1.

Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerative disease in which one of the defining characteristics is global cognitive decline including memory,¹ orientation, judgment, and reasoning. Amyloid-beta 42 ($\mathrm{A}\beta 42$) is a peptide composed of 42 amino acids that is believed to be related to the development of AD.² $\mathrm{A}\beta$ peptides can bind to various biomolecules, such as proteins, lipids, and proteoglycans, and therefore cause toxic effects to neurons by perturbing the properties and functions of plasma membranes.^3–5 The $\mathrm{A}\beta$ peptides can assemble into various degrees of polymerization, and the conformations of oligomers, proto-fibrils, and fibrils are found to exist in brain lesions of AD patients. Both $\mathrm{A}\beta$ fibrils and oligomers are demonstrated to be neurotoxic. $\mathrm{A}\beta$ oligomers are also shown to correlate with cognitive impairment and induce synaptic dysfunction,⁶ apoptosis, and mouse abnormal behaviors.⁷ It has been verified that the membrane stiffness of Neuro-2a (N2a) neuroblastoma cells is increased by $\mathrm{A}\beta$ oligomers.⁸ In addition, $\mathrm{A}\beta$ pore-like oligomers and $\mathrm{A}\beta$ channels have been observed on lipid bilayers.⁹ Therefore, we may conjecture that the $\mathrm{A}\beta$ peptides have the ability to vary other mechanical characteristics of cell membranes.

Recently, a number of groups have demonstrated that the stimulation of electrical fields (EFs) on neuronal cells can affect specific cell behaviors, including migration directions,¹⁰ cell orientation,¹¹ growth of neurites,^12,13 and neuron–neuron contact.¹⁴ Owing to the nonintrusive nature of the EF stimulation on live cells, EFs are believed to be developed into potential therapeutic applications for a variety of neurological diseases. Because both the EF and $\mathrm{A}\beta$ peptides show evident variations on neuronal cells, it is intriguing to know how cells respond to these two external stimulations simultaneously.

In the present work, we used the noninterferometric wide-field optical profilometry (NIWOP) technique to measure the membrane roughness of N2a cells under both the treatments of $\mathrm{A}\beta 42$ peptides and EFs. The NIWOP technique has been used to measure the dynamical membrane ripples on live cells.^15–17 Compared with atomic force microscopy, the NIWOP technique is faster and noncontact, and therefore the membrane topography as well as the cell viability is not perturbed during the measurement. In addition, because the NIWOP setup is constructed on a conventional optical microscope, it is compatible with current cell culture devices and easy to conduct various treatments during the observation.

2.

Materials and Methods

2.1.

Cell Culture Device Capable of Conducting EF Stimulations

We remodeled an ordinary 100-mm culture dish for the EF stimulation according to the design described by McCaig et al.¹⁸ Figure 1 shows the layout and a photograph of this culture device. The volume of the cell chamber is $44.5\times 2.5\times 0.063{\mathrm{mm}}^3$. We did not coat any cell-adhesive proteins or molecules on the bottom of the culture dish. The acrylic sheets and double-sided tapes (PET8018, 3M Corp., St. Paul, MN, USA) were incised by a ${\mathrm{CO}}_2$ laser cutting machine. The EF was built through two agar bridges connecting the culture device and two bottles of phosphate-buffered saline (PBS). The anode was a silver (Ag) electrode, and the cathode was a AgCl electrode. When the direct-current (dc) power supply was turned on, the ${\mathrm{Cl}}^{-}$ ions in the cell culture medium were forced to move toward the anode and thus an ion current was formed. Because the resistance in the culture chamber was finite, the ion current produced a potential difference along the direction of the ion movement. Because the ion current was increased as the applied voltage, we could have an EF strength inside the cell culture region proportional to the voltage. The EFs were calculated as the potential difference divided by the distance between the two measuring probes inserted into the cell culture region. We tested eight devices with an applied voltage up to 25 V. A direct-current electric field (dcEF) strength as high as $500\mathrm{mV}/\mathrm{mm}$ could be built in these culture devices and the EF strength was linear with the applied voltage (data not shown). In the following experiments, we fixed the strength of EF at $300\mathrm{mV}/\mathrm{mm}$ because this EF strength led to obvious variations in membrane roughness and showed no degradation on cell viability.

