Microcavity Lasers for Cancer Cell Detection

Microcavity Lasers for Cancer Cell Detection

ME 381

Introduction to MEMS

Final Paper

12/6/2002

Aaron Gin

Kathryn Mayes

Ryan McClintock

Will McBride

1)Project Summary

With the advancement of medicine, high-speed processing of biological cells has become an increasingly important tool to quickly diagnose and treat a wide variety of diseases and illnesses. To further improve the identification of cells, particularly cancerous ones, the authors propose a microfabricated cavity laser device that is capable of differentiating between individual cells. The analysis is performed using stimulated or pumped laser emission from the resonant optical cavity of the device. This light is partially transmitted through the individual cells in the resonant cavity and is then analyzed by a variety of hardware to determine the size, shape and other characteristics of the cell. This technique is similar to flow cytometry, in which cells with a fluorescent tag are placed in a suspension fluid, such as a buffered saline and streamed through the path of a laser beam to determine, for instance, the concentration of cancerous cells. The refractive index difference of the cell with respect to the empty cavity can slightly change the emission spectrum from the laser. This unique spectrum can give important information to scientists and physicians about the biological sample.

The authors suggest leveraging microfabrication techniques to create a portable, inexpensive and disposable analysis device. In particular, epitaxial growth using molecular beam epitaxy (MBE) or metal-organic chemical vapor deposition (MOCVD), plasma-enhanced chemical vapor deposition (PECVD) and reactive ion etching (RIE) will be used to realize the microcavity laser and its on-chip microfluidic channels.

The objectives of this proposed effort include:

- successful fabrication of the device

- achieving real-time cell analysis

- development of a useful consumer medical device

Upon completion of these objectives, this effort should develop a very compact cytometry device, seen in Figure 1, that is faster and more accurate than current systems, and which can be used during surgery for in situ cell analysis. This will give physicians and scientists an invaluable tool for biological cell processing and may lead to rapid progress in other characterization fields.

Figure 1: Photograph of actual microcavity laser device, approximately the size of a nickel. From the Sandia National Laboratory homepage;

2)Table of Contents

1)Project Summary

2)Table of Contents

3)Project Description

a)Introduction

i)Motivations and applications

ii)What is cancer?

iii)Who is at risk?

iv)How is cancer traditionally detected?

v)The need for instantaneous classification of cells

vi)The Bio-Cavity LASER concept

b)Technical Overview

i)Why use MEMS?

ii)Optically-pumped vertical cavity surface emitting semiconductor lasers (VCSELs)

iii)Output dependence on cell shape

iv)System Overview

c)Fabrication of the Microcavity Laser

4)Technical Paper Review

a)Miniaturized imaging system

i)Introduction

ii)Optical Design Considerations

iii)Optical Design of the Miniature Microscope Objective

iv)Assembly of Miniature Optical Systems

v)Fabrication of Micro-Optics

vi)Patterning With Binary Photomasks

vii)Patterning With Greyscale Photomasks

viii)Hybrid Glass Material

b)MEMS Microcavity Laser

i)Introduction

c)Conclusion and Discussion

5)Future Work

6)References Cited

7)Biographical Sketches

a)Ryan McClintock

i)Education

ii)Appointments

iii)Publications

iv)Synergistic Activities

v)Collaborators & other Affiliations

b)Kathryn Mayes

i)Education

ii)Appointments

iii)Publications

iv)Collaborators & other Affiliations

c)Aaron Gin

i)Education

ii)Appointments

iii)Publications

iv)Collaborators & other Affiliations

d)William McBride

i)Education

ii)Appointments

iii)Publications

iv)Collaborators & other Affiliations

3)Project Description

a)Introduction

i)Motivations and applications

This project seeks to develop a fast reliable way to identify cells as either cancerous or non-cancerous. Cancer is a major medical problem in modern society. This project will work both to develop diagnostic techniques that can compete with the current biopsy procedure and the testing for cancer specific antigens. However, the real benefit of this project lies in real time quantification of cells during surgical removal of a cancerous mass. Currently surgeons must rely on skill in interpreting diagnostic images, and familiarity with the body to know how much material is necessary to be removed during a surgical procedure. Our proposal seeks to develop a Bio-Cavity laser based system that can provide instant feedback to the surgeon based upon the number of cancerous cells detected in cellular material removed during a procedure. The Bio-Cavity laser is an extremely sensitive MEMS biased device that uses differences in optical density of normal and cancerous cells to differentiate between the two.

ii)What is cancer?

