Cell Manipulation
Chapter 1: Motivation
Biomedical scientists need tools to insert genes, proteins, and other chemicals into living tissues at very specific locations.
Hand-drawn syringes with glass needles are currently used, but the damage they cause is often fatal to cells. Even when the cells survive the membranes are deformed over a large area. Deformation alters normal intracellular activity leading to treatment failure.
Other drawbacks are inaccuracy and a difficulty knowing when the needles break the surface.
Chapter 2: Micro-injection
A MEMS (Microelectromechanical System) micro-injector is smaller so it solves many of the problems a syringe had. The tip is half the size of a glass needle.
More precise injections are possible that target smaller cellular components. The delivery is accurate, and the deformation is reduced.
There is also another advantage we will look at in the next chapter.
Chapter 3: Force
The force needed to inject material into an embryo using a glass needle is almost 150 µN.
A micro-injector requires much less force so it causes less cellular damage.
The chapters to come will show how the micro-injector’s design allows it to measure this force and detect when injection occurred.
Chapter 4: Design
A silicon-nitride tip is attached to one of two silicon gratings. The tip is what actually breaks through the tissue. But the twin gratings are equally important because, as we will see, they allow the tip displacement and force to be precisely quantified.
Knowing how much force was necessary for a certain design will lead to improvements. And knowing surface strength profiles will give cell biologists more information on cellular surface mechanics.
Chapter 5: Fabrication
The micro-injector is made from silicon using the same technology used to make computer chips. Bands of silicon have been chemically etched away at regular intervals leaving the grating pattern.
The tip is around a thousandth of an inch wide and each band in the grating is one fifth the size of a human hair.
Chapter 6: Operation
During operation an embryo or cell is captured on an adhesive oil pad lying within a micro-channel.
Then it is mechanically pressed against the injector tip. The force pushes the top grating back, but the bottom grating doesn’t move at all. Four silicon springs bend backward to make this possible.
Chapter 7: Optics
When a fixed laser is focused onto the gratings the light is diffracted. A photodiode measures the diffraction intensity. As the top grating moves back the alignment pattern changes.
The intensity is at 100% when the gratings are in phase and zero when they are completely out of phase.
Chapter 8: Measurement
A digital oscilloscope graphs the intensity in real time while the top grating is pressed backwards. The gratings start in phase, go out of phase, return, and go out again as the grating is displaced further.
The width of one band is 10 µm. So we know that the grating moved 20 µm in the time the intensity takes to return to 100%. 10 µm to go out of phase plus 10 µm to return.
Chapter 9: Analysis
When the tip breaks the surface the top grating will snap back into its original position. This appears as a spike on the oscilloscope. Where the spike occurs reveals how far the tissue pressed the grating back.
Hooke’s Law is used to calculate how much force was applied: F = k×x The spring constant (k) is known and the displacement (x) is given by the oscilloscope measurements.
Chapter 10: Results
Applying Hooke’s Law we find that the average force for micro-injection is about 50 µN. Recall that a glass needle required 150 µN.
Chapter 11: Vibration
To further reduce the injection force the top grating can be vibrated at a high frequency. This method is different because the tissue is moved towards the injector but stopped before the cell is penetrated. Then the tip is vibrated faster and faster until it breaks the surface.
A piezoelectric plate is bonded to the top grating. When a voltage is applied to the plate it contracts. The voltage is turned on and off so the material contracts and expands rapidly. A vibrational micro-injector needs only a force of 2.7 µN.
Chapter 12: Other applications
A micro-injector could be used in an array configuration so that multiple embryos or cells could be injected at once. Each cell would be self-assembled onto an adhesion pad in a micro-fluidic channel. After the injection the chamber would be flushed and the process repeated.
The same system could also sweep over any surface mapping its profile much like an atomic force microscope does.
Chapter 13: Conclusion
The MEMS micro-injector is a powerful new tool that minimizes injection damage. Micro-injectors have been used to measure the penetration force under various physiological conditions. Future designs will enter the nanometer scale giving scientists new and exciting capabilities.
Near Field Imaging
Chapter 1: Motivation
Importance of microscopes in biological and medical analyses. Microscopes are necessary to view small particles, eg. DNA, neurons. The higher the resolution, the better the resulting image. Lenses are limited because of the diffraction of light. Scientists are interested in viewing more molecular structures rather than cellular (big) structures. We want to overcome the resolution limit to see much smaller object.
