Microcavity Lasers for Cancer Cell Detection

ME 381 Introduction to MEMS

Final Paper

12/6/2002

Aaron Gin Kathryn Mayes

Ryan McClintock Will McBride

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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; www.sandia.gov.

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2) Table of Contents 1) Project Summary ......................................................................................................................... 2 2) Table of Contents ........................................................................................................................ 3 3) Project Description....................................................................................................................... 4

a) Introduction .......................................................................................................................... 4 i) Motivations and applications ........................................................................................ 4 ii) What is cancer? .......................................................................................................... 4 iii) Who is at risk?............................................................................................................ 5 iv) How is cancer traditionally detected?............................................................................ 5 v) The need for instantaneous classification of cells .......................................................... 6 vi) The Bio-Cavity LASER concept.................................................................................... 7

b) Technical Overview .............................................................................................................. 8 i) Why use MEMS?........................................................................................................ 8 ii) Optically-pumped vertical cavity surface emitting semiconductor lasers (VCSELs) .......... 8 iii) Output dependence on cell shape.............................................................................. 11 iv) System Overview...................................................................................................... 12

c) Fabrication of the Microcavity Laser..................................................................................... 12 4) Technical Paper Review............................................................................................................. 15

a) Miniaturized imaging system ............................................................................................... 15 i) Introduction .............................................................................................................. 15 ii) Optical Design Considerations ................................................................................... 16 iii) Optical Design of the Miniature Microscope Objective ................................................. 16 iv) Assembly of Miniature Optical Systems...................................................................... 17 v) Fabrication of Micro-Optics ........................................................................................ 19 vi) Patterning With Binary Photomasks ........................................................................... 20 vii) Patterning With Greyscale Photomasks...................................................................... 21 viii) Hybrid Glass Material ................................................................................................ 22

b) MEMS Microcavity Laser .................................................................................................... 22 i) Introduction .............................................................................................................. 23

c) Conclusion and Discussion ................................................................................................. 24 5) Future Work .............................................................................................................................. 24 6) References Cited ....................................................................................................................... 25 7) Biographical Sketches ................................................................................................................ 26

a) Ryan McClintock ................................................................................................................. 26 i) Education ................................................................................................................. 26 ii) Appointments ........................................................................................................... 26 iii) Publications .............................................................................................................. 26 iv) Synergistic Activities ................................................................................................. 26 v) Collaborators & other Affiliations ................................................................................ 26

b) Kathryn Mayes ................................................................................................................... 27 i) Education ................................................................................................................. 27 ii) Appointments ........................................................................................................... 27 iii) Publications .............................................................................................................. 27 iv) Collaborators & other Affiliations ................................................................................ 27

c) Aaron Gin .......................................................................................................................... 27 i) Education ................................................................................................................. 27 ii) Appointments ........................................................................................................... 27 iii) Publications .............................................................................................................. 27 iv) Collaborators & other Affiliations ................................................................................ 28

d) William McBride .................................................................................................................. 28 i) Education ................................................................................................................. 28 ii) Appointments ........................................................................................................... 28 iii) Publications .............................................................................................................. 28 iv) Collaborators & other Affiliations ................................................................................ 28

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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 2 below 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 www.cancer.ie

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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 3 below 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

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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 www.cancer.med.umich.edu

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

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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 5 below 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 (www.sandia.gov) 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 6 below 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.

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Figure 6. Diagram outlining the operational principal of the Bio-Cavity laser. Image taken from Sandia National Laboratory’s (www.sandia.gov) 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

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hE∆

=ν

(Eq. 1)

where ?? 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.

Glass, fused silica or sapphire substrate

Insulating material patterned for channel

Upper mirror: Alternating high and low refractive index dielectric material

Active layer: MQW

Lower mirror: Alternating low and high refractive index semiconductor materials

GaAs, InP or other substrate

VCSEL output emission

AIR

Input from pump laser

Glass, fused silica or sapphire substrate

Insulating material patterned for channel

Upper mirror: Alternating high and low refractive index dielectric material

Active layer: MQW

Lower mirror: Alternating low and high refractive index semiconductor materials

GaAs, InP or other substrate

VCSEL output emission

AIR

Input from pump laser

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

hEE 13 −

=ν,

(Eq. 2)

exciting the electrons in the valence band up to E3. In Fig 8b, the electrons in the unstable energy band of E3 quickly fall to E2, emitting energy in the form of incoherent photons of frequency

hEE 23 −

=ν.

