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Cytometry - May 1981 - Hoffman - Flow Cytometric Electronic Direct Current Volume and Radiofrequency Impedance Measurements
Cytometry - May 1981 - Hoffman - Flow Cytometric Electronic Direct Current Volume and Radiofrequency Impedance Measurements
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CYTOMETRY Val. 1,No. 6, 1981
Copyright 0by the Society for Analytical Cytology Printed in U.S.A.
ORIGINAL ARTICLES
One of the fist physical parameters measured using flow version of an instrument previously described (5). To date,
cytometric methods was the electronic cell volume or Coulter other investigators have used ac techniques to detect large
volume (3) which detects the change in direct current (dc) organisms (2, 9) and rf sensing commercial instruments have
electrical resistance of a small sensing orifice caused by entry been developed by Toa Electronics, Ltd. (4) and Coulter
of a cell or other particle. Because of their highly insulating Electronics, Inc.*s3(21) for analysis of single cells or particles.
plasma membrane capacitance becomes a relatively low Our instrument uses phase-sensitive detection to measure rf
impedance, and the cell appears electrically as a conductive impedance properties of single cells and particles.
particle with a complex ac impedance (5,15-17). Based on the
plasma membrane capacitance becomes a relatively low Materials and Methods
impedance and the cell appears electrically as a conductive Description of Instrument Flow System:Figure 1 shows
particle with a complex ac impedance (5,15-17). Based on the the flow chamber, which is constructed from Polypenco
rationale that the rf impedance change due to entry of a cell Q200.5 (The Polymer Corporation, Reading, PA) a cross-
into a sensing oritice should give information about the elec- linked polystyrene with low rf losses. It has: an entrance cap;
trical characteristics of the cell membrane and/or cell interior two body sections; a 93-pm diameter by 150-pm long cylindri-
( 5 ) , we constructed a flow-system instrument that would cal sapphire orifice jewel (Richard H. Bird Co., Inc., Waltham,
detect the rf impedance properties of single cells. MA); and an exit cap. The plastic pieces screw together. The
The present instrument extends the technique of electronic sample enters the flow chamber through tubing (90%platinum
cell volume measurement (3, 8) by applying both ac and dc plus 10%iridium) in the entrance cap, and a sheath flow (12)
current through the sensing orifice, and it is an improved constrains the sample to the center of the sensing zone. A
manifold is located in the entrance cap to distribute the sheath
' Performed under the auspices of the US Department of Energy,
with joint support from Grant 22585 from the National Cancer Coulter WH, Hogg WR. US Patent No. 3,502,974, 1970
Institute, Department of Health, Education, and Welfare. ' Coulter WH, Ingram M, Ferro PV: U S Patent No. 3,836,849, 1974
377
378 HOFFMAN, JOHNSON AND BRITT
NUT
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.
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 since the counting rates used were typically a few hundred
cells/sec. The coefficient of variation for the Coulter volume
Plastic Microspheres:The resolution and linearity of the
pulse-height distribution was typically 2% for the microsphere
instrument were tested using uniform polystyrene micro-
data. The 0" rf parameter usually has a slightly smaller
spheres (Coulter Electronics, Inc., Hialeah, FL). Microspheres
coefficient of variation than the Coulter volume parameter,
of 9.55, 12.50, 15.61 and 19.52 pm diameter were used corre-
and coefficients of variation of 1.6% have been obtained for
sponding to volumes of 456.1, 1022.7, 1991.6 and 3894.4 pm3,
t,he 0" rf distributions of 12.50-pm plastic spheres. The mean
respectively.
channel numbers of the pulse-height spectra for the Coulter
Biologic Cells:Asynchronous Chinese hamster cells (V79)
volume and 0" rf parameters are plotted versus the volume of
were used in these experiments. Stock cultures were main-
the plastic spheres in Figure 3, illustrating that both param-
tained in exponential growth as monolayers in plastic tissue
culture flasks (Falcon, Oxnard, CA) at 37% C in a humidified eters were directly proportional to particle volume.
5% co2/95%air atmosphere using Gibco BME (G-133, Grand dc Resistivity and rf Impedance Measurements of
Island Biological Co., Grand Island, NY) nutrient medium Biologic Cells:Electronic dc resistance sensing ( i.e., Coulter
volume, reference 3) based on detection of dc electrical resist-
supplemented with 50 U/ml potassium penicillin, 50 pg/ml
ance changes in a small sensing orifice due to the passage of
streptomycin sulfate and 10%fetal calf serum (Flow Labora-
a cell or particle has been widely used for characterizing cell
tories, McLean, VA). Exponentially growing cells were rinsed
populations. Only limited data are available for rf impedance
with Earle's salts solution, then dissociated using 0.25%trypsin
measurements of mammalian cells using flow techniques3 (5,
(DIFCO, 0152-17, Detroit, MI) in phosphate-buffered saline
6, 10, 20-22). These data indicate that intact cells appear
solution (Mallinckrodt Na2HP04=7H20no. 7914; KH? PO4 no.
