Membr. Cell Biol., 2000, Vol.14 (3), pp.

367 382
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Spontaneous and Forced Oscillations of Cell

Membrane of Normal Human Erythrocytes:
Absence of Resonance Frequencies in a Range of
0.03500 Hz
V. L. Kononenko and J. K. Shimkus
Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygin
Street, Moscow, 117977, Russia
fax: (095) 137 4101; email: konon@sky.chph.ras.ru
The spectra of natural oscillations of human erythrocyte cell membranes were
studied experimentally and theoretically. The measurements were carried out at room
temperature for both single normal cells and erythrocyte rouleaux in a range of
0.03500 Hz. The spectra were measured at a resolution better than 1% using two
techniques: registration of spontaneous membrane oscillations induced by thermal
agitation in the surrounding medium and registration of the amplitudefrequency
characteristics of the forced oscillations of erythrocyte elongation induced by a
high-frequency electric field with the amplitude harmonic modulation. The spectra
measured by both techniques had no resonance frequencies and decreased monotonically with the frequency increase. These results are confirmed by the theory developed for the extracellular excitation mechanisms of membrane oscillations. The
spectra of active oscillatory biomechanical processes were measured for comparison.
These processes are ciliary beating of human bronchial epithelium and ciliary beating
and artificial periodic contractions of the cytoplasm of the ciliate Spirostomum
ambiguum. The quality of the resonance lines of the order of 1020 registered may
serve as estimates for the line width in search of the resonance oscillations in erythrocytes induced by active cell processes.
(Received 28 December, 1998)

Resonant mechanical oscillations of the cell or its components and the

possible functional role of these oscillations have been the subject of many
theoretical and experimental works [112]. The characteristic frequencies of
oscillation and relaxation motions of the cell envelopes, o and r, as a whole

367

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V. L. KONONENKO AND J. K. SHIMKUS

have been shown to be within the range of 103 106 Hz, and in most cases
o << r [8]. Only in rather exotic cases of some plant cells with the rigid
envelope or in animal cells treated to obtain an osmotically strained envelope,
their oscillation spectra can have resonance frequencies [8]. The theoretical
analysis of natural oscillations of the bilayer lipid membrane, considered as a
thin film of a viscous fluid, estimates the lower frequency limit to be ~100 Hz
for the oscillations of the bilayer thickness and ~5 10 3 Hz for the bending
oscillations of the bilayer at an invariable thickness. In this case, however,
estimates of viscous damping also result in the inequality o << r [9].
At the same time, several works reported rather low-frequency and highquality resonances in the oscillation spectra of the cell surface, subcellular
particles and biochemically modified liposomes [11, 12]. Thus, Savushkin
and Vasilenko [11] presented the oscillation spectra of the surface of intact
and red cobra venom-treated rat erythrocytes containing extremely narrow
peaks within the range of 2 17 Hz.
Experimental studies of the possible existence of natural high-quality oscillations of the mammalian red cell membrane are of special interest, because
these cells are the simplest from the point of view of mechanics. Being
nucleus-free, they can be considered as thin viscoelastic envelopes filled with
a viscous fluid, a solution of hemoglobin. Mechanical modules phenomenologically describing erythrocytes within the framework of such a model are
well known for human erythrocytes [13]. Mechanical oscillations of such
envelopes can be calculated either analytically, in the case of their spherical
shape [5, 7], or numerically, with reference to the real shape of an erythrocyte
[5]. Within the framework of such calculations, the eigen modes of oscillations of the surface of normal erythrocytes decay exponentially [3, 5, 7].
However, we should emphasize that this model is only the first approximation in the biomechanical description of erythrocytes. It does not take into
account at least two important factors: the inhomogeneous structure of the
cell membrane (which is, in fact, composite from the point of view of
mechanics) and the existence of active and passive mechanisms of smallscale changes of the equilibrium shape of the cell, of the type of crenation and
echinocytosis. These factors can lead to significant distinctions of a real
oscillation spectrum of the cell membrane from that obtained within the
framework of the homogeneous passive envelope model. Elucidation of this
necessitates experimental studies of the oscillation spectra of the erythrocyte
membrane in the low-frequency region and comparison with the conclusions
of the envelope model. This issue is also related to the problem of the
resonance line width which characterizes the active and passive oscillatory
processes in cells in the low-frequency range of the order of few or tens of
hertz.
This work studied experimentally the oscillation spectra of the membrane
of normal intact human erythrocytes. The spectra were obtained by two