Fig. 1

(a) A schematic diagram and a photograph of the culture device for the electrical field (EF) stimulation and noninterferometric wide-field optical profilometry (NIWOP) observation. (b) Setup for the EF treatment and optical observation on live cells.

2.2.

Setup of NIWOP

Figure 2 shows the setup of the NIWOP system. The NIWOP technique is based on differential confocal microscopy and wide-field optical sectioning microscopy.¹⁹ In short, we used the structured-illumination wide-field sectioning microscopy²⁰ to obtain an intensity axial response curve similar to that of confocal microscopy. Then we placed the cell dorsal surface into the linear region of this axial response curve; in this region the intensity is linearly proportional to the height of the sample. Therefore, we can obtain the heights of cell membranes after a calibration process.¹⁵ The height measurement dynamic range is determined by the width of the intensity axial response curve, which is set by the numerical aperture of the objective and the period of the projected illumination pattern.¹⁹ The accuracy of height detection is limited by signal fluctuation divided by the slope of the linear region of this axial response curve. In the structured-illumination wide-field sectioning microscopy, three images ($I_1$, $I_2$, $I_3$) with the illumination pattern shifted by one third of the pattern period are captured and processed with the square-law detection principle:

$$I_s=\sqrt{{(I_1-I_2)}^2+{(I_2-I_3)}^2+{(I_1-I_3)}^2}\mathrm{.}$$

Therefore the imaging speed is lower than that of conventional wide-field imaging. In the present work, the image acquisition rate of the NIWOP system was $12\text{images}/\min$ (equivalent to $36\text{frames}/\min$). With a $40\times$ NA 0.80 objective lens the depth accuracy is $\sim 52\mathrm{nm}$, and the measurement dynamic range is $\sim 3000\mathrm{nm}$. Other details of NIWOP measurement on cell membrane roughness can be found in our previous publications.^15–17 The NIWOP system was constructed on an upright microscope, and we used a spatial light modulator to generate the pattern for structured illumination. The microscope and the piezo-electric transducer (PZT)-driven vertical stage were placed in a temperature-controlled incubator, which kept the temperature at $37\pm 1°\mathrm{C}$ during the experiment.

Fig. 2

A schematic diagram of the setup of NIWOP. SLM, spatial light modulator; PZT, piezo-electric transducer.

2.3.

Cell Culture and $\mathrm{A}\beta 42$ preparation

The cells we used in the present work were from a mouse neuroblastoma cell line N2a. The culture medium was minimum essential medium alpha (12571, Gibco, Life Technologies, NY, USA) with 10% fetal bovine serum (FBS) and 1% antibiotic pen-strep-ampho. Before the experiments, we used 10 μM retinoic acid (R2625, Sigma-Aldrich, St. Louis, MO, USA) and reduced the concentration of FBS to 1% to differentiate the cells for neurite growth.

To prepare $\mathrm{A}\beta 42$ oligomers and fibrils, we used the following procedures that were modified from the protocols proposed by Dahlgren et al.²¹ The $\mathrm{A}\beta$ peptide was first dissolved by hexafluoroisopropanol (HFIP) in $2.5\mathrm{mg}/\mathrm{mL}$ to dissociate $\mathrm{A}\beta$ preaggregates. HFIP was evaporated in vacuum for $>3\mathrm{h}$ and $\mathrm{A}\beta$ films were dissolved in anhydrous dimethyl sulfoxide (DMSO). For $\mathrm{A}\beta$ oligomers preparation, $\mathrm{A}\beta$ stock in DMSO at 5 mM was refolded in Dulbecco’s modified Eagle medium/nutrient mixture medium (21041, Gibco, Life Technologies, NY, USA) with the concentration of 100 μM and incubated at 4°C for 24 h. The sample was centrifuged and the soluble portion was collected. For $\mathrm{A}\beta$ fibrils preparation, $\mathrm{A}\beta$ in DMSO at $10\mathrm{mg}/\mathrm{mL}$ was refolded in 10 mM sodium phosphate buffer, pH 7.4. The final $\mathrm{A}\beta$ concentration was adjusted to 50 μM and incubated at room temperature with agitation for several days. The fibril species was monitored in the presence of 5 μM of thioflavin T (ThT) to indicate mature fibril formation. In our experiments, the differentiated N2a cells were treated with 5 μM of $\mathrm{A}\beta 42$ fibrils or oligomers.

