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Electromechanical coupling of murine lung tissues probed by Piezoresponse Force Microscopy Peng Jiang, Fei Yan, Ehsan Nasr Esfahani, Shuhong Xie, Daifeng Zou, Xiaoyan Liu, Hairong Zheng, and Jiangyu Li ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 27, 2017

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ACS Biomaterials Science & Engineering

Electromechanical coupling of murine lung tissues probed by Piezoresponse Force Microscopy Peng Jianga,b,c,*, Fei Yanb,*, Ehsan Nasr Esfahanic, Shuhong Xiea, Daifeng Zoub, Xiaoyan Liud, Hairong Zhengb, Jiangyu Lib,c,# a

Key Laboratory of Low Dimensional Materials and Application Technology of Ministry of Education, School of Materials Science and Engineering, Xiangtan University, Yuhu District, Xiangtan, Hunan, 411105, China

b

Shenzhen Key Laboratory of Nanobiomechanics, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, University Town of Shenzhen, Shenzhen, Guangdong, 518055, China

c

Department of Mechanical Engineering, University of Washington, Seattle, WA, 98195, USA

d

College of Metallurgy and Materials Engineering, Chongqing Key Laboratory of Nano/Micro Composites and Devices, Chongqing University of Science & Technology, Shapingba District, Chongqing, 401331, China

*

Authors contributed equally to this work.

#

Authors to whom the correspondence should be addressed to; Email: [email protected].

Abstract Elastin is a major constituent of lung that makes up approximately 30% of lung’s dry weight, and the piezoelectricity of elastin is expected to be exhibited in lung tissues. Since hundreds millions of cycles of inhalation and exhalation occur in one’s life time, such piezoelectric effect leads to hundreds millions of cycles of charge generations in lung tissues, suggesting possible physiological significance. Using piezoresponse force microcopy (PFM), we show that the murine lung tissues are indeed piezoelectric, exhibiting predominantly first harmonic piezoresponse in both vertical and lateral modes. The second harmonic response, which could arise from ionic motions, electrochemical dipoles, and electrostatic interactions, is found to be small. The mappings of amplitude, phase, resonant frequency, and quality factor of both vertical and lateral PFM are also obtained, showing small fluctuation in frequency, but larger variation in quality factor, and thus energy dissipation. The phase mapping is confined in a small range, indicating a polar distribution with preferred orientation. It is also found that the

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polarity of the electomechanical coupling in lung tissues can be switched by an external electric field, resulting in characteristic hysteresis and butterfly loops, with a presence of internal bias in the polar structure. It is hypothesized that the piezoelectric charge generation during inhalation and exhalation could play a role in binding of oxygen to Hemoglobin, and the polarity switching can help damp out the possible sudden increase in air pressure. We hope such observation of piezoelectricity and its polarity switching in lung lay the foundation for the subsequent studies of its physiological significance.

Keywords: Biopiezoelectricity; Murine lung; Piezoresponse Force Microscopy; Elastin; Polarity switching


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1.

Introduction Piezoelectricity is a linear coupling between mechanical and electric fields, i.e.

electric charges can be generated by mechanical force in a piezoelectric materials, and mechanical deformation can be resulted from electric loading as well 1. The piezoelectric effect was discovered by Curie brothers in 1880, and it was initially limited to inorganic and synthetic materials. In 1940, the phenomenon was reported in wool and hair 2, and that was the first observation of piezoelectricity in biological systems. In 1957, Fukada published a landmark paper on the piezoelectric effect in bone 3, making the effect well known in biophysics community. Since then a wide range of tissues were found to be piezoelectric, including bones

3-5

, teeth

6,7

, muscles 8, nerves 9, blood vessel walls

10

, and skins

11,12

,

resulting in suggestions that piezoelectricity is a fundamental property of biological tissues and possesses important physiological functions

13-15

. For example, it was proposed that the

surface aspects of bone remodeling is governed in part by the piezoelectric polarization produced when the bone is deformed 16. In recent years, there have been renewed interests in piezoelectricity of biological systems thanks to the emergence of powerful piezoresponse force microscopy (PFM) that enables the probing of electromechanical coupling at the nanoscale