SPONTANEOUS AND FORCED MEMBRANE OSCILLATIONS

369

complementary methods: noise spectroscopy and active oscillatory action. In

the former case, we recorded a frequency spectrum of spontaneous bending
oscillations (flicker) of the erythrocyte membrane due to the thermal agitation
in the surrounding medium and, possibly, active processes in the cell. In the
latter case, erythrocytes were placed into an electric high-frequency field with
the amplitude harmonic modulation, and the amplitudefrequency characteristics (AFC) of forced oscillations of cell elongation under the influence of
the field were recorded. To obtain experimental data on the really possible
resonance line width of periodic biomechanical processes in the cells, we
measured the spectra of ciliary beating of human bronchial epithelium and
ciliary beating and periodic contractions of the cytoplasm of the ciliate Spirostomum ambiguum.
EXPERIMENTAL
The spectra of spontaneous oscillations of the membrane and the amplitude
frequency characteristics of forced oscillations of erythrocyte shape, and also
the spectra of ciliary beating of the epithelium and autooscillations of the
ciliate cytoplasm were registered using an MPV-3 Leitz optical microscopephotometer connected to a computer and an SK4-72/2 spectral analyzer. The
measurements were carried out at room temperature. The measuring setup
and the methods of measurements were described in [6, 14]. An erythrocyte
suspension was obtained before the measurements by sampling several drops
of the blood from an experimentors finger into 2 3 cm 3 of the suspension
medium. The erythrocytes were not washed out, because the practice had
shown a positive effect of the presence of blood plasma components in the
medium on the preservation of normal erythrocyte shape. In the registration
of the erythrocyte flicker, the medium was a standard physiological saline
with phosphate buffer at pH 7.2. In the registration of dielectrodeformation
oscillations of erythrocytes, we used an isotonic low-conductivity medium
containing 0.29 M of D-mannitol, 5 mM glucose and 1 mM phosphate buffer,
pH 7.2. To record the frequencies of ciliary beating of the epithelium, we used
human bronchial epithelium biopsies obtained in the course of a standard
clinical procedure of bronchofibroscopy by the method of diathermy biopsy
[15]. The biopsies were placed, transported from the clinic and studied in
medium 199 on Hanks solution at pH 7.2. The ciliary beating and oscillations of the cytoplasm of the ciliate Spirostomum ambiguum possessing a
contractile activity [16] were studied in the culture medium which contained,
in mM: 0.38 NaCl, 0.054 KCl, 0.034 CaCl 2, 0.027 MgCl2, 0.089 NaHCO3.
The ciliates of the clone used had an average size of about 800 m in length
and 120 m in diameter, which simplified their mechanical immobilization.
The flicker spectra were recorded both for single normal cells and erythrocyte rouleaux. In studies of individual cells, a drop of additionally diluted

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V. L. KONONENKO AND J. K. SHIMKUS