Chapter 2: Near field Scanning optical microscope (NSOM)
To achieve higher resolution than the diffraction limit, we can miniaturize the light source. To view the surface of a sample, a small light source is scanned over the area of interest. If the light source is small, it is effective only within a small area, which is called near field. During scanning, the distance between the light source and the sample is very important. Need to constantly keep the sample within the near field of the light source. This method is called near field scanning optical microscopy (NSOM). It will give us an image of the surface of a sample. The light source is typically between 50 and 200 nm.
Chapter 3: Design
To make the light source approach the sample, a probe is necessary. Conventional NSOM utilizes a probe made of drawn optical fibers to have a very small aperture. However, this probe is manually prepared and assembled. Therefore, it is not suitable for large throughput applications. We utilize MEMS technology to create silicon based mechanical probes which is integrated with an LED.
Chapter 4: Fabrication
The NSOM probe is fabricated using conventional microfabrication techniques of silicon. An SOI (silicon insulater) wafer is patterned by wet etching to create sharp, flat probe tips. The tip is cut using a focused-ion beam to create two electrodes and a narrow gap to trap the LED. The gap size is 150nm. Using the electrostatic force, nanoparticles in toluene solution are trapped in the gap. Because a nanoparticle is composed of a small number of atoms, its energy level is quantized, and energy is released in the form of light. Therefore, these nanoparticles act as a point light source, or LED.
Chapter 5: Operation
As stated previously, we need to keep the sample within the near-field of the light source. An actuator induce vibrations on the NSOM probe attached to a tuning fork. The tuning fork senses the oscillations of the probe tip. The probe is moved closer to the surface of the sample. The atomic force from the surface of the sample dampens the oscillations. Therefore, we can tell how close the probe tip is to the surface. We can keep the probe tip with the light source within 5 nm of the surface.
Chapter 6: Imaging
If the probe is kept at a constant distance while scanning, we can obtain topographical images of a surface. When a light source comes within the near-field of a sample, the sample is excited by the light, resulting in photoluminescence. Photoluminescence is measured by a photodetector to produce an image The excitation occurs in a very localized area of the sample resulting in high resolution optical imaging. The advantage of using NSOM is that ability to correlate topographical and optical imaging.
Chapter 7: Other Applications
Because the probe is made using semiconductor fabrication techniques, arrays of probes can be generated for high throughput testing. This probe may be used to study the structure of various cells to examine their morphology, for example, to detect abnormal cells in a neuron cell culture.
Cancer Diagnostics
Chapter 1: Cancer, The Global Menace
Cancer is the second most common cause of death worldwide. According to Cancer Research UK, over 10 million new cases of cancer are diagnosed annually, and over 6.5 million deaths are reported. Over 60% of these deaths occur in developing or underdeveloped nations with limited access to effective cancer-screening technologies. In the United States, cancer accounts for more than half a millions deaths a year, almost one-fourths of all mortalities. Cancers of the lung, stomach, liver, bowels, and breast are the most common causes of death. The threat that cancer poses shows no signs of relenting in the near future. Cancer-related deaths are estimated to rise worldwide to 9 million by 2015, and 11.4 million by 2030.
Chapter 2: Portable, Early Detection
Despite these dire predictions, scientists have found that many cancers can be treated effectively if detected at an early stage in its progression. Studies have shown that survival chances can improve from less than 10% when detected after the cancer has spread to over 90% if detected when the cancer is still confined to a local region. However, most cancer patients live in underdeveloped countries, unable to afford advanced cancer screening technologies. Average annual healthcare spending per person in an African nation like Ethiopia is a paltry four dollars compared to over four thousand dollars in the United States. The need for a low-cost screening technique to aid cancer patients suffering in poor nations is paramount. An ideal screening system would provide accurate diagnosis in a single sitting, require minimal infrastructure, be battery-powered, and could be transported to rural villages to the patients in need.
Chapter 3: Visual Markers of Cancer
A good screening technique should look for key identifiers of cancer, and detect them with extremely high sensitivity and accuracy. Building a screening system then involves understanding these identifiers. 85% of all cancers originate in the epithelium, the outermost layer of skin that covers our entire body. The epithelium is usually around 200 micrometers thick, and the cells within the epithelium are around 10 micrometers in diameter. Studies have found that cancerous cells in the epithelium are characterized by their abnormal size and shape, and unusually large nucleus. These key markers of cancer can therefore be identified by studying images taken with a microscope at very high resolution.
Chapter 4: Confocal Microscopy
Confocal microscopy is one such optical imaging technique capable of resolving sub-cellular features in the epithelium. Invented in 1961 by Marvin Minsky at Harvard University, confocal microscopy uses a small pinhole to pick out light from only a small area of the sample, and block light coming from other out-of-focus areas of the sample under investigation. By moving the pinhole, or by moving the position of the sample, or by scanning the illuminating beam across the sample, a point-by-point image of the entire object can be reconstructed from the confocal microscopy system.