Humans start off as a single cell and throughout life it is necessary for cells to repeatedly divide in order to provide for growth. In addition, cells routinely become old and die naturally, and are thus replaced by newly formed cells. Cellular reproduction requires copying of the genetic material that describes the cell and its operation. There are inherent error-checking mechanisms, however, occasionally a mistake is made during reproduction of the generic material. Sometimes these errors can be fatal to the cell, in which case it merely dies, other times the body’s immune system is able to find and kill the defective cells. However, occasionally the genetic code that controls cell death and reproduction is damaged. This is what causes cancer. It can lead to uncontrolled cellular reproduction, and the body’s immune system cannot always intervene early enough, and thus a tumor develops. Figure 2below shows how an error during normal cell division can produce a cancerous cell (shown in yellow) that can ultimately lead to a tumor forming.

Figure 2. A. Normal cells, B. Cells reproduce to compensate for normal cell death, C. During cell reproduction a error is made in one of the cells (shown in yellow), D. This cell goes into over-drive reproducing uncontrollably, if the body's immune system cannot regain control the mass will continues to grow and go malignant and spread to other parts of the body. Image courtesy of Irish cancer society

iii)Who is at risk?

Despite the advances of modern medicine cancer poses a very real threat to humans. The American Cancer Society estimates that slightly fewer than 1 in 2 men and over 1 in 3 women will develop some form of cancer during their life. We have made significant medical advances in the treatment of cancer, however many people are still dying every year from various forms of cancer. Figure 3below shows average fatalities from cancer in various part of the body based upon data collected beginning in 1930.

Part of the problem lies in our current medical techniques. There are a variety of non-invasive techniques that target quickly multiplying cells and can reduce the size of a tumor, often to the point where it is no longer noticeable. However, even if surgery is elected we currently have no way of knowing for certain that all of the cancerous cells have successful been removed. It only takes one cancerous cell to begin a new cancerous growth. Typical we don’t think of people as being cured of cancer, but merely in remission; meaning there doesn’t appear to be any external signs of cancer but that we have no guarantee it will never resurface.

Figure 3. Average cancer rates by location in the body for males and females from 1930 through 1998. Image taken from the American Cancer Society’s Facts and Figures 2002 edition.

iv)How is cancer traditionally detected?

Trained personnel screen individuals for cancer by routine examination of the body, diagnostic imaging of cancer-prone regions, and testing for specific antigens produced by the body in its fight against certain specific forms of cancer. However these methods only provide cause for suspicion. In order to confirm that an abnormal growth is cancerous, a biopsy is necessary. This involves physically removing a sample of the suspect growth, and sending it to a laboratory for examination.

The most rudimentary analysis is to stain the cells and classify them under an optical microscope. Figure 4 shows two groups of cells, the one on the left is healthy, and the one on the right is cancerous. The disordered growth, larger number of cell currently involved in reproduction, and the odd shapes of the individual cells are the key factors analyzed to reach this conclusion. The problem is that this technique requires a highly trained eye; most doctors are not trained in cellular analysis, and thus must rely on an external laboratory to provide analysis. The inherent processing delays cost patients valuable time. Our proposed device will be simple to use, instantaneous, and reasonably priced such that it may one day be found in every doctor’s office.

Figure 4. Left) tissue sample collected from a normal male prostate. Right) tissue collected from a cancerous growth in a male prostate gland. Image courtesy of University of Michigan Cancer Center

The other more technically sophisticated analysis technique is flow cytometry. Flow cytometry is a powerful technique used to provide information on the chemical and structural makeup of a cell. Its power comes from chemical florescence; either florescence intrinsic to various cellular components, or the ability to tag specific cells with florescent dyes. It is also capable of measuring the scattering and transmission of a cell. Its many capabilities include the ability to differentiate cancerous from non-cancerous cells with a high degree of accuracy. This technique is problematic due to the fact that it is a macroscopic device relying on a focused laminar fluidic stream, probed by either multiple lasers or a tunable laser, and analyzed using a small array of lenses and detectors. Our proposed device sacrifices size and complexity by focusing only on cancer detection, and uses MEMS to simplify the fluidics and optics of the system. NASA and the American Cancer Society have reduced the size from that of about a pool table down to a tabletop unit, however the cost is still prohibitive.

The other major problem with both of these detection techniques is their requirement for a macroscopic sample. In order to collect a sufficient sample, patients must be subjected to a rather large needle that physically extracts a large number of cells from the center of the cancerous mass. This leaves a small hole in the surrounding tissue; it also disturbs the tumor, and can allow cancer cells from the tumor to migrate to other parts of the body. Because of the use of MEMS, our device requires only a few cells to produce results. This means a smaller needle is required, which helps minimize the trauma to the tumor and surrounding cells.