(Eq. 3)

Figure 8c shows the electrons holding in E2 since it is a more stable energy level. This allows E2 to collect many electrons, resulting in a population inversion between E1 and E2. Finally, in Figure 8d a photon with frequency

hEE 12 −

=ν

(Eq. 4)

impinges on the sample stimulating the emission of several electrons in E2 which in turn each stimulate several other electrons in E2 for an avalanche effect emitting many coherent electrons with frequency ? = (?2-E1) / h.

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a b

dc

E1

E2

E3

E1

E2

E3

E1

E2

E3

E1

E2

E3

a b

dc

E1

E2

E3

E1

E2

E3

E1

E2

E3

E1

E2

E3

Figure 8. Optically pumped stimulated emission. Adapted from Kasap.

The active layer provides the structure where the photons are generated. It can be constructed of bulk GaAs or InGaAs with a thickness of 50-150 nm or a single quantum well (SQW) or multiple quantum wells (MQW). A MQW is the best choice for better efficiency. The quantum wells (QW) can have a thickness between 5 and 30 nm separated by higher band gap barrier layers with a thickness of up to 250 nm. Quantum wells increase the efficiency because they further restrict the allowed energies of the electrons to more specific energy levels by the small scale of the active layer thickness. The QW is able to trap and hold electrons in a small area better than bulk, enhancing population inversion. Figure 9 shows the energy band diagram for a MQW structure.

Active Layer Barrier LayerE(conduction band)

E(valence band)

E

Active Layer Barrier LayerE(conduction band)

E(valence band)

E

Figure 9. MQW band structure. Adapted from Kasap.

The upper and lower mirrors need to be highly reflective in order to trap the photons for lasing. Lasing occurs when the distance between the upper and lower mirrors is a multiple of the emitted quarter wavelength. The photons bounce back and forth between the mirrors gathering more and more coherent photons from further emission from the semiconductor until a threshold is reached and the laser beam is strong enough to emit light through the mirrors, at which point the output power is high. The mirror structures are Bragg reflectors: alternating layers of high and low index of refraction material so that at the interface between each high to low index material most of the light is reflected back. After several periods where most of the light is reflected at each interface, 95-99% of the original beam will be reflected back to the cavity. The thickness of each layer must be designed according to the equation:

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22211λ

=+ dndn

(Eq. 5)

where n1 is the refractive index of material 1, d1 is the thickness of material 1, n2 is the refractive index of material 2, d2 is the thickness of material 2 and ? is the emission wavelength. The top mirror must be made out of a dielectric material that will be transparent to the pump laser’s wavelength. The bottom layer is made out of 28.5 periods of alternating low index AlAs of thickness 620nm and high index Al( 0.2) Ga (0.8) As with a thickness of 715 nm for a wavelength of emission of 850 nm.

iii) Output dependence on cell shape When a sphere or cell or other small dielectric body enters the channel in the VCSEL, it slightly alters the effective length of the lasing cavity. This causes the laser to laze at a slightly different wavelength. The amount of change can determine the size of the body in the channel. For example, if the body in the channel is simply a dielectric sphere, the output dependence on size can be described as:

( )d

pLxxn

π

ξλ

2

00104

−

=∆

Eq. 6

Where ?? is wavelength shift, ? is a geometrical factor averaged over the spherical volume and has a value = 1, n is the refractive index of the sphere, L is the effective cavity length, p is the longitudinal mode index, xln is the nth 0 of the lth Hankel function and d is the sphere diameter. Figure 10 shows the spectrums of a spheres with d = 6 µm (bottom), 10 µm (middle) and 22 µm (top).

Figure 10. Spectrum of VCSEL with 3 different sized dielectric spheres in the channel. From Meissner, et al.

The incredibly small wavelength shift does not have a noticeable impact on the functionality of the VCSEL; that is, it will still lase even though the mirrors were designed for a different wavelength. The shift in cavity length, caused by slightly bending the light path through the sphere (or cell), directly causes the shift in output spectrum. Spheres of known diameter were used to demonstrate the reliability of the VCSEL’s wavelength shift depending on the size of the body in the cavity, but the goal is to tell the difference between cancerous cells and benign cells. Cancerous cells would have a larger nucleus and cytoplasm than benign cells;

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therefore they should emit a different spectrum. By being able to recognize the difference between a normal and abnormal cell spectrum, doctors will be able to tell if the cell is cancerous.

iv) System Overview

Figure 11. System overview. Adapted from Gourley.

