2.1.

Nanoscale Optical Imaging of Transparent Nanospheres Under Live Cell Conditions

The imaging system is adapted from an inverted COI system with a live cell incubation system operated at 37°C and 5% ${\mathrm{CO}}_2$ [Fig. 1(a)]. Neuroblastoma cells (SHSY5Y) were grown in a culture dish with an in vitro electric field stimulator assembly (Fig. 2). The stimulator is connected to positive and negative electrodes that receive programmed signals from a pulse generator. For NOI [Fig. 1(b)], the conventional optical condenser and halogen light source of the COI system [Fig. 1(c)] were replaced by a high-resolution dark-field condenser and an illuminator (white LED, halogen, or metal halide). In NOI, the condenser was put into the culture dish with cell media and positioned at a submillimeter distance ($d_{\mathrm{NOI}}$: 10 to 1000 μm) above the cells, while the condenser of the COI was positioned several centimeters ($d_{\mathrm{COI}}$: 2.5 to 4 cm) from the cells. The resolution of the NOI system was demonstrated using transparent polymers that are similar to transparent cells, 100 nm standard polystyrene nanospheres ($\text{mean diameter}=97\pm 3\mathrm{nm}$). The nanospheres were clearly seen with a large FOV using NOI [Fig. 1(d)]. Due to the Brownian motion of nanospheres in water and high-scattering effect in NOI dark-field imaging, the apparent size of the nanospheres was $469\pm 84\mathrm{nm}$ (sample number, $n=112$). In contrast, with bright-field COI (the most widely used imaging technique in cell biology), the nanospheres were not visible in the phase-contrast images [Fig. 1(e)]. For direct comparison of the two systems under dark-field conditions, we viewed nanospheres using a high numerical aperture ($\mathrm{NA}=1.2$ to 1.4) in both NOI and COI (Fig. 3).

Fig. 1

Comparison of nanoscale optical imaging (NOI) and conventional optical imaging (COI) systems for real-time imaging of living cells. Illustrations of (a) a live cell imaging microscope equipped with a live-chamber controller; (b) the NOI system with a nano-optical dark-field condenser, specific illuminators, and a live-chamber system; and (c) the COI system for phase-contrast imaging with a conventional optical condenser and a live-chamber system. Images of standard polystyrene polymer nanospheres ($97\pm 3\mathrm{nm}$) observed by (d) NOI with an image size of $468.75\pm 83.85\mathrm{nm}$ and (e) COI. The distance between the high-resolution condenser and the object, $d_{\mathrm{NOI}}$, was approximately 10 to 1000 μm, and the distance between the condenser and the object, $d_{\mathrm{COI}}$, was approximately 2.5 to 4 cm.

Fig. 2

Real-time dynamics of cell coupling mediated by F-actin under electrical field stimulation in a NOI system. (a) Side-view of the graphene electric field stimulator (GEFS) showing plated neural cells. (b) An illustration of the cell-coupling mechanism between neurons. To induce cell coupling, a weak electrical field ($4.5\mathrm{mV}/\mathrm{mm}$) was applied, and the field-of-view (FOV) was observed in real time. (c) Time-lapse images of field-induced cell interactions at 1-min intervals. The arrows demonstrate the early appearance of F-actin fibers at the onset of cellular contact. Scale bar (c)$=10\mu \mathrm{m}$.

Fig. 3

Comparative images of 100 nm standard polystyrene nanospheres under COI and NOI with a high numerical aperture (NA) (1.2) dark-field condenser. Fixed: nanospheres fixed on the cover glass are visualized in dark-field view using COI (a) or NOI (b). Moving: nanospheres in the water are shown in a captured image cut from the video of either COI (c) or NOI (d).

Fixed samples of nanospheres could be viewed using either technique, but visualization of mobile nanospheres in water was significantly better with NOI [Fig. 3(d)] than with COI [Fig. 3(c)].

2.2.

