d-Glucose-Induced Second Harmonic Generation Response in

J. Phys. Chem. B 2009, 113, 2513–2518 D-Glucose-Induced

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Second Harmonic Generation Response in Human Erythrocytes

Dmitry Lev, Alexander Puzenko, Alexandra Manevitch, Zacharia Manevitch, Leonid Livshits, Yuri Feldman, and Aaron Lewis* The Department of Applied Physics, The Hebrew UniVersity of Jerusalem, 91904 Jerusalem, Israel ReceiVed: April 10, 2008; ReVised Manuscript ReceiVed: NoVember 5, 2008

The first experimental results of the nonresonant second harmonic generation (SHG) studies of human erythrocytes membrane exposed to various glucose concentrations in phosphate buffered saline (PBS solution) are presented in this article. It is shown that the SHG signal from the membrane can be altered as a function of glucose concentration. The link between the variation of the SHG intensity and the membrane dielectric permittivity with glucose is established both theoretically and experimentally by comparison with time domain dielectric spectroscopy (TDDS) measurement data. 1. Introduction An important technique for investigating the properties of biological cell membranes is generation of the second harmonic signal.1 Today, resonant SHG is being recognized as one of the most sensitive techniques for membrane potential measurements.2-4 In such an application of SHG, a dye is inserted into the membrane and is used to give a resonant second harmonic signal for monitoring alterations in membrane potential.3-5 This paper focuses on the nonresonant SHG from cell membranes. The object of our study is the human erythrocyte, which is a unique biconcave cell that lacks a nucleus. The SHG signal is thus associated with the noncentral symmetry properties of the membrane surfaces and depends on its dielectric properties. This is the reason that it is possible to measure a nonresonant SHG signal of erythrocyte suspension. It was shown that the signal intensity in our experiments is correlated with the concentration of D-glucose in the vicinity of the erythrocyte membrane. Moreover, it was observed that the results are consistent with the studies provided by time domain dielectric spectroscopy (TDDS). The ability to see a similar effect with femtosecond laser excited nonresonant SHG could open new avenues of monitoring glucose concentrations within blood vessels. It is known that transmembrane transport of glucose molecules within human erythrocytes triggers numerous biochemical and biophysical processes. It includes conformational changes of glucose transporter 1 (GLUT1),6-10 glycolytic cascade, or associated activities of several membrane proteins11-18 that may affect the membrane dielectric properties, including the nonlinear response in the optical range. In particular, membrane hyperpolarization that was previously observed in high exterior glucose concentration19 could be a logical reason for the monitored alterations of the SHG signal. Traditionally, the dielectric properties of cells were studied using the dielectric spectroscopy (DS) measurements of a cell suspension.20-22 The dielectric response is characterized by a pronounced dielectric relaxation in the 100 KHz to 10 MHz range due to interfacial polarization at the membrane surfaces.21-24 Usually this relaxation process is characterized by high dielectric strength and was termed the β-relaxation.20 * To whom correspondence should be addressed. Phone: +972-2-6584764; fax: + 972-2-679-8074; e-mail: [email protected].

In this paper we present the theoretical and experimental justifications that the nonresonant SHG signal from the human erythrocytes membrane can be altered as a function of D-glucose exterior concentration. 2. Experimental Procedures Cell Preparation. Fresh human erythrocytes from healthy donors were obtained in heparinized tubes from the Hadassah University Hospital (Jerusalem, Israel) an hour prior to experimentation. The cells were washed in phosphate-buffered saline (PBS, 137 mM NaCl, 8.9 mM Na2HPO4, 1.5 mM KH2PO4, and 2.7 mM KCl in double deionized H2O, pH 7.4, 290 mOsm) and centrifuged three times for 5 min at 300×g. The washed cells were kept in PBS buffer at a 50% hematocrite at room temperature for up to 2-4 hours before the suspensions were finally resuspended in PBS prepared with various additional concentrations of the D- glucose and L-glucose varying from 0 to 20 mM. Immediately before individual measurements, the cell suspensions were isolated from PBS buffer and simultaneously supplemented with one of nine different sugar concentrations ranging from 0 to 20 mM (i.e., 0, 2, 5, 8, 10, 12, 15, 18, and 20 mM) in diluted PBS buffer. To keep the final osmolarity constant in assay (equivalent to 100% PBS), corresponding concentrations of L-glucose were added to the suspensions. The sugar-supplemented suspensions were incubated for 5 min at room temperature before the measurements. The size and shape of the cells were examined with a microscope (IX70 Olympus, Japan) and captured with a DVC-1300 digital camera. Their size was also independently determined by a Coulter Counter.25 No size differences were observed for cells exposed to the studied (0-20 mM) D-glucose concentrations (8.2 ( 0.2 µm mean diameter and 89.1 ( 1.8 µm3 mean volume). Volume fractions of erythrocytes (or in other words, hematocrite) were controlled at 5 ( 0.5%.25-27 SHG Measurements. A Ti:Sapphire laser (Mira, Coherent Radiation, Santa Clara, USA) was pumped by the 8 W power of a solid state laser (Verdi V8, Coherent, Santa Clara, USA) that was used as an excitation source. The laser operated in the mode-locking regime and emitted ultrashort pulses of 60 fs at a wavelength of 780 nm with a frequency of the 76 MHz. The laser beam was directed to the back port of the microscope (Olympus, Japan). To cut the unwanted signals on the second harmonic frequency before they reached the sample, the filter

