Cell sample
We used mouse fibroblast L929 cells (ATCC, USA) cultured in Dulbecco’s modified eagle medium (DMEM; Invitrogen Life Technique, US) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, US) and 1% penicillin/streptomycin (Invitrogen Life Technique, USA) at 37 °C in a humidified atmosphere containing 5% CO2. The cell samples with cell densities of 1 × 104/mL on a 35 mm diameter cell culture petri dish (NUNC, Denmark), were washed with phosphate buffered saline (PBS, Sigma-Aldrich, US) three times and then treated with different PFA solutions (C
PFA = 10−5, 10−4, 10−1, 1, 4, 8 and 10%) for 5 min. Before AFM and SICM experiments, the treated cell samples were again washed three times with PBS.
SPM apparatus
A commercial SPM system (NX-Bio, Park Systems, South Korea) equipped with an inverted optical microscopy (Nikon Corp., Japan) which is designed specifically for biological applications was employed in this study. The SPM system not only achieved a soft material sample such as cell surface information using SICM, but it also obtained mechanical properties of a sample using AFM. All experiments using live cells were performed in a customized live cell chamber (Live Cell Instrument, South Korea). The live cell chamber was adjusted to 37 °C with 5% CO2 and 95% humidity, readily providing the specific environmental conditions needed to sustain cell cultures. Within this environment, live cell SICM imaging or AFM experiment was conducted for extended timeframes via optical monitoring methods, including optical phase contrast and digital image correlation microscopy.
SICM measurement for cell imaging and fluctuation analysis
The operation of SICM relies on an ion current that flows between an electrode inside a nano-pipette and an electrode located in an external bath solution. This ion current provides a feedback signal used to maintain the tip-sample distance and allow for the nano-pipette to scan topographical information (Fig. 1b). In spite of low lateral resolution (10–20 nm) [22], SICM offers useful topographical measurement without applying any mechanical force onto the sample surface.
SICM imaging and ion current-distance (I–D) curve experiments were performed using customized SPM system with 100 nm inner diameter nano-pipette fabricated from borosilicate capillaries (inner diameter 0.6 mm, outer diameter 1.0 mm, World Precision Instruments, USA) using a CO2-laser pipette puller (Sutter Instruments, USA). The cell topographic images were obtained with the so-called hopping mode [23], in which the nano-pipette approached sample surfaces with the pre-set threshold of 1.2%.
The apparent fluctuation amplitude of cell apical surfaces, a
m, was estimated from the I–D curve of SICM measurement [16, 19]. The measured ion current with cell apical surface fluctuation was assumed to be a convolution of the non-fluctuation-based ion-current relation, I
0, and the existing probability of cell surface position at z, P(z).
$$\left\langle I( {z - z_{0} ,\delta z^{2}_{s} }) \right\rangle = \mathop \smallint \limits_{ - \infty }^{\infty } I_{0} \left( {z - z_{0} } \right) P\left( {z_{s} - z_{0} , \delta z^{2}_{s} } \right) {\text{d}}z_{s}.$$
(1)
The z and z
s
are the position of the pipette and the sample, respectively. The \(z_{0}\) and \(\delta z^{ 2}_{s}\) are the time-average position of cell surface and the deviation of the sample fluctuation. The non-fluctuation ion-current relation is approximately expressed as the following form [19]:
$$I_{0} \left( {z - z_{0} } \right) = I_{sat} \left( {1 + \frac{\zeta }{{z - z_{0} }}} \right)^{ - 1} ,$$
(2)
where I
sat
is the reference current when the pipette is far enough from the sample surface, and ζ is a constant from the pipette geometry. It is here assumed that the cell fluctuation obeys the Gaussian distribution,
$$P\left( {z_{s} - z_{0} , \delta z^{2}_{s} } \right) = \frac{1}{{\delta z_{s} \sqrt {2\pi } }}\exp \left( { - \frac{1}{2}\left( {\frac{{z_{s} - z_{0} }}{{\delta z_{s} }}} \right)^{2} } \right).$$
(3)
The I–D curves measured at around the cell center were fitted to the Eq. (1) to estimate the apparent fluctuation deviation \(\delta z^{ 2}_{s}\)
\(I_{sat}\) and \(\zeta\) were determined experimentally to be \(I_{sat}\) = 1 nA and \(\zeta\) = 4.9 × 10−2 nm, respectively, which were estimated from the SICM measurement on a glass substrate [16]. According to Eq. (3), the fluctuation amplitude of apical cell membrane, a
m is defined as the Gaussian distribution, P, with the root mean square (RMS) displacement of cell surface fluctuation, \(\langle \delta z^{2}_{s} \rangle^{{\frac{1}{2}}}\).
AFM measurements for Young’s modulus of cells
The force curve measurements of AFM were performed to estimate Young’s modulus of cells. We used a commercial AFM cantilever (Biolever mini, Olympus, Japan) with less than 0.09 N/m of a nominal spring constant. Because a cantilever with a small spring constant makes a relatively large deflection for a small force, the cantilever used in this study provides reliable data of the cell surface structure. The spring constant of the AFM cantilever was calibrated using the thermal vibration method [24]. AFM cantilever was cleaned using ethanol and exposed to UV light for 30 min to remove contamination on the AFM cantilever and tip. We measured more than 50 force curves with 512 data points. The force curves were analyzed with a Hertz model using a commercial SPM data analysis program (Park Systems, South Korea). We assumed the AFM tip shape is four-sided pyramid with a half cone angle α, so that the force on cantilever F is expressed as.
$$F = \frac{E}{{1 - v^{2} }}\frac{\tan \alpha }{\sqrt 2 }\delta^{2} ,$$
(4)
where E is the Young’s moduls, ν is the Poisson’s ratio and δ is the indentation (depth). ν and alpha were set to be 0.5° and 35°, respectively. The scan rate of the AFM cantilever and the maximum loading force were set to be 1–2 µm/s and 3–8 nN, respectively.
Cell viability assay
To evaluate the viability of cells with PFA treatment, we used a LIVE/DEAD® Viability/Cytotoxicity Kit (L3224; Invitrogen life technique, USA). Briefly, the PFA-treated cells were immediately incubated using the live and dead stain fluorescence dye for 10 min. Then the final 2 µM calcein AM and 4uM EtD-1 mixture solution were added to the PFA-treated cell sample. A commercial fluorescence microscope (Nikon Corp., Japan) was used to obtain fluorescence images of cells where green and red colors represented live and dead cells, respectively.