suspension was placed on a slide between two standard plastic cover glasses
with vaseline oil applied over the perimeter to prevent microflows and desiccation of the preparation. In such microchamber, cells could preserve their
normal discoid shape for up to 24 h. After the suspension precipitated, cells
weakly attached to the bottom of the chamber in one or two points of their
lower circumference of the maximal thickness were sampled for registration.
The attachment was checked by small sharp movements of the microscopic
stage, which led to the displacement and elongation of the cells attached. This
approach made it possible to eliminate the possible contribution of the
Brownian motion of a cell as a whole to the registered membrane flicker
spectrum, but still to preserve the biconcave shape of the cell to the maximal
extent. The spectra were registered by an objective lens 100/1.25 PHACO
in two optical modes: phase contrast sensitive to changes of the thickness of
an object, and backward scattering of laser irradiation sensitive to the shift of
its boundaries. The theory of microphotometric registration of the flicker in
these modes has been developed in [17, 18]. The intensity of the probing
radiation after its passing through a cell within the limits of the opening of the
measuring diaphragm was registered. The projection of the measuring
diaphragm on the cell surface was a square of 1.31.3 m2 in its centre. Thus,
in the registration of the flicker of individual cells their plane of symmetry
was perpendicular to the optical axis of the microscope, and the fluctuating
component of the signal was defined by the oscillations of the central part of
the cell disc. Due to the random character of the flicker, we registered and
averaged several hundred spectra of individual temporal samplings of the
signal in succession to obtain a statistically significant spectrum. The
frequency spectrum of the sampling obtained as a discrete set of values of the
signal from an analogue-to-digital transducer was calculated by fast Fourier
transformation. The flicker spectra within a broad frequency range of
0.03500 Hz were obtained as a result of registration and unification of
spectra in several narrower overlapping ranges.
To register the flicker spectra of erythrocyte rouleaux, a sampled drop of
blood was placed directly between two cover glasses and sealed by vaseline
oil over the perimeter. In observations, such a preparation represented a
system of linear and linearly branched erythrocyte rouleaux lying on the
bottom of the microchamber. For measurements, linear rouleaux were taken;
the projection of the measuring diaphragm was fixed on the axis of a
rouleaux. The planes of symmetry of the cells forming a rouleaux were in this
case parallel to the optical axis of the microscope, and the flicker signal was
formed by oscillations of the rims of these cells.
Forced oscillations of erythrocytes were studied in a microchamber
equipped with flat electrodes with a gap of ~80 m between them. The
electrodes were parts of a razor blade turned by their sharp edges towards
each other and pressed to a standard cover glass along its longer side. The

SPONTANEOUS AND FORCED MEMBRANE OSCILLATIONS

371

inter-electrode space was filled with a small amount of diluted erythrocyte

suspension in a solution of sugars, the electrodes were covered with a
rectangular segment of a cover glass and vaseline oil was applied over its
perimeter to obtain a closed volume. An a.c. voltage with a frequency of
2 MHz and amplitude of 2.5 V was applied to the electrodes. The amplitude
of the field was modulated by low frequency changed discretely within the
range of 0.01 1000 Hz at a modulation depth of 40%. The cells polarized
under the action of the high-frequency field were pulled into the region of a
maximal inhomogeneity of the field close to the sharp edges of the electrodes
and proved to be pressed to them. The action of the field on the polarization
charges induced by it at the opposite borders of a cell defined an elongation
of a cell, which depended on the field amplitude squared. The harmonic
modulation of the amplitude of the field leads to forced oscillations of the
elongation of an erythrocyte. The dependence of the amplitude of the
oscillations on the frequency of modulation of the field (AFC) was registered
in phase contrast, with an objective lens 40/0.60, which had the working
distance of up to 2 mm. The projection of the measuring diaphragm as a
narrow rectangle of 12.5 m2 was oriented along the field and was
overlapped by the image of the erythrocyte by about half. This provided a
linear dependence between the displacement of the edge of the cell and the
change of the light flux through the diaphragm. The variable component of
the photodetector signal was processed by a spectral analyzer, and the amplitude of the first harmonic was registered.
The frequencies of ciliary beating of the bronchial epithelium cells and of
the spectra of contractions of the ciliate cytoplasm were registered similarly,
by the frequency analysis of the fluctuations of the light flux within the limits
of the rectangular measuring diaphragm, projected to the preparation. The
size of the diaphragm projection was varied in different experiments to cover
several to many oscillating cilia. A system of two cover glasses separated by
a 2 mm-thick ring spacer and sealed with vaseline oil was used as a microchamber for bioptic specimens. In experiments with the ciliates, to immobilize them and to establish the mode of periodic contractions of the cytoplasm, we used a system of two cover glasses, one of which was melted at
edges. After a drop of the medium with the ciliates was placed between the
cover glasses and excess fluid was removed, the space between the glasses
reduced to a minimum (determined by the uneven edges) under the action of
capillary forces, and the glasses slightly spread the ciliates in between them.
This enabled us at the first stage of measurements to register the frequencies
of beating of peristomal and motor cilia of an immobile ciliate. Subsequently,
about 1 h after the specimen was prepared, such compression led to a sharp
bending of the ciliate body and the emergence of a region of autooscillations
of the cytoplasm, evidently, due to the contractile reaction in response to a
mechanical stimulus.