10.1021/jp803106y CCC: $40.75  2009 American Chemical Society Published on Web 01/30/2009

2514 J. Phys. Chem. B, Vol. 113, No. 8, 2009

Lev et al.

Figure 2. Real part of complex dielectric permittivity vs frequency for a 5% biconcave erythrocyte suspension in PBS buffer with 10 mM D-glucose at 25 °C.

system at a nonuniform time scale of up to 5 µs (frequency range from 200 kHz up to 2 GHz).25,28 The effects of electrode polarization were subtracted within time domain according to the fractal approach in the time window from 2.36 to 2.92 µs for all cells and ghosts suspensions.25 The conductivities of the erythrocyte suspensions (σsus) and buffer (σb) were evaluated in the same time window. Finally, dielectric spectra of the system under investigation were obtained through Fourier transformation.29 3. Results and Discussion

Figure 1. Schematic representation of the experimental setup.

was placed in front of the microscope port. As the beam entered the microscope it was focused into the sample by a 50× objective with numerical aperture (NA) of 0.45. The average power that hit the sample was 5 mW (or pulse power density 1.4 · 1015 [W per pulse/m2]). The forward propagating signal that came from the blood cells was collected by the 50× objective with NA of 0.45. The beam was focused onto the entrance slit of the monochromator (HORIBA Jobin Yvon Inc., USA) that was set to detect the SHG signal (half-wavelength light). This signal was transferred to the current by the photomultiplier (PMT, Hammamatsu 931B, Japan) that was placed right behind the monochromator. The signal from the PMT was increased by the analog processor (Stanford Research systems, USA) and monitored with an oscilloscope (Tektronix TDS 520, UK). It is known that the SHG signal has a very narrow bandwidth, so to determine the maximum signal we changed the monochromator settings to around the half-wavelength (390 nm). To increase the electronic sensitivity of the signal the output of the analog processor was set to give a logarithm of the modulus of the input signal, amplified by a gain factor of 2. Thus, the monitored signal was connected to the real signal by the following formula: Iobs ) 2ln(Ireal) (see Figure 1). TDDS Measurements. The TDDS measurements were performed by a time domain dielectric spectrometer (Dipole TDS Ltd., Jerusalem; total measuring inaccuracy 5%) using a 0.09 pF polished golden plate sample holder with the recording