7100 and NaC12). Chinese hamster cells (line CHO) were electrically as conductive particles having impedance proper-
ties which are related to size and in general corresponds to
maintained free of contamination with Mycoplasma in F-10
cellular buoyant density3 (10, 21). In the present study we
medium (Schwartz/Mann, Ham modified, cat. no. 7712-21)
supplemented with antibiotics (100 U/ml of penicillin G, 100 measured the flow-through dual-parameter dc resistance and
O", 4.5 MHz rf impedance properties of single mammalian
pg/ml of streptomycin sulfate) and 10%calf and 5%fetal calf
cells sampled from suspension cultures or dissociated from
sera. Cultures of exponentially growing cells were grown in
spinner flasks and analyzed as saline washed cells. monolayer and spheroid populations. Figure 4 schematically
Several Murine cell types were analyzed including em- illustrates the general pattern we observed: ( a ) that the rf
bryonal carcinoma cells (line ECC); differentiated teratocar- impedance for intact mammalian cells is smaller than the rf
cinoma cells (lines PYS and SE); Chinese hamster lung (line impedance for plastic microspheres having the same dc vol-
V279-171b); and mechanically dissociated cells from solid ume; and ( b ) the contour patterns observed tended to be
dependent on cell-type and proliferative state. This is due to
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 0
...
....
cells, representative samples of 70% ethanol-fixed and 2% z
buffered glutaraldehyde-fixed CHO cells and human eryth- -I
..
W
rocytes were compared with unfned cells. To assess the effects z
of physical and chemical agent-induced damage on the dc z
resistance and ac impedance properties of mammalian cells
a .* V79-171b
I
we compared control V79 cells versus X-ray exposed at 400 0
v
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
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).
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 383
have described opacity as the ac resistance divided by dc and measuring the size of small nematodes. Rev Sci Instrum 46:
resistance (10,21).We use the term relative opacity as defined 517, 1975
3. Coulter WH: High speed automatic blood cell counter and cell
above since there have been no complete Coulter opacity size analyzer. Proc Natl Electron Conf 12: 1034, 1956
instrument descriptive studies published to date that would 4. Helleman PW, Benjamin CJ: The Toa microcell counter. 1. A
allow a critical comparison of opacity and the Los Alamos study of the correlation between the volume of erythrocytes and
instrument measured relative opacity data.) Thus the relative their frequency distribution curve. Scand J Haematol6: 69, 1969
opacity can be used as a measure of the difference in particle 5. Hoffman RA, Britt WB: Flow-system measurement of cell imped-
ance properties. J Histochem Cytochem 27: 234, 1979.
resistivity at high and low frequency electrical current. Ho- 6. Hoffman RA, Swartzendruber DE: Electrical impedance analysis
mogeneous particles and plastic microspheres have a relative of single murine teratocarcinoma cells. Exp Cell Res 122: 426,
opacity value of 1.0, whereas mammalian cells with intact 1979
plasma membranes will have values less than 1.0. Table 1 7. Irimajiri A, Doida Y, Hanai T, Inouye A: Passive electrical
properties of cultured murine lymphoblast “L5178Y” with refer-
summarizes the measured relative opacity values for various ence to its cytoplasmic membrane, nuclear envelope, and intra-
cell types analyzed at O”, 4.5 MHz rf in the present study and cellular phases. J Membrane Biol38: 209,1978
examples of values for fixed cells. In general, we observed 8. Kachel V: Electrical resistance pulse sizing (Coulter sizing). In:
relative opacity values for lymphocytes having high nuclear/ Flow Cytometry and Sorting, Melamed MR, Mullaney PF, Men-
cytoplasmic ratios to be 0.75-0.82. Erythrocytes were usually delsohn ML (eds). John Wiley & Sons, New York, 1979,p. 61-104
9. Kindlman PJ, Applewhite PB, Merowitz HJ: Capacitive detection
around 0.7; cultured differentiated mammalian cells ranged of very small aquatic animals. Rev Sci Instrum 39: 121, 1968
from about 0.63 to 0.68; and undifferentiated cells appear to 10. Leif RC, Guarino V, Lefkove N, Pell L, Rodriguez C: Electrical
have lower values than differentiated cells (Table 1,reference impedance (Coulter effect) measurements of single human eryth-
6). Ethanol-fixed cells were located essentially on the plastic rocytes (abstr). Fed Proc 38: 1062, 1979
11. Meistrich ML, Trostle, PK: Separation of mouse testis cells by
microsphere reference line, and hence had relative opacity equilibrium density centrifugation in renografin gradients. Exptl
value = 1.0;glutaraldehyde-fixed cells showed scattered values Cell Res 92: 231, 1975
from 0.60 to 0.95. The uncertainty for the mean relative 12. Mullaney PF, Van Dilla MA, Coulter JR, Dean PN: Cell sizing:
opacity data for a given run was 2%, while the day-to-day a light scattering photometer for rapid volume determination.
