Research Progress
Activities
1. Confirm the validality of cancel cell's (HeLa cell) and HUVEC cells’ surface charge characteristics via electrostatic cell–nanoparticle interaction method
For this goal, we continued the research on a single cell's surface charge mapping using the electrostatic cell–nanoparticle interaction illustrated in Fig. 1. A micro device was made to separate the cells from the unbonded NPs. The channel consisted of one inlet reservoir, one connecting channel with a space height of ~2um, and the outlets. Once loaded into the device, the cells were captured at the bottom of the inlet reservoir: the lower height of connecting channel prevented the cells from moving to the outlet, while the unbonded NPs with the sub-micrometer size were guided to the outlet. After the separation, the cells were positioned on the inlet reservoir’s bottom (at the edge between the inlet reservoir and connecting channel). A fluorescence microscope imaged every single cell from the back of the glass substrate. We took 100 Z-stack images of each captured cell at various focal depths with a Fluorescence Microscope. The cell surface area profile and fluorescence distribution can be calculated from the Z-stack-based three-dimensional data. Thus, each pixel’s fluorescence intensity was then transferred to the surface charge mapping from the calibration curve shown above.
Figure 1. Illustration of the method for a single cell's surface charge mapping. The cell suspension is mixed with nanoparticles, followed by incubation, washing of unbonded nanoparticles, and capturing cells. The positively charged nanoparticles can bind onto the wall of the cell due to the static electrical force. Z-stack images of cells bonded with nanoparticles are captured by a fluorescent microscope, which is processed to map the fluorescent distribution and thus the surface charge mapping of single cells.
2. Research a new cell surface charge mapping method using a photoelectrical imaging system
In Year 3 we have been developing a label-free optoelectronic imaging system based on indium tin oxide (ITO)-coated glass substrates for mapping the surface charge distribution of single living cells in a serum-free saline solution. Cells can be placed upon an ITO layer, in contact with metallic microelectrode array. Incident light will be focused on the microelectrodes on the ITO surface. The metal micro-electrodes are made of Chromium and patterned to 2μm×2μm squres or 1-2 μm nickel micro-particles. Photocurrent images are obtained by capturing the reflected light from the microelectrodes through a charged-coupled device (CCD) under an applied bias voltage or surface charge/potential of a cell. The voltage or potential difference applied on the microelectrode array will affect the photoelectric conversion of the ITO layer, so that the potential change on the microelectrode can be obtained by measuring the light intensity change of the reflected light. These images can then be processed using the Matlab program to generate the light intensity change mapping and the surface charge mapping.
Figure 3. Illustration of the method for mapping a single cell's surface charge. The metallic micro electrodes were patterned on the ITO layer using either Nickel microparticles or metallic thin films. The WE, CE and RE represent the working electrode, counter electrode and reference electrode, which is the components of the three-electrode system. Light will illuminate the micro-electrode array, and the reflected light will be recorded by CCD camera. The light intensity distribution will be processed to map the the surface charge of a single cell.
Principle and Experimental Setup
In this system, photoexcited charge carriers will be transferred across the ITO-electrolyte interface, increasing the photocurrent flow in the external circuit. Therefore, as the potential applied on the ITO surface changes, the intensity of the reflected light will change. For example, when the potential difference between the solution and ITO is in a sine wave change, the reflected light received by the CCD also produces a corresponding sine result. In the experiment, 4×4 cm ITO-coated glass slide was washed with ethanol and deionized water; after air-drying, the ITO-coated glass was kept at room temperature before use. The PDMS chamber consisted of one large open inlet reservoir. The setting is shown in Figure 3. Led lamps (max. 25 mW) were used to illuminate the ITO and generate charge carriers. The external voltage can be sinusoidally and continuously modulated with different potential bias for measurement. Incident light was focused onto microelectrodes patterned on ITO-coated glass using an objective lens, and the reflected light will be captured by a CCD camera. The paterned microelectrodes allows us to characterize the surface chrger distribution on the cell surface. Working electrode (WE), counter electrode (CE) and reference electrode (RE) are used in the measurement cell, to mimic the cell surface potential aand calibrate the relation betweeb the light intensity and the surface otential. Once the calibration is established, a cell will be sited on the metal micro-electrode array, the light will illuminate these electrodes and the reflected light will be recorded. The light reflectance intensity will be compared with the ones without cell. Then the surface potential distribution will be converted into surface charge distribution according to Gouy−Chapman theory.
