Comparison study of live cells by atomic force microscopy, confocal microscopy, and scanning electrochemical microscopy

Abstract

In this report, three kinds of scanning probe microscopy techniques, atomic force microscopy (AFM), confocal microscopy (CM), and scanning electrochemical microscopy (SECM), were used to study live cells in the physiological environment. Two model cell lines, CV-1 and COS-7, were studied. Time-lapse images were obtained with both contact and tapping mode AFM techniques. Cells were more easily scratched or moved by contact mode AFM than by tapping mode AFM. Detailed surface structures such as filamentous structures on the cell membrane can be obtained and easily discerned with tapping mode AFM. The toxicity of ferrocenemethanol (Fc) on live cells was studied by CM in reflection mode by recording the time-lapse images of controlled live cells and live cells with different Fc concentrations. No significant change in the morphology of cells was caused by Fc. Cells were imaged by SECM with Fc as the mediator at a biased potential of 0.35 V (vs. Ag/AgCl with a saturated KCl solution). Cells did not change visibly within 1 h, which indicated that SECM was a noninvasive technique and thus has a unique advantage for the study of soft cells, since the electrode scanned above the cells instead of in contact with them. Reactive oxygen species (ROS) generated by the cells were detected and images based on these chemical species were obtained. It is demonstrated that SECM can provide not only the topographical images but also the images related to the chemical or biochemical species released by the live cells.Key words: live cells, atomic force microscopy, confocal microscopy, scanning electrochemical microscopy.

Scanning Electrochemical Imprinting Microscopy: A Tool for Surface Patterning

“…A wide variety of local surface modifications have been accomplished by the feedback mode. These include metal deposition, 31 [59][60][61][62][63][64][65][66][67][68][69] and, in a few cases, also modification. 70 Here we present an approach, termed scanning electrochemical imprinting microscopy, which combines the elements of SPM and printing.…”

Filming a live cell by scanning electrochemical microscopy: label-free imaging of the dynamic morphology in real time

“…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.…”

“…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.…”

“…(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]. …”

“…The resolution of SECM was about 5 μm because a 5.0 μm diameter Pt ultramicroelectrodes (UME) was used in the experiment. The SECM principle, instrumentation, operating procedures, and fabrication of 5 μm Pt UME were described in previous publications [10,12]. …”