17-21

. The technique has

been applied for functional imaging of individual collagen type I fibrils 22,23, dried tendon and isoelectrically focused collagen hydrogels 24, self-assembled peptides nanotubes (PNTs)25,26 , and nacre under flexural stresses

27

, among others, revealing correlation between

piezoelectric response and structural heterogeneity of the biological materials. Furthermore, it was discovered that the polarity of electromechanical coupling in some of the systems, such as clamshell

28

, γ-glycine

29

, and aortic walls

30

can be reversed by external electric field,

analog to ferroelectric switching in some inorganic piezoelectric materials 1. It turns out that the polarity of elastin can be switched

31,32

, but not that of collagen

31,33,34

, and subsequent

studies revealed that in aortic wall, electromechanical coupling and its switching correlates with the early stage development of atherosclerosis, making it possible to investigate the initiation and mechanism of artery hardening using PFM 35. Elastin is a major constituent of lung that makes up approximately 30% of lung’s dry weight

36,37

. Therefore, lung tissue is expected to possess strong electromechanical coupling,



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and its polarity is expected to be switchable. With the average respirator rate in the range of 12 to 18 breathes per minute for a healthy adult 38, hundreds millions of cycles of inhalation and exhalation occur in one’s life time, corresponding to hundreds millions of cycles of piezoelectric charge generations of lung tissues. Yet quite surprisingly, piezoelectric effect in lung tissues has never been reported yet, to our best knowledge, and its physiology significance has not been studied. Hence, in this paper, we investigate the electromechanical coupling and its switching in murine lung using PFM technique. 2.

Materials and methods

2.1

Sample preparation The murine lung tissues were derived from sacrificed and dissected C57 mice or SD

rats, obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), and the procedures of all animal experiments were approved by Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences Animal Care and Use Committee. Lung tissues were fixed in 4% paraformaldehyde solution for 24 h, and then dehydrated in a graded distilled water/ethanol series with increased ethanol concentrations (70%, 80%, 95% (І), 95% (ІІ), 100% (І), and 100% (ІІ)) for 15 minutes each, followed by washing with a graded ethanol/hexamethyldisilazane (ethanol/HMDS) series with increased HMDS cencontrations (30%, 50%, 70%, and 100%) for 15 minutes each. They were then dried overnight in room temperature. Dehydrated tissues were cut from the lower part of the lung close to the center into sections perpendicular to the vertical axis of the lung, as shown in Figure S1a in the Supporting Information (SI). The rugged edges of the sections, including those with bronchioles, were then removed, as shown in Figure S1b, resulting in specimens approximately 6 mm wide, 3 mm high, and 0.9 mm thick. The specimen was glued onto silicon wafer sputtered with gold/platinum using silver paint for subsequent PFM studies, as shown in Figure S1c. 2.2

Morphology characterization The morphology of lung tissues was characterized by scanning electron microscopy

(SEM, FEI Sirion SEM) and atomic force microscopy (AFM, Asylum Research, MFP-3D-Bio). For SEM, sample was sprayed with a thin gold layer to make sure that the


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surface topography can be imaged with a focused beam of electrons. For AFM, tapping mode topography was obtained during the scan of 2×2 µm2 and 1×1 µm2 areas using an ASYELEC-01 cantilever probe with the spring constant of 2 N/m and resonance frequency in air around 60 kHz.

Figure 1. Schematics of PFM; (a) AFM system setup; (b) enhanced piezoresponse amplitude near cantilever-sample resonance; (c) DC wave form on top of AC excitation for switching PFM; and (d) characteristic hysteresis and butterfly loops associated with polarity switching. 2.3

Piezoresponse force microscopy Piezoresponse force microscopy (PFM) experiments were carried on MFP-3D-Bio