We conducted the observation on $\mathrm{A}\beta 42$ with fluorescence microscopy and transmission electron microscopy (TEM). Fluorescein FAM-labeled $\mathrm{A}\beta 42$ peptides were purchased from Biopeptide (San Diego, CA). For TEM imaging, the $\mathrm{A}\beta 42$ samples were deposited on 400-mesh Formvar carbon-coated copper grids (EMS Electron Microscopy Sciences, Hatfield, PA, USA) for 5 min, negatively stained by 2% acetate, and rinsed by ${\mathrm{ddH}}_2\mathrm{O}$. The samples were examined with a Hitachi H-7000 transmission electron microscope (Chiyoda, Tokyo, Japan) with an accelerating voltage of 75 kV. Figure 3(a) and 3(b) shows the TEM micrographs of $\mathrm{A}\beta 42$ fibrils and oligomers, respectively. The fibrils usually formed planar aggregates, while the oligomers dispersed evenly in the field of view and therefore could hardly be identified. Figure 3(c) displays optical images of the N2a cells treated with 5 μM $\mathrm{A}\beta 42$ for 2.5 h. Before the optical observation, we removed the culture medium containing the $\mathrm{A}\beta 42$ peptides, washed the cells by PBS, and then refilled the device with the fresh medium. The fluorescence images confirmed that both the fibrils and oligomers could adhere onto the cells. Because of the larger areas of the fibril aggregations, the visibility of the fibrils was higher than that of the oligomers. Nevertheless, the actual sizes of the fibrils and oligomers were much smaller than the diffraction limit, and hence the fluorescence images could not be used as a quantitative assay of the amounts of $\mathrm{A}\beta 42$ peptides on the cell membranes.

Fig. 3

(a) Transmission electron microscopy (TEM) micrograph of Amyloid-beta 42 ($\mathrm{A}\beta 42$) fibrils. (b) TEM micrograph of $\mathrm{A}\beta 42$ oligomers. (c) Optical images of N2a cells treated with the $\mathrm{A}\beta 42$ peptides for 2.5 h. The fluorescence signal is from the FAM fluorescein labeled on the $\mathrm{A}\beta 42$ peptides. DIC, differential interference contrast.

3.

Results and Discussion

Figure 4 shows the membrane topography on the N2a cells obtained by NIWOP. Because the height variations on the whole cell are often larger than the NIWOP measurement dynamic range, we arbitrarily selected a $10\mu \mathrm{m}\times 10\mu \mathrm{m}$ region at the edge of each cell, and calculated the standard deviations of the membrane height in this region as the estimation of membrane roughness. For each condition of experiment, we captured one NIWOP image per minute at the same field of view for 3 h. With both the treatments of EFs and 5 μM $\mathrm{A}\beta 42$ fibrils or oligomers, the cells were alive during all the experiments.

Fig. 4

The topography of Neuro-2a (N2a) cells measured by NIWOP. The left column contains the results measured after 0.5 h of treatments; while the right column shows the results after 2.5 h. In each column, the regions in the dashed boxes ($30\mu \mathrm{m}\times 30\mu \mathrm{m}$) in the left panels are magnified in the right panels: (a) 5 μM $\mathrm{A}\beta 42$ fibrils treatment, (b) 5 μM $\mathrm{A}\beta 42$ oligomers treatment, (c) 5 μM $\mathrm{A}\beta 42$ fibrils and $300\mathrm{mV}/\mathrm{mm}$ direct-current electric field (dcEF) treatment, (d) 5 μM $\mathrm{A}\beta 42$ oligomers and $300\mathrm{mV}/\mathrm{mm}$ dcEF treatment.