Label-Free NOI of F-Actin in Living Cells

We compared NOI and COI images of identical areas of living cells [Fig. 4; low-magnification large FOVs are shown in Fig. 4(a)–4(d) and 4(i)–4(l)]. The dark-field NOI image clearly shows neural cell nanostructures that are hard to see in the COI image, including nanofiber bundles in the filopodia, intracellular microvesicles, and the nuclear membrane. NOI can better resolve detailed nanofiber structure than COI. For example, the number of visible nanofibers was greater in NOI images [Fig. 4(e)] than in COI images [Fig. 4(f)], and the fibers were longer in the NOI images. More importantly, we can use NOI to view the finer nanofibers at junctions between interacting cells [Fig. 4(m) and 4(n)]. The differing capabilities of NOI and COI to reveal the structure at the cell’s contact face are shown in Fig. 4(o) and 4(p). Overall, the NOI system shows great potential as a high-resolution large FOV tool to study the fine structure and interacting behavior of the living cells.

Fig. 4

Microstructures of filopodia (a)–(h) and cell junctions (i)-(p) in living cells. Filopodia, upper row: NOI (a) and COI (b) images of identical areas of filopodia at the cell edge. The dashed boxed outline indicates a region targeted for further magnification. Second row: (c) and (d) are the magnified regions of (a-1, the blue dashed box) (b-1, the red dashed box), respectively. Third row: similarly, (e) and (f) are the magnified images of (c-2) and (d-2), respectively. NOI (e) shows the microfibers more clearly than does COI (f). Cell junctions, upper row: (i) and (j) show the cell connections imaged by NOI (i) and COI (j), respectively. As before, a dashed boxed outline indicates a region targeted for further magnification. Second row: (k) is a magnified image of (i-1, the blue dashed box), and (l) is a magnified image of (j-1, the red dashed box), respectively. Third row: (m) and (n) are magnified images of (k-2) and (l-2), respectively. The microfiber connections at the junction are visible in more detail in (m) than in (n). The drawing of the cell boundary (g)–(h) and microfibers (o)–(p) show the distinct formations in morphology. $\text{Scalebars}=50\mu \mathrm{m}$: (a), (b), (i), and (j); 25 μm: (c), (d), (k), and (l); and 10 μm: (e), (f), (m), and (n).

F-actin and tubulin are the most abundant cytoskeletal proteins in neuronal cells. To identify the observed filopodial nanofibers in cells examined with the NOI system, we stained the neuronal cells with anti-F-actin, anti-tubulin antibodies (red), and 4′,6-diamidino-2-phenylindole (DAPI) (blue), and then imaged by fluorescent microscopy (FM) (Fig. 5). The nanofibers located by NOI at the cell edges correspond with the location of F-actin staining [Fig. 5(a) and 5(b)], but not with location of tubulin staining [Fig. 5(c) and 5(d)]. F-actin is a long, helical filament made of globular-actin (G-actin) subunits. These monomers are approximately 7 nm in diameter with the twist of the helix repeating every 37 nm.³³ F-actin must be observed usually after staining via FM, since the fine structure is too small to be seen by COI. In addition, these nanostructures are more difficult to image in living cells because the filament structure is dynamic.

Fig. 5

Identification of nanofibers in fixed-cell images from NOI (a, c) through the comparison with FM imaging (b, d): direct comparisons are shown between (a) and (b) and (c) and (d). FM images show staining with either anti-F-actin antibody (b) or with anti-tubulin antibody (d). $\text{All scale bars}=10\mu \mathrm{m}$.

Filopodia forms at the interacting junction of two cells (Fig. 6). Images of identical fixed and stained neural cells were used to directly compare NOI [Fig. 6(a), 6(c), and 6(e)] and FM [Fig. 6(b), 6(d), and 6(f)]. At the cell-surface edge, the protruding fine nanofibers seen using the NOI system correspond to the F-actin stained filopodial structures in the FM images [Fig. 6(c) and 6(d) to 6(e) and 6(f)]. Unstained F-actin was easily visualized by NOI because of enhanced light-scattering effects, a main signal for dark-field imaging. The nuclear membrane was also well distinguished in NOI. In contrast, using ordinary optical imaging techniques, DAPI-stained nuclei cannot be used to view nuclear membranes because DAPI stains only the DNA. However, using NOI, unstained nuclear membranes were clearly observed [Fig. 6(g) and 6(h)].