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Figure 1. Amplitude spectra of spontaneous oscillations of the membrane of

a single erythrocyte (1, laser irradiation; 2, phase contrast) and an erythrocyte
rouleaux (3, phase contrast) measured in the lowest frequency range.

RESULTS
Figures 13 present typical spectra of spontaneous (flicker) and forced
oscillations of erythrocyte shape, measured for single cells and erythrocyte
rouleaux. As the registration was based on the discretization of the signal, the
spectra are presented as discrete sets of respective Fourier coefficients to
directly indicate the value of spectral resolution. For convenience of
comparison, each spectrum is normalized to the amplitude at a lowest
frequency. The flicker spectra of single cells were measured in the mode of
the alternate registration of signal samplings from the same cell in phase
contrast and laser irradiation. The measurements showed that the spectra of
single signal samplings were extremely irregular, and no less than 200
samplings were required to obtain a sufficiently reproducible averaged
spectrum. The flicker spectra in Figs. 13 are averaged from 400 samplings
for single cells and from 800 samplings for rouleaux. The data in different
figures refer to different erythrocytes and different rouleaux. The spectra
measured in the mode of laser irradiation, have a smaller slope and,
respectively, significantly larger spectral amplitudes as compared with those

SPONTANEOUS AND FORCED MEMBRANE OSCILLATIONS

373

Figure 2. Amplitude spectra of spontaneous oscillations of the membrane of

a single cell, measured in a broad frequency range (for designations, see the
legend to Fig. 1). Solid lines indicate the calculated spectra.

measured in phase contrast. The averaged spectra measured for different

normal cells differ by the slope in the double logarithmic scale: (0.40.6)
below 10 Hz and (0.81.2) over 50 Hz. The flicker spectra of aggregated
erythrocytes damp stronger as the frequency increases. However, all the
spectra have the shape of monotonically damping curves without any
resonance lines, as shown in Fig. 2.
Figure 3 presents amplitudefrequency curves of erythrocyte elongation
oscillations under the action of a high-frequency field with a periodically
changing amplitude, measured at various pH values of the surrounding
medium. The AFC within a broad frequency range were obtained by
averaging many series of measurements carried out for single cells in several
narrower ranges and averaging the results for different cells. Both separate
AFC segments measured in various experiments and the averaged AFC of
forced oscillations contained, within the accuracy of measurements, no
resonance frequencies.
Figure 4 presents characteristic spectra of a periodic biomechanical process
associated with the motor activity of a cell, ciliary beating. The small relative

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V. L. KONONENKO AND J. K. SHIMKUS

Figure 3. An amplitude spectrum of spontaneous oscillations of the

membrane of a single cell (1, laser irradiation, pH 7.2) and the
amplitudefrequency characteristics of forced oscillations of erythrocyte
elongation under the action of an electric field (2, pH 7.4; 3, pH 6.5; 4, pH
6.1). The straight lines are approximations of the high-frequency regions; the
slope is 0.77 for pH 7.4 and 0.83 for pH 6.1.

line width of the spectra indicates a high spatial and temporal order of
oscillations of both the cilia of the bronchial epithelium cells, which provide
the transport function in the bronchi, and the peristomal cilia of the ciliate
Spirostomum ambiguum which perform the nutritive function. Autooscillations of the ciliary cytoplasm are characterized by narrower lines, and
are also much more inharmonious, which is indicated by a series of lines on
the spectra multiple to the fundamental frequency. The autooscillation mode
is not natural for the ciliate which, under normal conditions, performs a single
contraction in response to a mechanical stimulus [16]. The autooscillatory
mode observed in this work is, evidently, a reaction of the ciliary contractile
system to a prolonged compression, which leads to the irreversible changes
and death of the cell. Autooscillations develop not in all the cytoplasm but in
a narrow band directed from the anterior part of the ciliate body to the cytostome. The spectra recorded for adjacent regions of the cytoplasm at a
distance of about 50 m from the oscillating band had no resonances.

SPONTANEOUS AND FORCED MEMBRANE OSCILLATIONS

375

Figure 4. Amplitude spectra of ciliary beating of human bronchial epithelium

(a), peristomal ciliary beating (b) and autooscillations of the cytoplasm (c,
curve 1) of the ciliate Spirostomum ambiguum, and also a spectrum of a
non-oscillating region of the cytoplasm (c, curve 2).