Figure 5 shows the statistics of the membrane roughness of the N2a cells under various treatments. In order to ensure that the $\mathrm{A}\beta 42$ peptides had sufficient time to perform their reactions on cell membranes, we compared the membrane roughness at 0.5 and 2.5 h after the treatment. In Fig. 5, we see that both the $\mathrm{A}\beta 42$ fibrils and oligomers reduced the membrane roughness, which could result from the increase of membrane stiffness, as reported by Lulevich et al.⁸ At 0.5 h, the effect of oligomers was more significant than that of the fibrils. After 2.5 h of treatment, the fibrils reduced the membrane roughness by 18%, while the oligomers reduced the membrane roughness by 32%. The effect of $\mathrm{A}\beta 42$ oligomers on the roughness of N2a cell membrane was faster and stronger than that of $\mathrm{A}\beta 42$ fibrils. This result correlates well with the cytotoxicity of $\mathrm{A}\beta 42$ oligomer and $\mathrm{A}\beta 42$ fibrils.^8,21 These data suggest that the membrane roughness could be used as a cellular diagnostic parameter related to the effects of $\mathrm{A}\beta$ peptides.

Fig. 5

Membrane roughness of N2a cells with the treatments of $\mathrm{A}\beta 42$ fibrils, oligomers, and a $300\mathrm{mV}/\mathrm{mm}$ dcEF for 0.5 and 2.5 h. For each condition, $>10$ cells were measured in one experiment. Each experiment was repeated three times. Data show the $\text{mean}\pm \text{standard error}$ of the mean of the three experiments.

We also compare the effects of dcEF and the treatment with $\mathrm{A}\beta 42$. The $300\mathrm{mV}/\mathrm{mm}$ dcEF alone did not change the membrane roughness after 0.5 or 2.5 h of treatment. However, for the cells treated with $\mathrm{A}\beta 42$ peptides, after 2.5 h of treatment we found that the dcEF could make the roughness higher than the cells treated for 0.5 h. The effect of the dcEF was more significant on the oligomer-treated cells. The membrane roughness could be raised to the level of the control group after 2.5 h of the EF stimulation. In comparison, for the cells treated with $\mathrm{A}\beta 42$ fibrils, the membrane roughness was still lower than that of the control group at the 2.5th hour. This result indicates that the dcEF may counteract the change on membrane stiffness induced by $\mathrm{A}\beta 42$. Because the effect of $\mathrm{A}\beta 42$ oligomers was compensated more than that of the fibrils, we conjecture that the dcEF produces larger influences on the functions of the $\mathrm{A}\beta 42$ oligomers than on those of the fibrils. It has been known that $\mathrm{A}\beta$ oligomers can bind to cell membranes more effectively than the fibrils.⁵ Liao et al. had demonstrated that negatively charged gold nanoparticles can inhibit $\mathrm{A}\beta$ fibril formation and reduced the cellular toxicity of $\mathrm{A}\beta$ oligomers.²² Their results verified that $\mathrm{A}\beta$ aggregation could be affected by charged particles or molecules. We thus postulate that the dcEF effect on membrane roughness against the $\mathrm{A}\beta$ oligomers might also be related to the alteration of the $\mathrm{A}\beta$ aggregates and/or reduction of the cellular effects of $\mathrm{A}\beta$ peptides.