We recently investigated the specific influence of glucose on the dielectric properties of human erythrocytes in PBS suspensions using TDDS method.25-27 The membrane capacitance (which relates to dielectric permittivity and geometry of the cell membrane) is dramatically changed with D-glucose concentration. At the same time it was shown that the experiments with a nontransportable D-glucose enantiomer, L-glucose, induce minimal membrane alterations.25,26 The results indicate that the measured capacitance is directly correlated to sugar uptake by the erythrocyte. A typical example of dielectric spectra obtained for 5% of biconcave erythrocyte suspension exposed to 10 mM D-glucose after the electrode polarization correction is presented in Figure 2. The characteristic frequency of the dispersion (in the MHz region) and the values of complex dielectric permittivity have been found to be in a good agreement with previously reported results.25,30,31 To estimate the erythrocyte parameters, the observed β-dispersion, corresponding to the Maxwell-Wagner relaxation process, should be fitted using a modified approach for spheroid particle suspensions.25 The routine requires 11 variables, and 9 of them are obtained and fixed as follows: the cell axes (rx ) ry ) 4.1 µm and rz ) 1.3 µm) determined by microscopic measurements (for details see Experimental Procedures section); thickness of the erythrocyte membrane (d ) 3.1 nm)26; permittivity of the buffer (εb, estimated from a high frequency limit of the real part of the suspension permittivity) and cytoplasm (εcp ) 60);32 and dc conductivities of the membrane (σm ) 0)25 and the buffer (σb, obtained from the data treatment in time domain). The final results of the fitting are presented in terms of the membrane permittivity, εm, and the cytoplasm conductivity, σcp. The first experimental results of nonresonant SHG studies of human erythrocytes exposed to various concentrations of Dand L-glucoses in isotonic PBS buffer were compared with data obtained by the TDDS method.25 We found a significant increase

Nonresonant SHG from Cell Membranes

Figure 3. The normalized data of the membrane static dielectric permittivity (O) and SHG signal (9) vs D-glucose concentration. The linear fit for both data are shown by dashed and solid lines, respectively. For the normalization, the values of the membrane static dielectric permittivity and SHG at zero concentration of D-glucose were taken. Each point presents the averaging over more than 6 measurements. The samples from 3 donors were studied and each sample was measured at least twice.

J. Phys. Chem. B, Vol. 113, No. 8, 2009 2515

Figure 5. Schematic presentation of the transmission geometry for SHG at the front membrane interface. The surface nonlinear effect is specified by second order surface susceptibility χ(2) s . The media in the region z > 0 is the buffer solution with the linear dielectric permittivity εb(ω). The media in the region z < 0 is the membrane characterized by the linear dielectric permittivity εm(ω).

size of cell. Due to the special cell orientation, the larger part of the membrane surface is found to be normal to the laser wave vector. The anisotropy produced by the membrane surface is the cause of the noncentral symmetric properties of the media that is required for the generation of the second-order nonlinear SHG signal.33 In general, the nonlinear polarization spectra of a material could be expressed as

P(ω) ) χ(ω)(1) × E(ω) + χ(ω)(2) × E(ω) × E(ω) + ... (1) where P is the polarization vector induced by the vector of the external field E, ω is the angular frequency, and χ(n) is the n-th order nonlinear susceptibility tensor. The first term relates to the linear absorption and reflection of the electromagnetic waves described by the tensor of the linear complex dielectric permittivity: Figure 4. Calculation of the correlation coefficient R )(∑i n) 1 (xi jx)(yi - jy))/([∑ni ) 1 (xi - jx)2][∑ni ) 1 (yi - jy)2]) between the normalized static dielectric permittivity of the membrane and SHG intensity data, where is the R value displayed in the table is the correlation coefficient for the two data sets.

in the SHG intensity due to D-glucose concentration in the buffer that correlated with TDDS data (see Figures 3 and 4). Note that β-dispersion of cell suspension monitored by TDDS in the frequency range 105-107 Hz is strongly dependent on the membrane dielectric permittivity. The nonresonant SHG case, however, involves infrared (IR) frequencies (≈1014Hz). Moreover, there is the significant difference in the space scale of both methods. Dielectric spectroscopy (TDDS) operates with the macroscopic dielectric permittivity averaged over the macroscopic unit volume of suspension, whereas the secondorder nonlinear SHG signal appears on the mesoscopic scale that corresponds to the thickness of the erythrocyte membrane. In TDDS measurements the uniform distribution of the cell orientations in the macroscopic unit volume of suspension is realized. Thus, the averaging over the space leads to the central symmetry of the effective media. In the reported SHG experiment the single fundamental laser beam is focused on the one individual cell. The size of the focal spot is comparable with

ε(ω) ) ε
(ω) - iε

(ω) ) ε0[1 + χ(1)(ω)]

(2)

where ε0 is dielectric permittivity of free space. The second term in series expansion eq 1 corresponds to nonlinear effects such as SHG, sum, and difference frequency generation, Raman processes. The experimental conditions mentioned above allow us to consider the case of the normal incidence of the linear s-polarized laser beam (see Figure 5). According to the general phenomenological treatment of the surface SHG,34 for the normal incidence the power of the SHG signal generated by the nonlinear polarization sheet at the surface of the membrane slab34 can be presented as a sum of the contributions from the front and back membrane surfaces,

P(2ω) ) P(2ω)front + P(2ω)back

Here,

(3)

2516 J. Phys. Chem. B, Vol. 113, No. 8, 2009

P(2ω)front ) Q

P(2ω)back ) Q

| |

2√εb(2ω)

Lev et al.

