Existing methods for measuring surface charge did not depict physical information about the cell being measured. Using the Horn-Schunck image processing method, the captured images were automatically processed in real time to obtain the cell surface charge.
Application of surface charge
The above methods (PALS) provide the surface charge value of the measured particle without any images of the particle during the measurement process. Furthermore, the process of surface charge measurement using either blade counters or PALS is time-consuming for a complex sample.
Surface charge of biological cell
Therefore, surface charge measurement of biological cells serves a great potential in application of cancer research and other pharmaceutical applications. Cost per sample of these measurement techniques can be high, so in this study, a real-time, compact and cost-effective surface charge measurement method was built.
Aims and Objectives
The main objective of this study is the development and construction of a compact CCD microelectrophoresis system with image processing for surface charge measurement. To investigate cell surface bioburden using CCD-based microelectrophoresis with image processing method.
Introduction
Surface charge measurement
Phase analysis light scattering
This method measures the average Brownian motion of particles to estimate the average size and electrophoretic mobility of the entire multiparticle in an applied electric field. Note that the laser passes through the sample and the reference to compare the frequency shift of the laser source.
Coulter counter
When a particle passes through the narrow channel, the channel resistance changes and causes the channel voltage measurement to fluctuate. The measurement can be very precise; however, the measurement needs a long period of time to measure a group of cells.
Suspended microchannel resonator
This method measures changes in the resonant frequency of a hollow cantilever as suspended particles pass through the cantilever. In addition, the resonant frequency of the SMR system is very sensitive to the position of the particles in the cantilever. As the particle travels through the cantilever, the change in resonant frequency corresponds to the position.
The SMR system has demonstrated the ability to quantify the surface charge of particles by measuring the electrophoretic mobility of the particle. Comparison between the PALS method, Coulter counters and SMR is presented in terms of time of measurement, penetration and display of particle morphology.
Image processing of cell
Segmenting individual cells
In the process of cell detection, it generally consists of two main steps: (1) cell segmentation (separation from background), (2) cell association (recognition of cell between images). The result of the segmentation is to provide a new image containing labels indicating which segments of the image represent a single particle and which represent the background. One of the approaches for cell segmentation is the use of threshold value; in order to label pixels in the images, each pixel of image was compared where the pixels with values above the threshold as particle and values below the threshold as background (Ta et al., 2009).
This refers to the process of identifying and synchronizing the segmented cells from one frame to another to obtain the trajectory of the cells. However, this large amount of comparisons requires additional processing, which will add to the long processing time of the data.
Electric Double Layer
Electro-osmosis
Electroosmosis is defined as the movement of liquid relative to an applied potential across a membrane, microchannel or capillary tube. When an electric field is applied to a solution, the mobile portion of the EDL will migrate to the cathode or anode, depending on the EDL polarity. The migration of the ions from the EDL promotes viscous separation of the fluid molecules surrounding the EDL, which ultimately leads to bulk fluid movement, or electroosmotic flow.
The electroosmotic flow is independent of the size of the suspended particles in cases where the EDL is much smaller than the channel length. In this study, the channel size is relatively larger compared to the cell size, so the effect of electroosmosis on surface charge measurement was minimal.
Electrophoresis
Surface charge equation
Since the surface charge of cells is mainly contributed by the ionizable organic matter such as acids and bases. Consequently, the surface charge of the cell is highly influenced by the pH of the electrolyte in the buffer solution being measured. Considering these reasons, it is desirable to calculate surface charge measurement from EPM.
𝐸0 (2.4) Surface charge of the particle is calculated with the Gouy-Chapman theory (equation 2.5), where σ is the surface charge and κ is the Debye-Huckel parameter.
Surface charge of cell
Surface charge of cancer cell
Studies by the group of researchers, Dolowy, 1984, show that the surface charge of biological cells increases during tumorigenesis and decreases during necrosis. The results are compared between the surface charge of cancer cells and the normal cells. The surface charge of the cancer cell is higher in positive charge in pH 3 and reached isoelectric point at pH 5.
The surface charge of cancer cell and normal cell becomes negative charge at pH 4 onwards. The surface charge of both cells are similar in the graph pattern, but the surface charge of cancer cells is higher compared to the normal cell surface charge.
Introduction
Experimental apparatus and method
Compact CCD microscope
Compact CCD camera is attached to the top of the microscope as shown in Figure 3.1). The lens of the CCD camera is reversed and attached with the CCD sensor. The sample chamber is constructed with an empty hole in the center of the chamber to allow light to shine through to the sample.
This setup allows the camera to capture the image of stable samples back-illuminated by the light source. In addition, a collimator lens (with NA of 0.50) is inserted into the optical path between the light source and the CCD camera to align the light emitted by the LED.