Fig. 6

Direct visualization and comparison of F-actin in the same fixed neural cells between NOI (a) and fluorescent microscopy (FM) (b). In FM, F-actin is stained red, and DNA is stained blue. The dashed boxed outlines 1, 2, or 3 indicate regions targeted for further magnification. Further comparisons of NOI and FM images are shown in (c) and (d), (e) and (f), and (g) and (h), which are magnified images of (a-1) and (b-1), (a-2) and (b-2), and (a-3) and (b-3), respectively. The yellow outline of the nucleus in the NOI image (g) is overlaid on the 4′,6-diamidino-2-phenylindole (DAPI) image in the FM image (h). $\text{Scale bars}=10\mu \mathrm{m}$: (a) and (b); 5 μm : (c), (d), (e), (f), (g), and (h).

2.3.

NOI for the Study of Stimulus-Triggered F-Actin Dynamics

To further demonstrate the benefits of NOI for high-resolution large FOV and real-time imaging, we studied fast F-actin dynamics. In the filopodia, F-actins polymerizes to form cytoplasmic projections that are involved in cell migration, division, and differentiation, and that establish cell junctions and cell shape.³⁴ Previously, using a COI system for live cell imaging, a weak nEFS field ($4.5\mathrm{mV}/\mathrm{m}$) enhanced cell coupling through more frequent cell membrane contact (lamellipodial contact).³² Cell coupling was mediated by a vigorous generation of actin filaments, and RT-PCR showed an increase in the mRNA level of F-actin.³²

In this study, we used NOI to observe the interactive dynamics of nEFS-enhanced cell coupling (Fig. 2). Figure 2 depicts the experimental conditions for accelerating F-actin dynamics with electrical field stimulation. First, our in vitro graphene electric field stimulator (GEFS) is illustrated in side view [Fig. 2(a)]. Second, the $\text{transparent}\pm \text{graphene}$ electrodes can be visualized with a phase-contrast optic image with the cells. Third, the neural cell-coupling behavior is promoted by nEFS. Finally, field-induced F-actin formation progresses via several steps, as shown in schematic [Fig. 2(b)] and in NOI real-time images [Fig. 2(c)].

The cells show a wavering behavior in the observation period before stimulation (1 to 2 min prior). For the 2 min following nEFS, early contact is formed by a few actin filaments that repeatedly make and break contact. With nEFS inducing the cells to interact more quickly and strongly, filopodial contact was enhanced first (from 3 to 4 min) followed by the lamellipodia (from 5 to 7 min). In Fig. 2, the arrows show the appearance of the F-actin nanofibers that make cellular contact. F-actin readily initiates cell coupling with the assistance of a weak electric field created by nEFS. The NOI system offers an enhanced ability to view the fast changing F-actin structure (at the nanoscale level) in unstained living cells in real time.

4.1.

Nanoscale Optical Imaging

Our NOI system was created by modifying a conventional Leica microscope (Leica Microsystems GmbH, Wetzlar, Germany) with a high-resolution dark-field condenser (CytoVivia, Inc., Auburn, Alabama) and an illuminator system (white LED and halogen light; Dolan-Jenner Industries Inc., Boxborough, Massachusetts/metal halide light; Welch Allyn Inc., Skaneateles, New York). The microscope’s conventional optical condenser ($\mathrm{NA}=0.55$) was replaced by a high-resolution dark-field condenser ($\mathrm{NA}=1.2$ to 1.4). This system provides 100 nm resolution improving the contrast and signal-to-noise ratio.²⁸ The system’s resolution was confirmed by imaging polystyrene spheres with 100 nm standard monodisperse nanospheres [Fig. 1(d); $\text{mean diameter}=97\pm 3\mathrm{nm}$; Thermo Scientific, Inc., San Jose, California]. As shown in Fig. 1, the high-resolution dark-field condenser can be immersed in cell media. As a result, it could be positioned between 10 and 1000 μm directly above the neural cells plated in the culture dish. After each completed NOI experiment, the condenser was rinsed three times with distilled water and 70% ethanol, and then sterilized by UV for 30 min. A live-chamber system (TC-L, Chamlide, Seoul, South Korea) with a silicone top cover was used to maintain the environment at 37°C and 5% ${\mathrm{CO}}_2$ during the experiment. Images of the living cells were recorded using a CCD camera (Leica Microsystems GmbH, Wetzlar, Germany).