Chapter 5: In Vivo Detection
The standard method to examine a biological sample of a patient to test for diseases is to cut out a piece of the tissue to be examined from the patient, and place it under a microscope. This procedure is called biopsy. Sometimes, however, it may be difficult to obtain a biopsy from an internal organ, or the patient may be hesitant to undergo an expensive, time-consuming surgical procedure to test for a disease that may not exist at all. Ideally, we would like to obtain the required images without removing any tissue from the patient. This implies the use of a small endoscope that can be easily sent into the body to image the tissue inside the patient. An in vivo imaging technique of this type can dramatically reduce screening costs and time spent in diagnosing each patient.
Chapter 6: The Microscanner
When imaging the epithelium inside the patient for signs of cancer, it has been found that the best way to obtain a high-resolution image is to scan the illumination across the sample. A beam can be scanned across a sample by allowing it to fall on a mirror that can be rotated about two axes. However, since the mirror has to fit inside an endoscope and sent inside the body, it has to be extremely small. We have hence developed a “micro-scanner”. The microscanner is a mirror measuring 700 micrometers long and 500 micrometers across that can be rotated about two axes. By vibrating the mirror at a very high speed about one axis, and rotating it very slowly about the other axis, we obtain a raster scan pattern. The raster scan pattern is an efficient way to scan the beam of light across to obtain an image of the tissue.
Chapter 7: Vertical Comb Drives
We have designed tiny actuating systems that pull the mirror and make it rotate about the two axes. These systems are known as vertical comb drives. A vertical comb drive consists of two sets of combs made of conducting silicon – one comb, called the rotor comb, is attached to the mirror and placed higher than the other comb, called the stator comb. When a voltage is applied to the stator comb, a positive charge develops on its outer surface. These charges attract the negative charges in the rotor comb towards the stator comb. The attraction force is determined using Coulomb’s law for electrostatic force. Two sets of small silicon rods attached to the mirror, called torsion rods, ensure that the Coulomb force causes the mirror to rotate about the desired axes.
Chapter 8: Silicon Micromachining
The micromirror is fabricated by a process called silicon micromachining. The shapes of the stator combs are micromachined into a layer of silicon 30 micrometers thick on top of a silicon chip. Another silicon chip with a layer of silicon dioxide underneath is bonded on top of the first silicon chip, ground and polished to form a second silicon layer 30 micrometers thick. The silicon dioxide acts an insulating layer and prevents short-circuiting between the two conducting silicon layers. The features of the rotor combs, the micromirror, and torsion rods are defined in this second layer. The lower stator combs are then further trimmed to exactly match the position of the rotor combs. This automatic comb alignment procedure ensures stable operation of the microscanner. A layer of silver is coated on the mirror to improve its reflectivity to over 95%. Finally, tiny wires, one-fourth the diameter of a single strand of human hair, are attached to the silicon pieces to provide electrical connections to the vertical comb drives.
Chapter 9: Microscanner Performance
After fabrication, the microscanners are rigorously tested for reflectivity, mechanical robustness, reliability, scanning speed and angle, and power consumption. Precise, repeatable operation over a long period of time is required before the microscanners can be deployed in a clinical setting, or in the field. A large scanning angle allows imaging over a wider field of view, while fast scanning of the mirror allows us to capture video at a higher frame rate and of better quality. We carefully designed the microscanner using computer simulation tools to have adequate mirror size for good imaging, while maintaining large scanning angles, high resonant frequencies and low power consumption. These parameters are critical to building a high-resolution in vivo microscope that can be battery-powered.
Chapter 10: CancerScope
After testing, the microscanner is placed in a specially designed micro-electro-mechanical systems package fitted with flexible electronic systems and miniature optics for high-resolution imaging. The flexible electronics allow the package to be sent deep into the body while still maintaining electrical contact with the outside world. The miniature optical elements are fitted into ports specially designed to automatically align them to the micromirror. The entire package is connected to electrical ground to ensure patient safety at all times. The end tip of the endoscope is 4.7 millimeters in diameter while allows it unrestricted access to many of the typical sites of cancer in the human body.
Chapter 11: A Ray of Hope
Cancer screening technologies, like the one we are developing, can dramatically change the lives of millions of cancer patients around the world, and their families. The low-cost, portable operation of these advanced micro-electro-mechanical systems will provide even the poorest of cancer patients around the world access to the highest quality of point-of-care diagnostic techniques. Early detection of cancer allows chemotherapies to be much better targeted and effective, perhaps offering our friends a fighting chance, a ray of hope, in their battle against one’s the world’s greatest threats.