βk ) (1 - Rk)/Rk

2√εm(2ω)

·

√εb(2ω) + √εm(2ω) √εb(2ω) + √εm(2ω) 2 2√εb(ω) 2 (4) √εb(ω) + √εm(ω)

(

)|

2√εb(2ω)

√εb(2ω) + √εm(2ω)

(

·

Rk )

∑

2√εm(2ω)

k)x,y,z

√εb(2ω) + √εm(2ω) 2√εm(ω)

√εb(ω) + √εm(ω)

)| 4

2

(5)

2 2 Q ) [(32π3ω2)/(c3A)]|χ(2) s | P (ω); A is the area of the spot size of the laser beam; c is the velocity of light; χ(2) s (ω) is the second order surface nonlinear susceptibility; P(ω) is the incident power, εb(ω) and εm(ω) are the complex dielectric permittivity of the buffer and membrane at the light source frequency ω, respectively; εb(2ω) and εm(2ω) are the correspondent permittivity at the second harmonic 2ω. Note that the values of the exterior buffer and cytoplasm εcp(ω), εcp(2ω) dielectric permittivity are similar, that is, εb(ω) = εcp(ω) and εb(2ω) = εcp(2ω).35 These assumptions are used in eq 5 for evaluating the contribution P(2ω)back from the back membrane surface. Let us consider the dispersion of the linear complex dielectric permittivity εb(ω) and εm(ω), that we defined as permittivity of the buffer and membrane (see Figure 5). It is known that the dielectric permittivity of the cell suspension in the low frequency range, due to the interface polarization (β-process) is well approximated by the Maxwell-Wagner mixture formula.36 To evaluate the dielectric properties of biconcave erythrocytes, the model for nonspherical cell suspensions can be described by the following formulas:36

εpk(ω) - εb(ω) 1 ε(ω) ) εb(ω) + pεb(ω) 3 R ε (ω) + (1 - Rk)εb(ω) k)x,y,z k pk (6)

βkεm(ω) + εcp(ω) - βkυ(εm(ω) - εcp(ω)) εpk(ω) ) εm(ω) βkεm(ω) + εcp(ω) - υ(εm(ω) - εcp(ω)) (7) where εcp(ω) and εm(ω) are the complex permittivity of cytoplasm and cell membrane, respectively; and υ is the volume ratio of the inner ellipsoid to the outer ellipsoid. Taking into account that membrane thickness d is very small compared to the axis, rx, ry, and rz, υ can be approximated as

(

υ≈ 1-

and

)(

)(

d d d 11rx ry rz

)

(8)

∫0∞ (r2 +ds s)r k

(10)

s

Rk ) 1 and rs)√(r2x + s)(r2y + s)(r2z + s) (11)

Below, we will consider the real part of the membrane dielectric permittivity ε′m(ω) ) Re[εm(ω)] that for ω f 0 converges to the static value ε′m. Using TDDS measurements and relationships eqs 6-11, one can evaluate the static dielectric permittivity of the membrane ε′m. Recently, we found a strong correlation between the changes in the values of ε′m and the extracellular D-glucose concentration. However, with an increased frequency of up to ω ) ω0 (the laser source), the dielectric permittivity of the membrane is forced to decrease through the various relaxation processes of its polar components. This is illustrated in Figure 6 at low and high frequencies where the data exists and is graphically illustrated for intermediate frequencies where the exact dispersion is not known. However, the specific information regarding the dielectric permittivity decreasing in this intermediate frequency regime is not essential to this paper. Thus, the ratio between the high and static frequency dielectric permittivity of the membrane can be defined as

∆ε
m(ω0, G1, G2) ) K(ω0)∆ε
m(ω ) 0, G1, G2) (12) where ∆ε′m( ω ) 0,G1,G2) ) ε′m(G1) - ε′m(G2) and ∆ε′m( ω0,G1,G2) ) ε′m(ω0,G1) - ε′m(ω0,G2). Let us set in eq 12 G2 ) 0 and G1 ) G;