AFM using an ARROW-CONTPt-50 cantilever probe made of silicon, with the tip of the probe coated with Pt/Ir to make it conductive. The spring constant of cantilever is 0.2 N/m, and its resonance frequency in air is 14 kHz. The electromechanical coupling of the samples was probed by applying an AC voltage to the sample surface through the conductive probe, exciting a piezoelectric vibration that can be measured by the photodiode, as shown in Figure 1a. In order to enhance sensitivity, the AC voltage was applied near the resonant frequency ω0 of the cantilever-sample system, and the deflection was measured at the corresponding frequency using lock-in amplifier, resulting in piezoresponse that is magnified by orders of



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magnitude, as shown in Figure 1b, wherein both phase and amplitude of the piezoresponse can be decoded. This corresponds to first harmonic response that is related to linear piezoelectricity. Meanwhile, the cantilever can also be driven at ω0/2 while the deflection measured at ω0, resulting in second harmonic measurement that is related to quadratic electromechanical response. The combination of first and second harmonic responses can help us understand the mechanisms of electromechanical coupling in lung tissues. In addition, two deformation modes of PFM have been carried out, one is the vertical PFM (VPFM) that measures the out-of-plane normal displacement of the sample, which is associated with piezoelectric coefficient d33; and the other is in-plane lateral PFM (LPFM) that measures the shearing displacement of the sample associated with piezoelectric coefficient d15. Both VPFM and LPFM amplitudes were recorded in terms of voltage output from photodiode, and VPFM responses were usually calibrated to covert the voltage (mV) to displacement (pm). In addition to piezoresponse measurement, it is also possible to manipulate the polarity of the sample by applying a sequence of DC voltages in triangle saw-tooth form on top of the AC voltage, as shown in Figure 1c. If the polarity of the sample can be switched, then characteristic phase-voltage hysteresis loop and amplitude-voltage butterfly loop can be obtained, as shown in Figure 1d. In order to minimize electrostatic interactions between the charged probe and the sample, the DC voltage was stepped back to zero between each incremental step. This is the so-called “Off” state, during which the piezoresponse was measured from AC excitation. A harder ASYELEC-01 probe was used in this measurement to reduce the crosstalk. All the experimental curves reported here were acquired in the second cycle of DC voltage to allow them to be stabilized. More detailed information regarding PFM techniques and measurements can be found in a recent review 21. 3.

Results and discussions

3.1

Structure We first examine the microstructure and morphology of lung tissues in terms of SEM

image and AFM topography mapping. Elastin fibers are ubiquitous in the lung

39

, and the

SEM image in Figure 2a shows that the lung tissues are fibrous network with randomly oriented nanofibers. This morphology is also confirmed by low resolution AFM topography


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over a 2×2 µm2 area, as shown in Figure 2b, wherein porous fiber network is evident. The high resolution AFM topography scan over a 1×1 µm2 area is shown in Figure 2c, revealing globular type of features that was also observed in aortic wall

30,40

, indicative a hierarchical

structure of the fibers.

Figure 2. Microstructural morphology of murine lung tissue; (a) SEM image of mice lung tissue; (b) and (c) low- and high-resolution AFM topography of rat lung tissue acquired in 2×2 µm2 (b) and 1×1 µm2 (c) areas. 3.2

Linear and quadratic piezoresponses We then examine the piezoresponse of rat lung tissues as probed by PFM. All the

PFM measurements were taken on the fixed, dehydrated and dried samples, since PFM on wet sample is rather challenging due to electric conduction. Nevertheless, we believe that there is no qualitative PFM difference between wet and dry lung tissues, since it has been reported that the piezoelectric responses in wet and dry bones are not noticeably different 41. It is now well known that PFM responses can also arise from electromechanical mechanisms other than piezoelectricity, such as electrochemical dipoles electrostatic interactions

42

, ionic motions

43

, and

44

, among others, and thus it is important to identify the dominant

mechanism that is responsible for the piezoresponse observed in lung tissues. This can be accomplished by comparing first and second harmonic PFM responses in both vertical and lateral modes, as demonstrated by Chen et al. 45, since mechanisms other than piezoelectricity is often nonlinear in the nature, resulting in a prominent second harmonic response. To this end, AC excitation was applied at either ω0 (first harmonic) or ω0/2 (second harmonic) in our experiments, while the piezoresponse is measured at ω0 for maximum sensitivity at the resonance. As seen from Figure 3, under all four AC voltages of increasing magnitudes we