In the cell culture device shown in Fig. 1, the potential change in the cell culture region was built by the flow of ${\mathrm{Cl}}^{-}$ ions. It is thus intriguing to raise the discussion about the roles of ${\mathrm{Cl}}^{-}$ ion channels (also called ClC channels) in such externally applied electrical stimulations. At present, the knowledge about the influence of ClC channels on ${\mathrm{Cl}}^{-}$ ion flux in the dcEF is still unclear. Vieira et al. have suggested that at corneal wounds an inward ${\mathrm{Cl}}^{-}$ ion flux produced by the ClC-2 channel may enhance wound healing,²³ probably by way of the electrotaxis effect of the nearby epithelial cells. Nevertheless, the role of the ClC-2 channel in an externally applied dcEF has not been reported yet. Moreover, to the best of our knowledge, the relation between membrane roughness and ClC channels has not been discussed in literature. In the present work, the dcEF could have triggered some cellular responses or have changed the conformation of some proteins in the ClC channel. However, these responses were not manifested in significant changes in membrane roughness, as shown in Fig. 5, where the treatment of a $300\mathrm{mV}/\mathrm{mm}$ dcEF alone did not cause detectable changes in membrane roughness with the present NIWOP system (of which the height sensitivity is $\sim 52\mathrm{nm}$). In our setup, the externally applied dcEF induced a steady ${\mathrm{Cl}}^{-}$ flow that passed alongside the cell membrane without generating ${\mathrm{Cl}}^{-}$ concentration gradient across the membrane. With the present result, we could only postulate that the dcEF did impose synergic effects on membrane roughness together with $\mathrm{A}\beta 42$. It is also known that $\mathrm{A}\beta$ peptides produce cation (such as ${\mathrm{Ca}}^{2+}$) channels on lipid bilayer membranes.⁵ Therefore, the effect of external dcEFs on $\mathrm{A}\beta 42$ could also be related to cations. Obviously, more studies are necessary to correlate the transmembrane ion flux, $\mathrm{A}\beta 42$, and membrane roughness under the stimulation of dcEFs.

The measurement of the membrane fluctuations on live cells provides interesting and useful information about the cellular status. In addition to the NIWOP technique, there are more and more noncontact optical methods useful for quantifying cellular thickness or membrane topography. For example, Ding et al.^24,25 proposed a Fourier transform light scattering (FTLS) method that can reveal the dynamics of intracellular cytoskeletons in a label-free manner. The FTLS method provides reliable phase and amplitude measurements by using a common-path Mach–Zehnder interferometer setup in addition to a standard optical microscope.^24,25 Recently, Atilgan and Ovryn²⁶ implemented a scanning phase-shifted laser-feedback interference microscopy (psLFIM) to observe the dynamics of the retracting lamella of live cells. The psLFIM detects small variations of the refractive index of the sample in a spatially resolved way. With a well-designed algorithm, the local topography and reflectivity of the sample can be resolved from a set of interference measurements with five shifted phases.²⁶ Both the FTLS and the psLFIM techniques could be used as NIWOP in the present work to quantify membrane roughness. On the other hand, because the NIWOP technique relies on neither temporal nor spatial coherence to obtain the membrane topography, it provides better simplicity of setup and compatibility with existing microscopes.

4.

Conclusion

In this work, we demonstrated that high-sensitivity optical profilometry was useful for the diagnosis of cellular effects of $\mathrm{A}\beta 42$ peptides and dcEFs. The results indicated that both $\mathrm{A}\beta 42$ fibrils and oligomers reduced the membrane roughness of live N2a cells. The effect of the oligomers was stronger and faster than that of the fibrils. Under $\mathrm{A}\beta 42$ treatment, the dcEF increased the membrane roughness in comparison with the cells treated with $\mathrm{A}\beta 42$ only. The efficacy of the dcEF was more obvious on the cells treated with the $\mathrm{A}\beta 42$ oligomer.

Interactions with $\mathrm{A}\beta$ peptides and plasma membranes result in alternations of biophysical as well as biochemical parameters of cell membranes. Because the NIWOP observation is label-free, the measurement on membrane roughness could be used as a convenient tool to diagnose how the chemical and physical stimulations simultaneously affect the membrane properties on live cells. This technique may be helpful for the research about evaluating the effects of various types of treatments to the neuronal cells undergoing neurodegenerative diseases.