Mohd Ridzuan Ahmad

A Review of Cell Adhesion Studies for Biomedical and Biological Applications

Cell adhesion is essential in cell communication and regulation, and is of fundamental importance in the development and maintenance of tissues. The mechanical interactions between a cell and its extracellular matrix (ECM) can influence and control cell behavior and function. The essential function of cell adhesion has created tremendous interests in developing methods for measuring and studying cell adhesion properties. The study of cell adhesion could be categorized into cell adhesion attachment and detachment events. The study of cell adhesion has been widely explored via both events for many important purposes in cellular biology, biomedical, and engineering fields. Cell adhesion attachment and detachment events could be further grouped into the cell population and single cell approach. Various techniques to measure cell adhesion have been applied to many fields of study in order to gain understanding of cell signaling pathways, biomaterial studies for implantable sensors, artificial bone and tooth replacement, the development of tissue-on-a-chip and organ-on-a-chip in tissue engineering, the effects of biochemical treatments and environmental stimuli to the cell adhesion, the potential of drug treatments, cancer metastasis study, and the determination of the adhesion properties of normal and cancerous cells. This review discussed the overview of the available methods to study cell adhesion through attachment and detachment events.

In Situ Single Cell Mechanics Characterization of Yeast Cells Using Nanoneedles Inside Environmental SEM

In this study, characterization of cellular mechanics of W303 yeast cells was conducted using nanoneedles inside an environmental SEM (ESEM). This enhanced ESEM system comprises a standard ESEM instrument as a nanoimaging tool, a cooling stage as a humidity controller for cellular biology and 7 degrees of freedom and linear actuators as nanomanipulator/effector. Four types of nanoneedles were used in our experiments, i.e., silicon (Si), titanium (Ti) coated Si, and two types of tungsten (W) nanoneedles. The Si and Ti nanoneedles were fabricated using 2 N/m spring constant cantilevers. While the W nanoneedles were fabricated using 0.09 and 2 N/m spring constant cantilevers (W0.09 and W2 nanoneedles). The Si, silicon-titanium (Si-Ti), W0.09, and W2 nanoneedles are suitable to be used for local stiffness characterization of single cells. This capability can be used in future for fast disease detection since disease cells may show different cell mechanics properties as compared to the normal cells. The Si-Ti and W2 nanoneedles can penetrate the cell without cell bursting, and this could be important in single cell surgery in future to avoid cell damage.

The Effects of Cell Sizes, Environmental Conditions, and Growth Phases on the Strength of Individual W303 Yeast Cells Inside ESEM

We performed in situ measurements of mechanical properties of individual W303 wild-type yeast cells by using an integrated environmental scanning electron microscope (ESEM)-nanomanipulator system. Compression experiments to penetrate the cell walls of single cells of different cell sizes (about 3-6 mu m diameter), environmental conditions (600 Pa and 3 mPa), and growth phases (early log, mid log, late log and saturation) were conducted. The compression experiments were performed inside ESEM, embedded with a 7 DOF nanomanipulator with a sharp pyramidal end effector and a cooling stage, i.e., a temperature controller. ESEM itself can control the chamber pressure. Data clearly show an increment in penetration force, i.e., 96plusmn2, 124 plusmn10, 163plusmn1, and 234plusmn14 nN at 3, 4, 5, and 6 mu m cell diameters, respectively. Whereas, 20-fold increase in penetration forces was recorded at different environmental conditions for 5 mu m cell diameter, i.e., 163plusmn1 nN and 2.95plusmn0.23 mu N at 600 Pa (ESEM mode) and 3 mPa (HV mode), respectively. This was further confirmed from quantitative estimation of average cell rigidity through the Hertz model, i.e., ESEM mode (3.31plusmn0.11 MPa) and HV mode (26.02plusmn3.66 MPa) for 5 mu m cell diameter. Finally, the penetration forces at different cell growth phases also show the increment pattern from log (early, mid, and late) to saturation phases, i.e., 161plusmn 25, 216plusmn15, 255 plusmn21, and 408plusmn41 nN, respectively.

Nanoindentation Methods to Measure Viscoelastic Properties of Single Cells Using Sharp, Flat, and Buckling Tips Inside ESEM

In this paper, methods to measure viscoelastic properties of time-dependent materials are proposed using sharp, flat, and buckling tips inside an environmental SEM. Single W303 yeast cells were employed in this study. Each of the tips was used to indent single cells in a nanoindentation test. Three loading histories were used: 1) a ramp loading history, in which a sharp indenter was used; 2) a step loading history, in which a flat indenter was implemented; and 3) a fast unloading history, in which a buckling nanoneedle was applied. Analysis of the viscoelastic properties of single cells was performed for each of the loading histories by choosing an appropriate theory between the correspondence principle and the functional equation. Results from each of the tests show good agreement, from which strong conclusion can be drawn.