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applied, the first harmonic VPFM responses measured are always one order of magnitude higher than the second harmonic responses, suggesting that the linear piezoelectricity is the dominant electromechanical mechanism in lung tissue. While Vegard strain arising from ionic motion may also be linear to the applied voltage, recent theoretical analysis suggests that it plays a key role only at low voltage under 1 V, beyond which quadratic electrostriction dominates

46,47

. This voltage is much smaller than the voltage we applied, and thus we can

safely conclude that the piezoresponse in lung tissue is dominated by linear piezoelectricity, especially when no ionic species are expected to be present in the tissues. In addition, both first and second harmonic responses versus the driven frequency can be fitted nicely by the damped harmonic oscillator model

48

, allowing us to determine the quality factor and the

intrinsic piezoresponse amplitude.

Figure 3. Linear and quadratic VPFM responses of rat lung tissue under four AC voltages; (a) 11 V; (b) 16.5 V; (c) 22 V; and (d) 27.5 V.


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Figure 4. Linear and quadratic LPFM responses of rat lung tissue under four AC voltages; (a) 11 V; (b) 16.5 V; (c) 22 V; and (d) 27.5 V. Similar trend is also observed between first and second harmonic LPFM responses shown in Figure 4, that the linear response dominates electromechanical coupling, though it is observed that the damped harmonic oscillator model does not fit the torsional motion of the cantilever

well.

Indeed,

for

electromechanical

coupling

originating

from

either

electrochemical dipoles or electrostatic interactions, the lateral response is expected to be minimal due to the spherical symmetry involved, while piezoelectricity has inherently lower symmetry that often results in lateral response. The strong first harmonic response in LPFM thus further support the idea that the electromechanical response we observe in lung tissues is piezoelectric in nature. These two set of data thus suggest that the lung tissue is intrinsically piezoelectric, as expected from its elastin constituents, and the linearity of the electromechanical behavior is further confirmed by Figure 5, wherein both intrinsic VPFM and LPFM responses versus the applied AC excitation are plotted, after being corrected by the quality factor using damped harmonic oscillator model 48 and averaged over ten points at



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each voltage. Good linear and quadratic variation is observed in first and second harmonic responses, respectively, for both VPFM and LPFM, and the magnitude of linear responses are much higher than quadratic one, especially for LPFM, indicating that the piezoresponse is dominant in-plane. From the slope in Figure 5a, the effective piezoelectric coefficient is estimated in the order of 0.1 pm/V.

Figure 5. Averaged first and second harmonic responses (n=10) with error bars for vertical (a) and lateral (b) PFM responses versus AC voltages. 3.3

PFM mappings After confirming the linear piezoelectric nature of electromechanical coupling in rat

lung tissues, we examine its VPFM and LPFM responses over a 1×1 µm2 area using dual AC resonance tracking (DART) technique 49. The AC voltage applied to excite the piezoresponse is 20 V for VPFM and 30 V for LPFM, to ensure the sensitivity. During scanning, resonant frequency often shifts due to change in contact stiffness of the sample, and DART allows us to track resonance and thus eliminate the crosstalk with topography. Since phase and amplitudes at each point are measured under two different frequencies across resonance, it also allows us to solve for quality factor and resonant frequency at each point and yield corresponding mappings. In Figure 6, mappings of VPFM amplitude corrected by quality factor, phase, resonant frequency, and quality factor are shown, all overlaid on three-dimensional (3D) topography. Note that amplitudes mapping indicates the magnitude of piezoelectric responses, phase mapping reveals the orientation distribution of polarization, resonant frequency correlates with the contact stiffness, and quality factor is related to energy dissipation, respectively. To visulize the distributions of these quantities more clearly,


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corresponding histograms are also shown (Figure 6e). Some variation of amplitude mapping (Figure 6a) is observed, ranging approximately from 4 to 12 pm, showing different piezoelectric responses in different area. The phase (Figure 6b) is largely consistent, falling within approximately a 50o-range, indicating that the polar axis is aligned with preferred orientation. Small variation of frequency mapping (Figure 6c) is also observed, ranging from 62 to 68 kHz, and lower resonant frequencies, and thus softer spots, often correspond to higher PFM responses in Figure 6a, as expected. Quality factor, on the other hand, has much larger variation from 100 to 400 (Figure 6d), suggesting different energy dissipation in different area.