Individual frequencies of ciliary beating and especially the halfwidth of the

resonance lines differ noticeably for different cells, and also for the
registration regions on a preparation. In this work, we would only give
concrete magnitudes of these values for the spectra presented in Fig. 4. They
were obtained based on the approximation of the resonance lines by Gaussian
curves. In the spectra of ciliary beating of the bronchial epithelium (Fig. 4 a)
the centre of the first line is at 14.340.02 Hz, the halfwidth of the line is
0.820.05 Hz, the quality of the line (the ratio of its position to the halfwidth)
is 17.4. The centre of the second line is at 26.820.05 Hz, the halfwidth of
the line is 1.000.11 Hz, the quality, 26.9. For the spectra of peristomal
ciliary beating of the ciliate Spirostomum ambiguum (Fig. 4b) these values
are, respectively, 6.540.05 Hz, 0.95 0.11 Hz and 6.9. The spectra of auto-

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V. L. KONONENKO AND J. K. SHIMKUS

oscillations of the cytoplasm for the same species (Fig. 4a, curve 1) contain a
series of lines, for the first four of which the above parameters are 6.430.02
Hz, 0.500.05 Hz and 12.9 (line 1); 12.75 0.02 Hz, 0.64 0.03 Hz and 20.1
(line 2); 19.80.06 Hz, 1.310.15 Hz and 14.7 (line 3); 25.100.19 Hz,
2.300.71 Hz and 10.9 (line 4). Thus, the quality of the lines in the spectra of
natural oscillations of the biomechanical processes in the cells, having the
characteristic frequencies within the range of several up to tens of hertz, can
be characterized by the magnitudes of the order of 10 20.
DISCUSSION
The flicker phenomenon is of special interest in elucidation of the existence
and mechanisms of resonance oscillations of all the erythrocyte membrane or
its particular structures. This is determined, first of all, by the possibility of
studying the oscillatory response of the cell without any external effects. The
observation conditions are also optimal for the registration of possible manifestations of active cell processes in membrane oscillations. On the other
hand, the stochastic nature of the flicker determines the irregular structure of
the spectra of individual signal samplings. This complicates the reliable
detection of the possible resonance frequencies, especially in the case of the
low quality of the lines and in the upper limit of spectral resolution of the
instrument. Thus, the flicker spectra of a single cell in Fig. 2, obtained in both
optical modes, have non-monotone regions near 1 Hz. Along with this, the
given spectra also have non-correlating features, and on the whole, the
non-monotone regions of the spectra in Fig. 2 for one erythrocyte do not
correlate with these for another cell in Fig. 1. The flicker spectrum of yet
another erythrocyte (Fig. 3) has no special features at all. Finally, the flicker
spectra for erythrocyte rouleaux, obtained under conditions of a significantly
greater signal-to-noise ratio and at a much larger number of averagings,
contain no non-monotone regions, either (Figs. 1 and 2). Thus, we can
conclude that the results of membrane flicker studies indicate the absence of
resonance frequencies of erythrocyte oscillations.
These results are supported by experiments on the registration of an
erythrocyte response to a determinate oscillatory effect (Fig. 3). In fact, such
measurements are a standard method for studies of the resonance characteristics of an oscillatory system. The amplitudefrequency characteristics
measured are of a typical relaxation character at all pH values of the medium
used. This is indicated by the presence of linear (in the double logarithmic
scale) regions with a slope close to 1 within the frequency range above 15
Hz (Fig. 3). Note that both methods used are physically similar and are complementary approaches to the studies of the oscillatory response of erythrocytes: the former registers the response to a randomly changing force; the
latter, to a determinate stimulus. This similarity is also indicated by the close