Bringing the nanolaboratory inside electron microscopes

A nanolaboratory is one of the systems to realize various nanoscale fabrications and assemblies to develop novel nanodevices to integrate borderless technologies based on a nanorobotic manipulation system. We have presented the nanolaboratory inside electron microscopes including a transmission electron microscope (TEM), scanning electron microscope (SEM), and environmental-SEM (E-SEM) for three-dimensional (3D) and real-time nanomanipulation, nanoinstrumentation, and nanoassembly. The following is a presentation of our current work of nanomanipulation and nanoassembly based on the hybrid nanorobotic manipulation inside a TEM and an SEM toward carbon nanotube (CNT) applications. Single cell stiffness measurement has been also presented based on the nanorobotic manipulation system inside an E-SEM.

Instantaneous and Quantitative Single Cells Viability Determination Using Dual Nanoprobe Inside ESEM

We performed single pulses current measurement on single cells using dual nanoprobe through environmental scanning electron microscope nanomanipulator system. The ability to characterize the electrical property of single cells can be used as a novel method for cell viability detection in quantitative and instantaneous manners. The nanoprobe was successfully fabricated using focused ion beam tungsten deposition and etching processes. The characteristics of the nanoprobe were examined from the energy dispersion spectrometry and noise analyses. In this paper, for the first time, the electrical property of single cells under their native condition was presented. In order to apply this method for cell viability detection, two types of cells were used, i.e., dead cells and live cells. The results showed that there is a significant difference on the electrical measurement data between dead and live cells.

Buckling Nanoneedle for Characterizing Single Cells Mechanics Inside Environmental SEM

We propose a buckling nanoneedle as a force sensor for stiffness characterization of single cells. The buckling nanoneedle was easily fabricated by using focused ion beam etching from a commercialized atomic force microscope cantilever. There are notable advantages of using buckling nanoneedle for single cells stiffness characterizations. First, severe cell damage from an excessive indentation force could be prevented. Second, large variations in single cells stiffness property could be easily detected either from the dented mark on the cell surface after the indentation and/or by comparing the buckling length of the nanoneedle during the indentation. The calibrations of the buckling nanoneedle were done experimentally and numerically. The calibration results from both methods showed a good agreement. The calibration data show the relationship between the indentation force and the buckling length of the nanoneedle. This relationship was used for obtaining force data during a nanoindentation experiment between a buckling nanoneedle and single cells. We performed in situ measurements of mechanical properties of individual W303 wild-type yeast cells by using a buckling nanoneedle inside an integrated SEM (ESEM)-nanomanipulator system. Finer local stiffness property of single cells was compared at different pressure and different temperature ranges. This detection method of the stiffness variations of the single cells could be applied in the future fast disease diagnosis based on single-cell stiffness analysis.

Effect of ambient humidity on the strength of the adhesion force of single yeast cell inside environmental-SEM

A novel method for measuring an adhesion force of single yeast cell is proposed based on a nanorobotic manipulation system inside an environmental scanning electron microscope (ESEM). The effect of ambient humidity on a single yeast cell adhesion force was studied. Ambient humidity was controlled by adjusting the chamber pressure and temperature inside the ESEM. It has been demonstrated that a thicker water film was formed at a higher humidity condition. The adhesion force between an atomic force microscopy (AFM) cantilever and a tungsten probe which later on known as a substrate was evaluated at various humidity conditions. A micro-puller was fabricated from an AFM cantilever by use of focused ion beam (FIB) etching. The adhesion force of a single yeast cell (W303) to the substrate was measured using the micro-puller at the three humidity conditions: 100%, 70%, and 40%. The results showed that the adhesion force between the single yeast cell and the substrate is much smaller at higher humidity condition. The yeast cells were still alive after being observed and manipulated inside ESEM based on the result obtained from the re-culturing of the single yeast cell. The results from this work would help us to understand the ESEM system better and its potential benefit to the single cell analysis research.

Micro- and Nanomechatronics

The current and future technologies and issues of micro- and nanomechatronics, and nanobiotechnology based on the nanorobotic manipulation system are presented. The following are also discussed in detail: micromechatronics for industrial and research applications; nanomechatronics for industrial and research applications; single-cell surgery system based on the nanorobotic manipulation system; nanorobotic manipulation system; and single-cell analysis based on an environmental scanning electron microscope (E-SEM). Devices based on micro- and nanomechatronics technologies have great potential in advanced industrial applications that will realize high efficiency, high integration, high functionality, low-energy consumption, and low cost.

Nanofork for Single Cells Adhesion Measurement via ESEM-Nanomanipulator System

In this paper, single cells adhesion force was measured using a nanofork. The nanofork was used to pick up a single cell on a line array substrate inside an environmental scanning electron microscope (ESEM). The line array substrate was used to provide small gaps between the single cells and the substrate. Therefore, the nanofork could be inserted through these gaps in order to successfully pick up a single cell. Adhesion force was measured during the cell pick-up process from the deflection of the cantilever beam. The nanofork was fabricated using focused ion beam (FIB) etching process while the line array substrate was fabricated using nanoimprinting technology. As to investigate the effect of contact area on the strength of the adhesion force, two sizes of gap distance of line array substrate were used, i.e., and . Results showed that cells attached on the gap line array substrate required more force to be released as compared to the cells attached on the gap line array substrate.