Figure 6. VPFM mappings of rat lung tissue overlaid on 3D topography in a 1×1 µm2 area; (a) amplitude; (b) phase; (c) frequency; (d) quality factor; and (e) histogram distributions.



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Figure 7. LPFM mappings of rat lung tissue overlaid on 3D topography in a 1×1 µm2 area; (a) amplitude; (b) phase; (c) frequency; (d) quality factor; and (e) histogram distributions. The mapping of LPFM responses in a different area are shown in Figure 7. The corrected amplitude (Figure 7a), which is not calibrated due to accuracy issues, varies from 0.015 to 0.04 mV, and the phase distribution is similar to that of VPFM, falling into a 50o-range that is consistent with aligned polar axis. The frequency variation is 273 to 277 kHz, and the much higher frequency value than VPFM one is associated with the torsional vibration of cantilever. In Figure 7d, the quality factor is found to range from 50 to 500, and larger variation in quality factor is consistent with what we saw in VPFM.


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Figure 8. Switching characteristics of mice lung tissue, where butterfly loops (a and c) and hysteresis loops (b and d) of VPFM (a and b) and LPFM (c and d) are measured at same point. 3.4

Polarity switching It has been reported that the polarity of elastin can be reversed by external electric

field 31, 32, and such switching behavior can be suppressed by glucose 31 as well as affected by arteriosclerosis 35, suggesting potential physiological significance of polarity switching. It is thus interesting to examine whether the polarity of electromechanical coupling of lung tissues can be reversed or not, by applying a sequence of DC voltage up to 15 V, similar to those shown in Figure 1c. At the same time, AC voltage of 3 V was applied to excite the piezoelectric vibration for measurement at each DC voltage step, resulting in characteristic hysteresis and butterfly loops as shown in Figure 8, where 180o phase reversal is evident in both VPFM and LPFM. It is important to point out that since both VPFM and LPFM responses are predominantly linear in nature, as demonstrated earlier, the 180o phase reversal is a true indication of intrinsic polarity switching of the system. The coercive voltage is estimated from the loops to be 2.5 V on the positive side and -6.1 V on the negative side for VPFM, as well as 4.3 V on the positive side and -4.5 V on the negative side for LPFM, and slight asymmetry toward negative voltage is observed, indicating an internal bias in the polar structure. This also results in asymmetric butterfly loop with higher response under positive



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voltage. Similar switching behavior was also found in rat lung tissues, as shown in Figure S2, which appears to be dependent on cycling period that is often observed biological tissues. This is believed to be related to relaxation behavior of polarity switching, as reported by Liu et al. 30, that the switched polarization tends to relax back after removal of the DC voltage. 3.5

Discussion Elastin, as an extracellular matrix (ECM) protein, is present in all connective tissues

of vertebrates 50, rendering essential elasticity to aorta, lung, ligament, and skin subjected to repeated physiological stresses 37. In addition to its structural function, recent evidences also support the physiological significances of elastin, for example in vascular morphogenesis 52

51,

and homeostasis 53. Using PFM, we show that the lung tissues, consisting of large portion

of elastin fibers, exhibit strong electromechanical coupling that is dominated by the linear piezoelectricity, and the polarity of the electromechanical response can be switched by external electric field, analog to classical ferroelectricity. A human being experiences hundreds millions of cycles of inhalation and exhalation in one’s life time, and the observed piezoelectric effect suggests that hundreds millions of cycles of piezoelectric charges are generated by lung tissues during the process. While the physiological significance of electromechanical coupling has yet to be determined, a number of interesting questions can be asked. For example, whether piezoelectric charges play any role in binding of oxygen to Hemoglobin, which is polar, or whether the polarity switching induced by mechanical stress can help damp out the possible sudden increase in air pressure. These open questions remain to be investigated, and we hope that the observation of piezoelectricity and its polarity switching in lung lay the foundation for the subsequent studies. 4.