4.2.

Cell Culture

SHSY5Y human neuroblastoma cells (ATCC, Manassas, Virginia), an adhesive neural cell line, were grown in Dulbecco’s modified Eagle’s medium (Invitrogen, Grand Island, New York) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, Missouri) and 1% penicillin-streptomycin (Invitrogen) in a T75 flask (Nunc, Thermo Fisher Scientific, Roskilde, Denmark). Cells were subcultured every three days by resuspension in $1\times$ cell-dissociation solution (Sigma-Aldrich) with subsequent washing using $1\times$ phosphate-buffered saline (PBS; Invitrogen). To facilitate cell attachment and growth, the neural cells were plated either on a culture dish (60 mm diameter, Techno Plastic Products AG, Trasadingen, Switzerland) or on a laminin-coated (Roche Diagnostics GmbH, Mannheim, Germany) electric field stimulator. The plated cells were maintained in a 5% ${\mathrm{CO}}_2$ incubator (Sanyo Electric Co., Ltd., Osaka, Japan) for 24 h prior to the experiment. The cells were then examined using the COI or NOI system.

4.3.

Immunocytochemistry

After imaging, the neural cells were fixed for 20 min with a 4% paraformaldehyde solution (Biosesang, Seongnam, South Korea) and rinsed three times with PBS at room temperature. The fixed cells were treated for 1 h with a blocking solution containing 4% normal goat serum (Vector Laboratories, Burlingame, California), 0.2% Triton X-100 (Sigma-Aldrich), 2% bovine serum albumin (Sigma-Aldrich), and 2% FBS (Sigma-Aldrich). After three 5-min PBS washes, rhodamine-conjugated phalloidin (Cytoskeleton Inc., Denver, Colorado) was applied to the cells for 1 h to stain the F-actin. For visualizing the microtubules in the cells, anti-tubulin beta3 (Millipore, Billerica Massachusetts) was applied to cells for 1 h, and then the cells were incubated in Alexa Fluor 594 (Invitrogen). After completion of the procedure for F-actin or tubulin staining, the cells were washed twice for 5 min with PBS, and then treated with DAPI (Molecular Probes, Eugene, Oregon) in PBS for 20 min to stain the nuclei. After the last 5-min PBS wash, the cells were mounted in fluorescent mounting medium (Dako Cytomation, Carpinteria, California), and then covered with a cover glass (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany).

4.4.

Graphene Electric Field Stimulator

A transparent electric field stimulator was used to stimulate the neural cells. Procedures for manufacturing a GEFS system have been described elsewhere.³² In this study, we modified the system by using a larger culture dish (60 mm in diameter) in order to approach the high-resolution dark-field condenser with a submicrometer closure [Fig. 2(a)]. The neural cells on a cover slide were indirectly exposed to an electric field generated by two transparent graphene electrodes (gap distance approximately 280 μm) assembled under the cover slide.

4.5.

Noncontact Electrical Field Stimulation Paradigm

The GEFS electrodes were connected to a pulse generator. An electrical pulse was generated by a 9-channel programmable pulse stimulator (Master-9; A.M.P.I., Jerusalem, Israel) and two stimulus isolators (ISO-Flex; A.M.P.I.). Charge-balanced biphasic pulse trains with a frequency of 1 Hz and pulse width of 250 ms were applied repeatedly. The biphasic pulse trains were delivered 36 times every 100 s for 60 min. Each stimulation train was applied for 10 s at 90-s intervals for an hour of imaging time. The electric field strength was $4.5\mathrm{mV}/\mathrm{mm}$, and the neural cell behavior was recorded with the optical microscope every minute. A total of 60 time-lapse imaging frames were obtained during the electrical field stimulation.