Microelectrophoresis chamber with two electrodes
A glass-based channel with a rectangular cross-section (0.2 cm x 0.5 cm) and a length of 2 cm was made in the middle of the chamber.
DC power supply
Imaging system
- Calibration of the compact CCD microscope
- Experimental procedure and image data collection
- EPM and surface charge measurement
- Electrokinetic Transport Equations
- DC electric field displacement
- Image processing
- Yeast cell
- Cell culture procedure
- Normal human bone cell (hFob 1.19)
- Cancerous human bone cell (U2OS)
From the image processing process, the result of the velocity will be calculated after the cells are identified. Then the optical algorithm will calculate the average speed of the identified particles in the image. The cell speed and the direction of the mobilities are determined by the DC electric field.
After obtaining the image intensity value, the image will be passed to the filtering function. The bone cell is a type of sticky cell, so the cell will begin to attach to the bottom of the flask (Day 6 in Figure 3.14). The bone cell is a type of sticky cell, so the cell will begin to attach to the bottom of the flask (Day 6 in Figure 3.15).
The growth of the cancerous bone cell is significantly faster than the normal bone cell.
Conclusion
It takes twice as long for normal bone cells to double as for cancerous bone cells. At the same time, the growth of the normal bone cell population slows down during the 40th day of cell culture. As the bone cancer cell population continues to grow in the cell culture process.
Introduction
Surface Charge of Polystyrene Bead
The image of the beads is shown in Figure 4.2 where the polystyrene beads are spherical and stable in shape. At modified web camera magnification, 125 X, the measured EPM of this polystyrene bead is -1.17 µmcm/Vs at DC electric field of 10 V. The measured surface charge of the polystyrene bead using the Malvern instrument is -1 ,32 𝜇𝑚𝑐𝑚/𝑉𝑠 with 5% uncertainty.
On the other hand, the measured surface charge value of the polystyrene bead using the compact CCD microelectrophoresis system is -1.17 µmcm𝑉𝑠⁄ with an uncertainty of 10%. These spatial directional data, together with the applied electric field and the velocity of the beads, were used to calculate the electrophoretic mobility of polystyrene beads.
Image of yeast captured with SEM and compact CCD microelectrophoresis system
In Figure 4.5, the image of yeast cells is captured by a compact microelectrophoresis system of a CCD microscope. The compact CCD microscope microelectrophoresis system can acquire images of cells between 3 µm and 15 µm in size. The yeast cells in this image are measured with an average size of 4 µm in diameter. This cost-effective setup provides a clearer and more distinct cell outline, which is more suitable for measuring the surface charge of the biological cells used in this study.
In Figure 4.5, yeast cells are isolated from each other and cell aggregations are rare.
Surface charge of normal bone cell (hFob 1.19) and cancer bone cell (U2OS) are similarly shaped (Figure 4.8). The surface charge of the cells (normal and cancer) is positive at pH value < pH 4.4. The results show that the surface charge of the bone cancer cells and normal bone cells used in this study is positive at pH 4, which is 0.40 µmcm/Vs (cancer cell) and 0.54 µmcm/Vs (normal cell).
The surface charge of these two cells showed significant differences at the pH close to human blood (Haven, 1941). The surface charge of cancer cells is very negative compared to normal cells, and this can be explained through changes in the cell membrane during tumorigenesis.
Surface Charge of Cervical Cancer Cell (HeLa)
Studies had suggested that the phospholipid content of human breast cancer cells is increased compared to the normal counterparts (Podo et al., 2007; Sakai, 1992; Punnonen et al., 1989). Cancer cells replicate rapidly compared to the normal cell, hence an increased level of phospholipids stimulates cell membrane synthesis, which is closely related to the rapid replication process (Ruiz-Cabello et al., 1992). Whereas in acidic buffer medium (low pH) the charge of the membrane is due to the amino groups while in basic buffer solution with high pH value it is largely due to the carboxyl and phosphate group.
Surface charge of these cells is closely related to the amount of phospholipids on the membranes, where cells are found to be negatively charged in high pH values while they are positively charged at low pH values. Consequently, increased phospholipids in cancer cell can cause the surface charge of the cell to increase negatively in high pH buffer medium.
Conclusion
Recommendation
The designed compact CCD microelectrophoresis system with image processing has been shown and proven to be suitable for automated surface charge measurement of biological cells with size ranges from 4 to 20 µm. Automated D-analysis of cell migration and interaction in the thymus cortex from time-lapse sequences of 3-D multi-channel multi-photon images. Integrated measurement of the mass and surface charge of discrete microparticles using a suspended microchannel resonator.
Real-time surface charge measurement of discrete microparticles using microelectrophoresis and compact CCD microscope. The area of interest is sent to the subblock for average speed calculation.