Conclusions Using a series of PFM experiments, we show that the electromechanical coupling of

murine lung tissues are piezoelectric in nature, exhibiting predominantly first harmonic piezoresponses that are much larger than the second harmonic one. The mappings of piezoresponses show consistent polar orientation distribution and small fluctuation in elasticity, but larger variation in energy dissipation. There is an internal bias in the polar structure of lung tissues, though the polarity can still be switched, as indicated by the


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characteristic hysteresis and butterfly loops. Since hundreds millions of cycles of inhalation and exhalation occur in one’s life time, the piezoelectricity of lung tissues could be physiological significant. It is hypothesized that the piezoelectric charge generation during inhalation and exhalation could play a role in binding of oxygen to Hemoglobin, and the polarity switching can help damp out the possible sudden increase in air pressure. We hope such observation of piezoelectricity and its polarity switching in lung lay the foundation for the subsequent studies. Acknowledgments We acknowledge the support by National Key Research Program of China (2016YFA0201001), National Basic Research Program of China (973 Program, 2015CB755500), National Natural Science Foundation of China (Nos. 11627801 and 81571701)

and

Shenzhen

Science

and

Technology

Innovation

Committee

(JCYJ20160331191436180).

Supporting Information Schematic of sample preparation; butterfly loops of rat lung tissue. This material is available free of charge via the Internet.



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10.1016/S0369-8114(01)00147-X.



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Electromechanical coupling of murine lung tissues probed by Piezoresponse Force Microscopy

Peng Jianga,b,c,*, Fei Yanb,*, Ehsan Nasr Esfahanic, Shuhong Xiea, Daifeng Zoub, Xiaoyan Liud, Hairong Zhengb, Jiangyu Lib,c,#

For Table of Contents Use Only


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Figure 1. Schematics of PFM; (a) AFM system setup; (b) enhanced piezoresponse amplitude near cantilever-sample resonance; (c) DC wave form on top of AC excitation for switching PFM; and (d) characteristic hysteresis and butterfly loops associated with polarity switching.



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Figure 2. Microstructural morphology of murine lung tissue; (a) SEM image of mice lung tissue; (b) and (c) low- and high-resolution AFM topography of rat lung tissue acquired in 2×2 μm2 (b) and 1×1 μm2 (c) areas.


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Figure 3. Linear and quadratic VPFM responses of rat lung tissue under four AC voltages; (a) 11 V; (b) 16.5 V; (c) 22 V; and (d) 27.5 V.



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Figure 4. Linear and quadratic LPFM responses of rat lung tissue under four AC voltages; (a) 11 V; (b) 16.5 V; (c) 22 V; and (d) 27.5 V.


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Figure 5. Averaged first and second harmonic responses (n=10) with error bars for vertical (a) and lateral (b) PFM responses versus AC voltages.



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Figure 6. VPFM mappings of rat lung tissue overlaid on 3D topography in a 1×1 μm2 area; (a) amplitude; (b) phase; (c) frequency; (d) quality factor; and (e) histogram distributions.


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Figure 7. LPFM mappings of rat lung tissue overlaid on 3D topography in a 1×1 μm2 area; (a) amplitude; (b) phase; (c) frequency; (d) quality factor; and (e) histogram distributions.



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Figure 8. Switching characteristics of mice lung tissue, where butterfly loops (a and c) and hysteresis loops (b and d) of VPFM (a and b) and LPFM (c and d) are measured at same point.


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Figure S1. Schematic of sample preparation; (a) the location where sections were cut from lung; (b) the photo of lung section with dimensions labeled (top), the location where edges were cut from the section (middle), and the photo of the specimen (bottom); (c) the photo of specimen glued onto the silicon substrate for PFM measurement.



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Figure S2. Butterfly loops of vertical SS-PFM (a) and lateral SS-PFM (b) under different cycling period. Both figures were acquired from rat lung tissue.


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Electromechanical Coupling of Murine Lung ... - ACS Publications

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