf-imped-
ance information.
Conclusion
This report has described a flow instrument which simul-
taneously measures electrical dc resistance and rf-impedance
for analysis of single cells and particles. The dc resistance and
O”, 4.5 MHz rf-impedance signals of plastic microspheres were
proportional to particle volume, whereas the dual-parameter
contour patterns and relative resistivities of intact cells ap-
pears to be dependent on cell volume plus internal cell struc-
ture related to cell-type, proliferation state and in some cases
differentiation state. The instrument has potential use for
rapid, nondestructive assessment of radiation and drug-in-
duced cell damage not requiring fluorochrome staining.
Acknowledgments
The authors thank R. D. Hiebert and Z. Sander for stimulating
discussions during the initial stages of instrumentation development and
J. C. Martin, G. C. Salzman and J. H. Jett for their comments on the
manuscript. We thank Z. V. Svitra, D. E. Swartzendruber, R. J . Kissane,
H. A. Crissman and N. Tokita for their collaboration on various aspects
of the biologic and cytologic studies.
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Appendix
Theoretical Derivation of Pulse Amplitudes for Phase-Sen-
sitive rf Detection: The circuit shown in Figure 8 represents the
most general description for a half-bridge circuit. The change, AVO,in

384 HOFFMAN, JOHNSON AND BRITT
I A- I
FIG.8. Circuit for complex impedance analysis.
the voltage V , will be derived in terms of the circuit component values
and the small changes, ARoand ACo, in the circuit elements, R , and
C,, which represent the imepdance of the sensing orifice. If the input
impedance of the bridge preamplifier is sufficiently high (Fig. 2), then
the AVOcalculated for the half-bridge is also the signal detected by
the full-bridge preamplifier. We will assume that this is true. The
complex impedance of the ith RC pair is given by Z, = R J ( 1 + jw R,
C c ) where
, j =a and w = 271 X frequency. Voltage V , is V , = Z,E/
(Z, + Zl). The variation in V, for small variations in R , and C, is
calculated from the following partial derivates:
and
av,/ac,= - jw R,' aV,/aR,,
where Rp = ( R J t d / ( R ,+ R I ) ,C, = Co+ CI,r, = R,
C,, TI = R I CI,
and D = 1 + w2R$C9. Note that, if the impedance is inductive, C, or
Cl can be negative. The equations are more tractable if we define
Then aV,/dR, = ( a - j P ) E and aV,/aCo= (-wRo2P - jwRO2oc)E.
The
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variation in V, for small variation in R , and C, is
a v,
AVO= -ARo + -ACo
avo
aR, ac,
AVO= [(aAR, - wRO2PAC,)- j ( P A R , + wR,2aAC,,)]E.
If we define the phase of the complex signal voltage AV, with respect
to the driving signal E,then the signal in-phase with E , AV,, (OD), is
given by
AVo(Oo)= (aAR, - wRo2PAC0)]El. (-41)
The signal that is 90" out-of-phase with E, AVO(90°),is
AV,(90°)= - (PAR, + wR,*(YAC,)IEl. (A2)
1 El is the magnitude of the voltage E .
In general, the in-phase (0') and quadrature (90' ) signals given by
equations ( A l ) and (AZ),respectively, are dependent on both AR,
and ACo. However, when P = 0, the situation is simplified so that
AVO(0") = (Y ARo IE I and AVO(90")= - wRO2oACo I E I. One condition
for which /3 = 0 is when C, = C1 = 0 (i.e., when all elements in the
circuit are pure resistors at the frequency w ) . If CI # 0, it is still
possible to make P = 0 by a suitable adjustment of the other circuit
values. A method has been described4 for producing a nearly pure
resistance change, AR,, in the orifice. This would provide an easy way
to detect the condition /3 = 0, since in that case ACo = fl = 0, there
would be no signal for AVO (90') [see equation (AZ)].This might
make it possible to use the AVO (90")signal to detect capacitance
changes.
It is expected that the term wRO2ACo wiU always be less than ARo.
To make a more quantitative comparison, we note that, if initially
the impedance of the sensing orifice is a pure resistance, R,, then the
changes in resistance, AR,and reactance, AX, of the impedance Z, for
small changes AR,, and ACo are
and
The approximations hold when w ( R , + AR,)AC, < 1, which will
generally be true. Note that ARo5 AR and (wRo2AC,)/AR, < I AXIAR.
The quantities AR and AX can be calculated for theoretical models
for the impedance of cell suspensions (e.g.,see Fig. l b in Reference
5 which shows the results for such a calculation). In the case of this
particular model, I AX1 is always less than AR and, below a character-
istic frequency, fo, IAXI is much less than AR.At the characteristic
frequency, \AX\ is a maximum and, at this frequency, \Ax] 0.6 AR.
In general, it is expected that, for a given cell, there will be a
characteristic frequency for which the term wRo2AC,is a maximum,
but this term will always be less than AR,.If the PAR, term in Eq.
(A2) is not to interfere with the wRZaAC, term, it is necessary to
make P approach zero as nearly as possible.
Hoffman R A US Patent No. 4,224,567, 1980.