∑

where εpk(ω) is the equivalent complex permittivity of the shellellipsoid, which has three components εpk)x,y,z(ω) along the x, y, and z axes; Rk is the depolarization factor along the k-axis; εb(ω) is the complex permittivity of the buffer; and p is the volume fraction. It has been reported that εpk(ω) can be expressed as:

rxryrz 2

(9)

ε
m(ω0, G) ) K(ω0)ε
m(0, G) + F(ω0)

(13)

where F(ω0) ) ε′m(ω0,0) - K(ω0)ε′m(0,0) is independent of glucose concentration. We assume that in the interval [ω0,2ω0] the membrane relaxation does not show significant processes, that is,

ε
m(ω0, G) = ε
m(2ω0, G)

(14)

Moreover, in the IR frequency band the dielectric permittivity of the buffer solution (mixture of water, salt, and D-glucose) can be defined by the dielectric permittivity of pure water:37

ε
b(ω0) ) ε
w(ω0)

(15)

ε
b(ω0) ) ε
w(ω0) = ε
w(2ω0) ) ε
b(2ω0)

(16)

and

The value of the ε′w(ω0) can be estimated using the water refractive index nw = 1.33 in the IR band, ε′w(ω0) = nw2 = 1.77. The value of the cell membrane refractive index is nm=1.45,38 correspondingly ε′m(ω0) = nm2 = 2.1. Thus, the small relative deviation

Nonresonant SHG from Cell Membranes

J. Phys. Chem. B, Vol. 113, No. 8, 2009 2517 Since the normalization procedures of dielectric and second harmonics data are also linear, one can get

P(2ω0)| norm ) A + BG

(23)

where P(2ω0)|norm is the normalized intensity of the SHG, and B is the slope of the normalized static dielectric permittivity data. This calculated result demonstrates a linear dependence of the second harmonic signal vs glucose concentration, and it is in good agreement with the experimental data that is shown in Figure 3. 4. Conclusions Figure 6. Schematic presentation of the real part of the membrane dielectric permittivity dispersion for the two different buffer glucose concentrations G1 and G2. Relationship between the static dielectric permittivity and the dielectric permittivity of the membrane in the IR range is established by means of the frequency-dependent coefficient K(ω).

δε
(ω0) ε
w(ω0)

, 1, where δε
(ω0) ) ε
m(ω0) - ε
w(ω0)

(17) allows us to evaluate the intensity of the SHG signal using eqs 3-5 as a series expansion with regard to the small parameter, with accuracy of the first order terms;

(

P(2ω0) = Q 2 +

δε
(ω0) ε
w(ω0)

)

(18)

At the same time, the membrane static dielectric permittivity versus glucose concentration can be approximate by the linear law (See Figure 3),

ε
m(0, G) = R + β · G

(19)

where parameters are determined by fitting eq 19 to the dielectric data (See Figure 3). By substitution of eq 19 into eq 13, one can obtain the membrane permittivity trend in the IR range.

ε
m(ω0, G) = K(ω0)(R + β · G) + F(ω0)

(20)

Taking into account eq 17, the trend of the intensity of SHG signal P(2ω0) can finally be presented as follows;

(

P(2ω0) = Q 1 +

ε
m(ω0, G) ε
w(ω0)

)

(21)

Substituting eq 20 into eq 21 leads to the linear dependence on the glucose concentration as follows,

(

P(2ω0) ) Q 1 +

K(ω0)(R + βG) + F(ω0) ε
w(ω0)

)

(22)