v)The need for instantaneous classification of cells

There currently exists no cost effective real-time method to reliably classify a cell as either cancerous or benign. The human eye is incapable of resolving individual cells, and thus reliably differentiating cancerous from non-cancerous cells. Yet this is exactly what surgeons are asked to do during a cancer removal procedure. Further complicating matters is the problem of analysis time: currently cancer identification requires some sample preparation before results can be extracted. Flow cytometers are not found in most operating rooms. Often times the patients has already been sewn back up before analysis is complete. This means that the surgeon has no idea how much material must be removed in order to eliminate all of the cancerous cells. Certain parts of the body are extremely sensitive to excessive removal, the most problematic being the brain. With real-time feedback providing the surgeon information about the nature of the cells as they are removed, a surgeon now knows exactly how much material to remove, and what is safe to leave behind. Our device will revolutionize cancer removal surgery by bringing accurate cost effective real-time cancer analysis into the modern operating room.

vi)The Bio-Cavity LASER concept

During surgery or a diagnostic procedure, fluid is extracted from the body. Figure 5below shows three possible sample sources: A.) a scalpel with integral suction feed directly into the Bio-Cavity laser device; B.) a miniature probe inserted into the region of interest for extraction of cells. C.) a traditional sample, collected either from biopsy of during a surgical procedure. The sample flows down a capillary tube where it is fed into the device. Within the device the dimensions are chosen such that cells travel single file while being analyzed. The Bio-cavity laser consists of two mirrors (red and pink) between which both the flow of cells (blue) and a laser gain medium (magenta, in the figure below) are placed. The cells are drawn through the device using a micro-fluidic pump. An external pump laser is fed into the device, and the resulting optical signal is collected using a fiber-optic cable. A miniature spectrometer and a laptop computer analyze the signal.

Figure 5. Schematic diagram of Bio-Cavity laser system with interfacing peripherals. Image taken from Sandia National Laboratory’s ( March 23, 2000 News Release.

The external laser pumps the optical gain medium. The gain medium is capable of supporting lasing of a variety of possible modes, within a narrow wavelength band. Depending upon the finesse of the cavity formed by the two mirrors, and the optical properties of the material inserted between them, the cavity will favor different modes, or wavelengths of lasing. Figure 6below shows three different possibilities depending upon the material within the active region. The support fluid (shown by the blue curve), the intercellular cytoplasm (shown by the green curve), and the nucleus (shown by the red curve) all support different lasing wavelengths. The wavelength separation between these peaks is very small (the colors red, green, and blue do not correspond the their respective visible wavelengths), typically on the order of a few nanometers.

Figure 6. Diagram outlining the operational principal of the Bio-Cavity laser. Image taken from Sandia National Laboratory’s ( March 23, 2000 News Release.

By analyzing the response due to the nucleus relative to that of the cytoplasm, the Bio-Cavity laser provides information on whether the cell is currently involved in cell division or not. Cells in the process of dividing have significantly more nuclear material than normal cells. Cancer is characterized by rapid, uncontrolled cell division. This means that the average cancer cell has far more nuclear matter than a normal healthy cell. In this way the Bio-Cavity laser provides direct feedback on the nature of the cells in its cavity, either cancerous or non-cancerous.

b)Technical Overview

i)Why use MEMS?

This system is best constructed using MEMS technology for several reasons. The main advantages revolve around portability, convenience, and cost effectiveness. Making the system small enough to be portable means its use is not restricted to a specialty room. It can be used in any operating room, in any hospital, or even out in the field. Its small size also allows it to be integrated with a scalpel so that as a surgeon cuts away at cancerous tissue areas, they can see in real time if the tissue they are scraping is cancerous or benign. The small size means that a smaller sample is needed for the analysis as well, making it less intrusive for the patient. Also, keeping the system compact saves money, not just in real-estate considerations, but also in manufacturing. The system can be manufactured in bulk, and possibly all components (excluding the analysis components) could be integrated on a single chip.

While using MEMS technology seems to simply make the system more user-friendly, there is also a technical reason that it needs to be on the scale of microns. The cavity of the laser needs to be approximately the width of a single cell under analysis, so that the results are conclusive. The following sections will clarify this issue by explaining in more detail how the laser functions.

ii)Optically-pumped vertical cavity surface emitting semiconductor lasers (VCSELs)

The microcavity laser used in this design is an optically pumped VCSEL. Semiconductor lasers emit coherent photons when electrons from the conduction band recombine with holes in the valence band. Since there is an energy gap between the conduction band and valence band in semiconductors in which electron energies are forbidden, the fall from conduction band to valence band gives off energy. In the case of a direct band gap, this energy is generally in the form of photons with frequency

(Eq. 1)

where ΔΕ is the difference in energy between the conduction band and valence band and h is planck’s constant. Figure 7 shows the basic structure of the VCSEL.

Figure 7. VCSEL structure.

The following figure demonstrates how optically pumped stimulated emission occurs. In order to have population inversion (more electrons in energy level E2 than in energy level E1), a third unstable energy level is necessary. In Fig 8a, the photons from the pump source impinge on the semiconductor with frequency