The morphology of single live cells reflects the organization of the cytoskeleton and the healthy status of the cell [1]. Monitoring the morphological changes of live cells may provide dynamic information of the cell attachment or cytoskeleton organization [1]. The most commonly used cell imaging method is fluorescent microscopy, which is extremely sensitive and allows the visualization of particular structure or compounds inside the cells [2, 3]. On the other hand, the preparation of the specimen is relatively time-consuming, and the live cells are sensitive to photo-damage [3]. As an imaging method with high spatial resolution, atomic force microscopy (AFM) avoids staining process and photo-damage. However, its cantilever tip mechanically damages the soft cells thus is not suitable for time-lapse experiments [4]. As a non-invasive single-cell analysis method, scanning electrochemical microscopy (SECM) has been successfully applied to live cell-imaging due to its high temporal and spatial resolution [5–13]. This technique is based on the measurement of the electrochemical current flowing through the SECM tip, which is usually an ultramicroelectrode (UME). The current detected at the tip is dependent on the separation space between the tip and the cell, therefore morphological information of the live cell can be revealed by the electrochemically mapped images [7–9, 11]. Compared to fluorescent microscopy, sample preparation of SECM is simple without any staining or labeling procedure. Unlike AFM, the SECM probe does not need to touch the cell, thus it can carry on time-lapse measurement without mechanically scratching the cell. Nevertheless, most SECM imaging experiments were conducted with the addition of a certain redox mediator, which is usually non- physiologic and undesired [7–9, 11, 12]. In our previous study [14], we found that dissolved oxygen in the medium solution could be detected by SECM, which provides an opportunity of label-free imaging cellular morphology using dissolved oxygen as the redox mediator. Bladder cancer is the fourth most common cancer of men and the eighth most common cancer of women [15]. Like most of cancers, bladder cancer begins with the mutation of one single cell [16, 17]. Investigations of bladder cancer, especially the interaction with anti-cancer drugs, at the single cell level can provide new insight into its physiology, pathology and pharmacology, and promote the development of chemotherapy in response to single-cell behaviours [18]. Herein, the real-time morphological changes of single live T24 cells under non-physiological temperature are revealed by time-lapse SECM with dissolved oxygen as the indicator. While the reactive oxygen species (ROS) released by live cells may interfere with the detection of dissolved oxygen [12, 14, 19], we determined that under physiological conditions oxygen can be reduced at -0.455 V and hydrogen peroxide can be reduced at -0.745 V, while superoxide is oxidized at +0.055 V. Thus in this research the potential was set at -0.500 V to reduce the dissolved oxygen. We also found that the resting status when the T24 cells do not release ROS can last for up to 5 h [20], which sustains imaging T24 cells with only dissolved oxygen.
The typical SECM setup is illustrated in Figure 1.
The changing morphology of a T24 cell under room temperature was investigated by time-lapse SECM images scanned at about 0.8 μm above the nucleus of the cell with a 5 μm diameter Pt UME biased at -0.500 V vs. Ag/AgCl. (Figure 2b, d and 3) for oxygen reduction [14, 21]. The distance between the UME and the cell was calibrated with ferrocenemethanol after the time-lapse SECM experiment [12].
At -0.500 V vs. Ag/AgCl, the dissolved oxygen can be reduced at the diffusion controlled rate at the Pt surface of the UME [14, 21]. The time-lapse SECM images in Figure 2 present negative feedback as the current over the T24 cell is lower than the background current, which is the current measured over the Petri dish bottom. This indicates the cell is not releasing anything redox active, and the current flowing through the UME only results from the reduction of dissolved oxygen in medium solution [20]. The T24 cell blocks the dissolved oxygen from diffusing to the Pt surface of the UME (Figure 1), resulting in decreased current over the T24 cell, which is called negative feedback [22, 23] in SECM (Figure 2b, d and 3).
In comparison of the SECM images and the 50 × optical micrographs obtained with the same T24 cell (Figure 2), the SECM images present higher spatial resolution than the optical microscopic images. The nucleus (black area) could be clearly distinguished from the membrane (brown area) in the SECM images (Figure 2a and 2b). Conversely, in the first optical microscopic images, the great nuclear area (blue dashed area) and membrane (orange dashed area) can be vaguely distinguished (Figure 2c); 1 h later, only an outline of the cell (blue dashed area) can be seen (Figure 2d). The time-lapse SECM images in Figure 2 demonstrate that the cellular morphology has been changed within 1 h. The membrane has been shrunk and the nucleus has been rounded and compacted (Figure 2b). The morphological change observed in the time-lapse SECM images is in consistence with the time-lapse optical micrographs. It is plausible that in the SECM image in Figure 2a, the current over the black dashed area on the cell membrane is higher than the background current. However, the cross section line drawn from the red dashed position presenting the cellular topography along this position (inset in Figure 2a) shows that this area corresponds to the edge of the cellular membrane, and the current over this area (black triangle pointed current in the cross section line) is no higher than the background current. The color in this area is relatively brighter since the adjacent cell (purple dashed area in Figure 2c) has elevated membrane, and the color scale of a SECM image is adjusted laterally in WiTec software with which the SECM experiments were conducted.
Figure 3 clearly demonstrates the real-time morphological change of the T24 cells over 156 min under room temperature. We could easily distinguish the black condensing nucleus in the center and the significantly shrinking cellular membrane around the nucleus in each image, and observe the dynamic morphological changes of each part. The time-lapse cross section lines drawn from the color dashes in each image of Figure 3 reveal the real-time topographical change of the T24 cell. At the beginning (Figure 3a), the great nuclear area is elevated from the extended cellular membrane; the non-nucleus part of the great nuclear area (green triangles) is higher than the nucleus part (yellow triangles); the whole cell is flat but the surface is uneven and wavy (Figure 3a-d). Then the non-nucleus part of the great nuclear area gradually merges to the nucleus (Figure 3a-f), and cannot be distinguished from the nucleus 30 min later (Figure 3f). The extended cellular membrane slowly shrinks in a worm-like manner, that is, partially elevates and pushes forward into the great nuclear area, for 93 min (red arrows in Figure 3a-k). 93 min later, the cellular topography becomes steady and no obvious change was observed in the next 63 min; the total cell height is raised but the cellular surface is smooth (Figure 3l-p). As can be seen from the lowest current value in each cross section line, the cell height was found to fluctuate periodically in a small range during the 156 min. The real-time morphological changes observed here with time-lapse SECM are consistent with the morphological features of early apoptosis [24], which suggests that the prolonged exposure to room temperature may induce apoptosis of T24 cells. An additional movie file shows the dynamic morphology of the T24 cell in more detail (see Additional file 1).
Additional file 1: Dynamic morphology of a T24 cell under room temperature. temperature. (MOV 500 KB)