The first measurements of the nonresonance SHG from the human erythrocytes membrane show that the SHG signal is altered as a function of the glucose transmembrane transport. The signal from the erythrocytes membrane increases with glucose concentration in the buffer solution. Previously a similar effect was demonstrated by the TDDS method. We show in eqs 22-23 that there is a link between the static dielectric permittivity of the membrane and the SHG signal, and this is the basis of our ability to correlate the effects observed with TDDS. The analysis performed above allows us to connect the slow variations (trends) of experimental data obtained by both methods. Meanwhile, a high correlation factor between the two findings, (see Figure 4) confirms the sensitivity of the SHG signal to the fine polarized structure previously observed by the TDDS method.25 Moreover, due to the generation of the SHG signal directly from the membrane there is no need to use a complex model for nonspherical cell suspensions for the evaluation of the membrane dielectric permittivity by the TDDS method. As our data clearly shows the SHG signal that is sensitive to the glucose concentration may yield new avenues of glucose sensing in blood. References and Notes (1) Huang, J. Y.; Chen, Z.; Lewis., A. J. Phys. Chem. 1989, 93, 3314. (2) Huang, J. Y.; Lewis, A.; Loew., L. Biophys. J. 1988, 53, 665. (3) Bouevitch, O.; Lewis, A.; Pinevsky, I.; Wuskell, J. P.; Loew., L. M. Biophys. J. 1993, 65, 672. (4) Nuriya, M.; Jiang, Jiang; Nemet, B.; Eisenthal, K. B.; Yuste., R. Proc. Natl. Acad. Sci. USA. 2006, 103, 786. (5) Campagnola, P. J.; Mei-dei, Wei; Lewis, A.; Loew., L. Biophys. J. 1999, 77, 3341. (6) Carruthers, A. Physiol. ReV. 1990, 70, 1135. (7) Epand, R. F.; Epand, R. M.; Jung., C. Y. Protein Sci. 2001, 10, 1363. (8) Salas-Burgos, P.; Iserovich, A.; Zuniga, F.; Vera, J. C.; Fischbarg., J. Biophys. J. 2004, 87, 2990. (9) Heard, K. S.; Fidyk, N.; Carruthers., A. Biochemistry. 2000, 39, 3005. (10) Levine, K. B.; Cloherty, E. K.; Hamill, S.; Carruthers, A. Biochemistry. 2002, 41, 12629. (11) Parker, J. C.; Hoffman., J. F. J. Gen. Physiol. 1967, 50, 893. (12) Mercer, R. W.; Dunham., P. B. J. Gen Physiol. 1981, 78, 547. (13) Jiang, W.; Ding, Y.; Su, Y.; Jiang, M.; Hu, X.; Zhang, Z. Biochem. Biophys. Res. Commun. 2006, 339, 1255. (14) Tsai, I. H.; Murthy, S. N.; Steck., T. L. J. Biol. Chem. 1982, 257, 1438. (15) Jenkins, J. D.; Madden, D. P.; Steck., T. L J. Biol. Chem. 1984, 259, 9374. (16) Kliman, H. J.; Steck, T. L. J. Biol. Chem. 1980, 255, 6314. (17) Low, P. S.; Rathinavelu, P.; Harrison., M. L. J. Biol. Chem. 1993, 268, 14627. (18) Laughlin, M. R.; Thompson., D. J. Biol. Chem. 1996, 271, 28977. (19) Zavodnik, I. B.; Piasecka, A.; Szosland, K.; Bryszewska., M. Scand. J.Clin. Lab. InVest. 1997, 57, 59. (20) Schwan, H. P. AdV. Biol. Med. Phys. 1957, 5, 147. (21) Pethig, R.; Kell, D. B. Phys. Med. Biol. 1987, 32, 933.

2518 J. Phys. Chem. B, Vol. 113, No. 8, 2009 (22) Foster, K. R.; Schwan, H. P. Crit. ReV. Biomed. Eng. 1989, 17, 25. (23) Takashima, S. Electrical Properties of Biopolymers and Membranes; IOP Publishing Ltd.: Philadelphia, USA; 1989. (24) Feldman, Yu.; Ermolina, I.; Hayashi, Y. IEEE Trans. Dielec. EI. 2003, 10, 728. (25) Livshits, L.; Caduff, A.; Talary, M. S.; Feldman, Yu. J. Phys. D: Appl. Phys. 2007, 40, 15. (26) Hayashi, Y.; Livshits, L.; Caduff, A.; Feldman, Yu. J. Phys. D: Appl. Phys. 2003, 36, 369. (27) Caduff, A.; Livshits, L; Hayashi, Y.; Feldman, Yu. J. Phys. Chem. B 2004, 108, 13827. (28) Feldman, Yu.; Andrianov, A.; Polygalov, E.; Romanychev, G.; Ermolina, I.; Zuev, Yu.; Milgotin, B. ReV. Sci. Instrum. 1996, 67, 3208. (29) Axelrod, N.; Axelrod, E.; Gutina, A.; Puzenko, A.; Ben Ishai, P.; Feldman, Yu. Meas. Sci. Tech. 2004, 15, 1.

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