variability was about 5%. Therefore, within the variability of Rev Sci Instrum 40: 1029, 1969
13. Raju MR, Johnson TS, Tokita N, Carpenter S, Jett JH: Differ-
the measured values it can be concluded that for the cells ences in cell-cycle progression delays after exposure to 238Pu
studied (Table 1):( a )intact cells have relative opacity values particles compared to x-rays. Radiat Res 84: 16, 1980
ranging from about 0.5 to greater than 0.8, depending on cell 14. Salzman GC, Hiebert RD, Crowell JM: Data acquisition and
type; ( b ) blood cells tended to have higher values than cul- display for a high-speed cell sorter. Comput Biomed Res 11: 77,
tured somatic or tumor cells; and ( c ) the relative opacities of 1979
15. Schanne OF, Ruiz P-Cerretti E: Impedance Measurements in
gluteraldehyde-fixed cells were nearly the same as unfixed Biological Cells. John Wiley and Sons, New York, 1978
cells, whereas ethanol-fixation destroyed the cellular rf-imped- 16. Schwan HP: Electric properties of tissues and cell suspension. In:
ance information. Advances in Biological and Medical Physics, Lawrence JH, To-
bias CA (eds). Vol. 5. Academic Press, New York, 1957, p. 147-
Conclusion 209
17. Schwan HP: Determination of biological impedances. In: Physical
This report has described a flow instrument which simul- Techniques in Biological Research, Nastuk WL (ed). Vol. 6.
taneously measures electrical dc resistance and rf-impedance Academic Press, New York, 1963, p. 323-407
for analysis of single cells and particles. The dc resistance and 18. Sinclair W K Mammalian cell sensitization repair and the cell
cycle. In: Proceedings Fifth Int’l Congress of Radiation Research,
O”, 4.5 MHz rf-impedance signals of plastic microspheres were Nygaard, OF, Adler, HI, Sinclair, WF, (eds). Academic Press,
proportional to particle volume, whereas the dual-parameter New York, 1975, p. 742-751
contour patterns and relative resistivities of intact cells ap- 19. Steinkamp JA, Fulwyler MJ, Coulter JR, Hiebert RD, Horney
pears to be dependent on cell volume plus internal cell struc- JL, Mullaney PF: A new multiparameter separator for micro-
ture related to cell-type, proliferation state and in some cases scopic particles and biological cells. Rev Sci Instrum 44: 1301,
1973
differentiation state. The instrument has potential use for 20. Swartzendruber DE, Travis GL, Martin JC: Flow cytometric
rapid, nondestructive assessment of radiation and drug-in- analysis of the effect of 5-bromodeoxyuridine on mouse terato-
duced cell damage not requiring fluorochrome staining. carcinoma cells. Cytometry 1: 238, 1980
21. Thomas RA, Yopp TA, Watson BD, Hindman DHK, Cameron
Acknowledgments BF, Lief SB, Lief RC, Roque L, Britt W: Combined optical and
electrical analysis of cells with the AMAC transducers. J Histo-
The authors thank R. D. Hiebert and Z. Sander for stimulating chem Cytochem 25: 827,1977
discussions during the initial stages of instrumentation development and 22. Tokita N, Johnson TS, Hoffman RA, Raju M R Introduction of
J. C. Martin, G. C. Salzman and J. H. Jett for their comments on the cytokinetic perturbations and increased cell-killingof V79 cells by
manuscript. We thank Z. V. Svitra, D. E. Swartzendruber, R. J . Kissane, radiochemotherapy. Cell Tissue Kinet 12: 681, 1979
H. A. Crissman and N. Tokita for their collaboration on various aspects 23. Wolff DA, Pertoft H: Separation of Hela cells by colloidal silica
of the biologic and cytologic studies. density gradient centrifugation. I. Separation and partial syn-
chrony of mitotic cells. Cell Biol 55: 579, 1972
Literature Cited
Appendix
1. Buell DN, Fowlkes BJ, Metzger H, Isersky C: Cell cycle and
morphological changes during growth and differentiation of a rat Theoretical Derivation of Pulse Amplitudes for Phase-Sen-
basophilic leukemia cell line. Cancer Res 36: 3131, 1976 sitive rf Detection: The circuit shown in Figure 8 represents the
2. Byerly L, Cassada RC, Russell RL: Machine for rapidly counting most general description for a half-bridge circuit. The change, AVO,in
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
384 HOFFMAN, JOHNSON AND BRITT
I A- I
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
FIG.8. Circuit for complex impedance analysis.
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.
the voltage V , will be derived in terms of the circuit component values
It is expected that the term wRO2ACo wiU always be less than ARo.
and the small changes, ARoand ACo, in the circuit elements, R , and
To make a more quantitative comparison, we note that, if initially
C,, which represent the imepdance of the sensing orifice. If the input
the impedance of the sensing orifice is a pure resistance, R,, then the
impedance of the bridge preamplifier is sufficiently high (Fig. 2), then
changes in resistance, AR,and reactance, AX, of the impedance Z, for
the AVOcalculated for the half-bridge is also the signal detected by
small changes AR,, and ACo are
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