Findings
3. Confirm the validality of cancel cell's (HeLa cell) and HUVEC cells’ surface charge characteristics via electrostatic cell–nanoparticle interaction method
This method utilizes the electrostatic interactions between positively charged fluorescence nanoparticles and cells. We can measure the surface charge density distribution on a single cell’s surface via a typical fluorescence microscope. We conducted zeta potential measurement for both cells using light scattering method. The measured zeta potentials for these two cells were -13.8 mV and -17.1 mV. We also calculated the zeta potentials of each Hela and HUVEC cell from the measured fluorescence intensity. The results are shown in Figure 1. The average calculated zeta potentials of Hela and HUVEC cells were -13.2 mV and -16.4 mV, which match the light scattering method measurement reasonably well.
We measured 60 HUVEC cells and 60 Hela cells. Each cell Z-stack image was processed using the procedures described in section Testing setup and signal processing to map the surface charge distribution. Typical results of Hela cells and HUVEC cells are shown in Figure 2 and Figure 3, respectively. Both plots showed the method could measure the surface charge mapping and cell topography simultaneously. The surface charge mapping seems HUVEC cells have a more uniform surface charge distribution than Hela.The results show that some cells displayed a charge density differing from the average values. It indicates that the presence of some highly negatively charged areas on the cell surface.
Figure 1. Comparison of the measured average charge density for each cell type, and the corresponding zeta potential of HUVEC and Hela cells. Left:distribution of average charge density of cells. Right:boxplot of zeta potential converted from the average charge density of cells.
Figure 2. Typical measurement results of surface charge mapping of a HUVEC cell. (a) z-stacks images, (b) surface height mapping, (c) the surface height profile at the centerline located in the cell center in X-direction, (d) fluorescent intensity distribution, and (e) the surface charge mapping.
Figure 3. Typical measurement results of surface charge mapping of a Hela cell. (a) z-stacks images, (b) surface height mapping, (c) the surface height profile at the centerline located in the cell center in X-direction, (d) fluorescent intensity distribution, and (e) the surface charge mapping.
4. Research a new cell surface charge mapping method using a photoelectrical imaging system
a. Proving the relation between light intensity and surface potential
The reflected light from the microscope was recorded for 40 seconds each sample. The surface charge mapping is expected to have a spatial resolution of 2μm×2μm due to the size of the micro electrodes. We read the reflected brightness of each pixel of the time series image, so that we can get the changes in potential and reflected light. In the measurement, the light intensity reflected by the microelectrodes was significantly greater than that of the blank ITO surface (surface without microelectrodes). We calibrated the relation between the light intensity and the surface potential by applying known potential on the microelectrodes using the three-electrode system. The obtained result is shown in Fig. 4, showing: 1) as a sinusoidal excitation electrical potential (100 mV, 1 Hz) was applied, the light intensity also changed with the same sinusoidal shape; 2) when a 100mV voltage bias was set, the light intensity (∆R/R) showed a distinguished magnitude change. This indicates the current method has the fast reponse to capture the surface potential change.
Figure 4. Measured dtnamic light intensity in response to the surface potential applied on the metallic micro-electrodes.
b. Proving the resolution of the surface potential measurement
Furthermore, we proved the measurement resolution of the method. As shown in Figure 2, applying different voltages at different times resulted in changes in the reflected light intensity, and this potential-reflected light relationship was reversible under repeated cycles of potential changes, indicating that ITO was within the applied voltage range. Figure 2 shows that when the surface potentials were set to 201mV and 200mV, respectively, the difference in the magnitude of the light intensity change can be identified, indicating a 1mV measurement resolution can be obtained.
Figure 5. The potential-light intensity correspondence and its reproducibility were verified by changing the surface potential at different times. In (a~d), the applied voltage is 0mV for 0~10 seconds and 20~30 seconds, while the applied voltage is 201mV and 200mV for 10~20 seconds and 30~40 seconds, respectively. (e~h) , the applied voltage is 0mV at 0~10s and 20~30s, and 200mV and 201mV at 10~20s and 30~40s, respectively.
In summary, in Year 3 we confirmed the validality of the cell surface charge mapping based on nanoparticle-cell interactions, and invented a new cell surface charge mapping method based on photoelectrical imaging. In the last Year, we will continue the research on a the cell surface charge mapping enabled by photoelectrical imaging, using Hela and HUVEC Cell as sample cells.