SPONTANEOUS AND FORCED MEMBRANE OSCILLATIONS

377

shapes of the erythrocyte flicker spectrum during a laser irradiation and the
AFC of its forced oscillations (at pH 7.2) (Fig. 3). However, there are also the
differences in the information obtained on the oscillatory properties of the
cell envelope. During the registration of the flicker one probes a local (or ultimately, point) membrane region; therefore, the spectrum measured contains
the contributions of all modes of envelope oscillations irrespective of their
wavelengths. Herewith, for normal erythrocytes the choice of a concrete
probing site the centre of a cell as in this work, or its rim [10] has no
effect on the complete frequency composition of the spectrum recorded.
Under conditions of an average homogeneity of the cell membrane to the
scale of the size of the erythrocyte and the absence of any relation of the
details of its shape (concave regions, the rim) to the lateral structure of the
membrane, demonstrated by experiments on the rolling of the membrane
relative to the cytoplasm, this choice only changes the ratio of the spectral
amplitudes of the modes registered. In contrast with the flicker, a spatially
homogeneous modulated electric field induces oscillations of erythrocyte
elongation, which mainly correspond to the only longest-wave flicker mode
excited.
Another distinction, essential due to the nonlinearity of the mechanical
properties of erythrocytes [13], are large deformations of the cell in usual
electric field experiments as compared with spontaneous bending
deformations in the case of the flicker. Because of this, the mechanical
stresses which occur in the membrane in these two cases can be due to elasticity moduli of different nature and magnitude.
Thus, the measurements of the flicker spectra and the AFC of forced
oscillations cover a broad range of nonlinear deformation characteristics of
normal erythrocytes. The results of the measurements exhibit no resonance
frequencies of the oscillations. Still, in relation to the finite spectral resolution
of any frequency measurements, and also to rather small widths of most peaks
registered in [11, 12], the question arises as to the nature and width of the
resonance lines of assumed oscillations in erythrocytes. The methods of
optical registration used in this work and in [11, 12] are sensitive to optical
density fluctuations the product of the relative refraction index nr by the
thickness of the object. Therefore, two sources of resonance are possible in
principle: autooscillations of nr and mechanical oscillations of the membrane.
For erythrocytes, autooscillations of nr can be expected only within the limits
of the thickness of the membrane, h, because the intracellular solution of
hemoglobin is a passive medium. Respectively, the fluctuating component of
the signal is proportional to the sum of nru(t) + nr(t)h, where u(t) is the
displacement of the membrane and nr(t) are oscillations of nr. The spectral
amplitudes of the membrane flicker within the frequency range of ~10 Hz are
~0.1 m [3], the value of h is of the order of 0.01 m. Thus, even in a hypothetical case of nr(t) ~ nr the fluctuations of the refraction index would have

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V. L. KONONENKO AND J. K. SHIMKUS

given a 10 times lower contribution to the recorded spectrum and would have
been indiscernible against the background of the continuous spectrum of the
membrane flicker. Therefore, the observed or expected resonances in the case
of erythrocytes are, probably, due to mechanical oscillations. From the point
of view of the theory of oscillations, two drastically different situations are
possible: passive oscillations of the membrane (or its structural elements)
under the action of thermal agitation in the surrounding medium and autooscillations defined by active processes in the cell and accompanied by
energy losses. In the case of active processes, the problem of a possible width
of the lines requires the analysis of particular models of biomechanical autooscillations. With reference to erythrocytes or their structural elements, the
authors are not aware of such models. Therefore, a reasonable estimate would
be to use the experimental values of the quality of the lines obtained in this
work, which characterize ciliary beating and cytoplasm autooscillations of
active cells. This brings us to an estimate of the expected halfwidth of the
lines of no less than 0.5 1 Hz within the frequency range of about 10 Hz.
In the case of passive oscillations of the membrane the minimal line width
is, probably, determined by viscous friction in the surrounding fluid. These
oscillations were analyzed by the joint solution of the equations of the theory
of elasticity for a thin envelope and the hydrodynamics for a fluid outside and
inside the cell [2, 8]. For normal erythrocytes, the statement of the problem is
simplified due to the absence of a constant tension of the membrane, and also
due to the possibility of ignoring in the first approximation the shear
deformations and strains of the membrane owing to the excess of the free
surface of the erythrocyte as compared with a sphere of an equal volume [3].
At the same time, the solution of the problem is extremely complicated by the
complex shape of the cell [5]. Taking the real shape of the erythrocyte into
account, it is impossible to obtain analytical formulae for a flicker spectrum,
but they can be obtained within the framework of approximate models [3, 5,
7, 17]. The most adequate model is a modified plane model which considers
the erythrocyte as a system of two plane bending-elastic membranes of a
finite diameter. The membranes are separated by a layer of viscous fluid (a
hemoglobin solution) and placed into another viscous fluid (the surrounding
medium) [17]. As is shown in [18], the spectrum of bending oscillations of
such a system is determined by the following frequencies:
i
e
Kc

f = ----------- , ri = ----------- , re = ----------- , rb = ---------- ,

3
2
3
2
id
id
e d
i d

(1)

where d is the distance between the membranes; Kc and are the elasticity
modulus and the viscosity coefficient of the bending of the membrane; and
are the density and viscosity of the internal (i) and external (e) fluids. The

SPONTANEOUS AND FORCED MEMBRANE OSCILLATIONS

379

frequency f characterizes the elastic oscillations of the bending of the

membranes; and the frequencies ri, re and rb, the damping of these oscillations owing to the viscosity of the fluids and the material of the membrane.
The work [5] also considered shear deformations of the membrane, probably,
affecting the parameters of the lowest modes of the flicker. This brings us to
one more frequency, 1 = /id, which characterizes the elastic shear
oscillations of the membranes. For human erythrocytes, the parameters
determining these frequencies are as follows: Kc = 1.810 12 erg, =
6.610 3 dyn cm 1, d 1 m, i = 6 cP [13], 10 6 cP [3], e = 1 cP, i
e 1 g cm 3. In this case, f = 30 rad s 1, 1 = 1103 rad s 1, ri = 6106
rad s 1, re = 1106 rad s, rb = 1104 rad s 1. Thus, f, 1 << ri, re, rb
and the motion of the membrane is not oscillation but relaxation. The eigen
frequencies of the bending oscillations are formally pure imaginary. As is
shown in [18], they are described to a good accuracy by the expressions:
6
e 3 1
f Q
,
- 12 + 1 + ----- Q
s ( Q ) i -----------
i
2
3

f Q e
Q 1
- ----- + ------------- , Q = qd,
a ( Q ) i -----------2 Q + 4

(2)

where q is the wave vector of the bending mode, s(Q) is the dispersion law
of symmetric (s) modes of oscillations changing the thickness of the erythrocyte, a(Q) is the dispersion law of antisymmetric (a) modes not changing the
thickness [18].
The frequency spectrum of each mode has a Lorentzian shape, and the
registered spectrum of the power of the membrane flicker, S(), is the sum of
contributions of all modes [18]:
2

k B T d 4
A s, a ( Q, S )
s, a ( Q )
------------------------------------ -------------------------------------- ,
S ( ) = S e --------
4
2
2
K c L s, a , Q
+ s, a ( Q )
Q

(3)

where Se, d and L are the surface area, thickness and diameter of an erythrocyte considered as a plane round disc; kBT is thermal energy; As,a(Q, S) is
the instrumental function of the optical mode of registration for symmetric
and antisymmetric modes of the flicker; S is the area of the projection of the
measuring diaphragm of the microscope-photometer on the surface of an
erythrocyte during the registration of the flicker spectrum. Owing to the
constancy of the erythrocyte surface area at all non-destructing deformations
[13], the values of d and L are related by the expression Se = L(L/2 + d). The

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V. L. KONONENKO AND J. K. SHIMKUS

symmetry and the boundary conditions of the problem of the flicker are
similar to the known problem of the oscillations of a round membrane fixed
over the contour. Therefore, out of the entire continuous set of bending modes
possible for a infinite membrane system, only the modes with nodes on its
boundaries can be excited in the cell. This brings us to a discrete set of
allowed wave vectors Q = qd in (3): Qn,m = 2(d/L)n,m, where n,m is a root
of the Bessel function with the number m of the order n. The registered
spectrum (3) differs from the natural spectrum of the erythrocyte flicker by
the presence of the instrumental multiplier |As,a(Q, S)| < 1 in each
summand. As is shown in [17, 18], this multiplier decays as Q is increased,
thus decreasing the contribution of the highest modes of the flicker into the
registered spectrum; it also depends significantly on the type of the optical
mode of registration. For the phase contrast sensitive only to the changes of
the optical thickness of an object, the function As(Q, S) has the shape of a
cut-off filter of highest harmonics, and the function Aa(Q, S) 0, since antisymmetric bending modes do not change the thickness of a two-membrane
system. In the mode of laser irradiation sensitive to single displacements of
the membranes, As(Q, S) Aa (Q, S) 0 and the registered spectrum
contains all frequencies of the natural spectrum of the membrane flicker. This
explains the difference of the flicker spectra measured for the same erythrocyte in different optical modes (Figs. 1 and 2). The calculations using formula
(3) for Se = 134 m2 [13], d = 1.9 m and the above mentioned mechanical
parameters of the erythrocyte, give a satisfactory description of the measured
spectra (solid lines in Fig. 2).
The theory of deformation of erythrocytes by a homogeneous highfrequency electric field has been developed in [14, 19] using the ellipsoidal
approximation for the shape of the erythrocyte and neglecting the inertial
movement of the cell envelope and the adjacent layers of fluid due to the
small thickness of the membrane and a comparatively slow motion. In this
model, the deformation of the erythrocyte is described completely by the ratio
1 of the semiaxes of an ellipsoid of the cell shape, which are longitudinal
and transverse to the field. As theory shows [19], at the experimentally
realized elongation of a cell < 2.2 the deformations of the membrane are
mainly of the bending character with a small contribution of shear
deformations. The values of the elastic stresses occurring in this case in the
membrane are characteristic of the bending stresses and are small as
compared with the stresses which would have occurred in the purely shearing
elongation of the cell. The stress pattern and the ratio of the characteristic
frequencies of the elastic oscillations and the viscous dissipation are close in
this case to those typical of the flicker in the model, taking into account the
shear stresses [5] considered above. According to [19], the dependence of the
amplitude of forced oscillations of the envelope, A, on the frequency of
modulation of a high-frequency field, , is described in this range of

SPONTANEOUS AND FORCED MEMBRANE OSCILLATIONS

381

deformations by a relaxation curve A() = A(0)[1 + (eff)2] 1/2, where eff

by the order of magnitude is the ratio 0 = (s/) of the surface viscosity of
the membrane to the shear elasticity modulus. At s = 6104 dyn s cm 1 [13],
0 ~ 0.1 s which is consistent with the AFC parameters given in Fig. 3. Let us
now estimate the ratio of the frequency of oscillations and dissipations in the
case of more significant deformations of the membrane, when the stresses
arising in it are totally determined by a much greater shear elasticity modulus,
, and the oscillatory mode is more probable. For this purpose, let us
generalize the equation obtained in [19] by taking into account the inertial
term. As an upper limit estimate, let us consider that all the intracellular fluid
is involved in the oscillatory motion, and the dissipation is totally determined
by the friction in a considerably more viscous lipid bilayer of the membrane.
Taking into account the expressions [14] for the generalized forces of shear
elasticity and viscous friction in the membrane, we obtain:
2

s
F p ( , t )
rs
2
1

2
+ ------- + --------1 ----- = ------------------- , 2 = -------- , rs = -------- ,
2
2
i V
i V
i VS e
2

(4)

where F(, t) is the ponderomotive electric force acting on the cell envelope
[14], V = 94 m3 is the cell volume [13]. For the erythrocyte, 2 = 8103
rad s 1, rs = 7.5106 rad s 1, which implies overdamping and the absence
of the resonance frequency of oscillations of the envelope.
It is also significant that the theoretical values of the characteristic
frequencies of mechanical oscillations in erythrocytes (except the flicker
frequency, f) exceed the frequencies of the measuring ranges of [11, 12] and
of the present work by several orders of magnitude. This means that they can
not be observed within these ranges irrespective of their ratio to the dissipation frequencies.
Another source of resonances can potentially be oscillations of individual
structural elements of the membrane or vesicles bound to it, similar to the
vesicles emerging during the loss of part of lipid material by erythrocytes. In
this case, the width of the line can be assessed if we consider the oscillations
of a spherical particle of radius a and density in the fluid with the viscosity
coefficient in the presence of the Stokes friction force. The damping
coefficient is, evidently, 9/2a2 and is equal to 4.5 10 6 rad s 1 for a
characteristic size a ~ 1 m.
Thus, the experimental data obtained and the theoretical analysis indicate
the absence of resonance frequencies in the range of 0.0 3500 Hz associated
with the oscillations of normal human erythrocytes.
The work was supported by the Russian Foundation for Basic Research (grant
No 97-04-48855-a).

382

V. L. KONONENKO AND J. K. SHIMKUS

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