The functions of the pump laser and the cavity have already been discussed above. The mirrors direct the pump laser beam to the first beam splitter, which reflects a majority of the beam towards the focusing lens to the cavity. The output of the VCSEL is then also focused with the lens, most of the beam is allowed to pass through the first beam splitter, and then it is divided into two analysis beams by the second beam splitter. The photodetector is for microscopic analysis and the spectrum analyzer will look at the output spectrum from the VCSEL to display any shift. The display computer receives the outputs from the photodetector and spectrometer to digitize, record, and display the information recovered from the cells; it should also be able to compare the recovered information with a look-up table for identifying identity, size, shape, variants, composition, etc and for providing one or more output signals to the cell processing means for subsequent processing of the cells.

c) Fabrication of the Microcavity Laser The fabrication process for the microcavity laser can be summed up in three basic steps: - Molecular Beam Epitaxy (MBE) or Metal-Organic Chemical Vapor Deposition (MOCVD) epilayer growth of lower Bragg mirror and laser gain region. - Machining of the substrate to create microfluidic channels and laser microcavity. - Wafer bonding to glass and top Bragg reflector.

Pump Laser

Photodetector Display

Spectrometer Beam Splitters

Mirrors

Focusing Lens

Cavity Analysis Region

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GaAs or InP Substrate

Glass or Pyrex

a)

b)

c)

d)

e)

f)

GaAs or InP SubstrateGaAs or InP Substrate

Glass or PyrexGlass or PyrexGlass or Pyrex

a)

b)

c)

d)

e)

f)

Figure 12: Schematic diagram of major fabrication steps for the microcavity laser.

A III-V semiconductor substrate material such as GaAs or InP (Figure 12a) is loaded into an epitaxial growth system (typically MBE or MOCVD) where the distributed Bragg reflector is first grown. These systems allow the user to precisely control the growth parameters such as gas flow or beam flux, substrate temperature and pressure. Additionally, many systems, especially the MBE, use in situ monitoring systems such as reflection high-energy electron diffraction (RHEED) for epilayer monitoring and infrared pyrometers for accurate temperature control. These reactors offer nanometer growth control and often single atomic layer roughness. This laser design uses a Bragg mirror that contains alternate layers of high and low refractive index materials such as AlGaAs and AlAs. Researchers use 28.5 periods of AlAs/Al0.2Ga0.8As layers for lasing wavelengths near 850 nm. Each layer is designed to be a quarter-wavelength thickness to maximize the reflectivity in the device. The mirror structure is applied using MBE or MOCVD as shown in Figure 12b. At this wavelength, the mirror has a reflectivity of 95-99%. The function of this mirror is to form one side of a resonant cavity between adjacent mirrors aligned parallel to each other. These mirrors act to ‘excite’ the gain media by reflecting photons back and forth through the cavity many times. The laser gain medium is grown on top of the Bragg mirror using MBE or MOCVD, incorporating multiple quantum wells comprised of InGaAs and GaAs with well thickness around 5 nm. The schematic step can be seen in Figure 12c. The total thickness of the gain medium is about 150 nm. The use of multiple quantum wells helps to increase the overall cavity gain. The lasing wavelength is chosen such that the light can be at least partially transmitted through the cells under test. Wavelengths between 600 and 1500 nm are desirable for this reason. This initial growth process imitates the first few fabrication steps of Vertical Cavity Surface-Emitting Lasers or VCSELs.

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After epitaxial growth, plasma enhanced chemical vapor deposition or PECVD is used to deposit an insulator such as silicon dioxide or silicon nitride as shown in Figure 12d. The PECVD system generates a radio frequency (RF) plasma to increase the energy of the component species. This allows lower growth temperatures and better control over the deposition conditions. The insulating layer deposited can vary in thickness depending on the cells under study. The thickness is generally determined such that one cell has just enough space to pass through the cavity. Any additional space could allow multiple cells to pass the analysis region at the same time or may introduce other measurement problems. Typically, 1 µm is deposited and is patterned using standard photolithography and chrome mask. Next, the pattern is transferred using a buffered oxide etch (BOE) such as buffered HF for silicon dioxide, or SF6 (sulfur hexaflouride) to remove Si3N4. This pattern transfer creates both the microfluidic channels and the laser microcavity as seen in Figure 12e. The channels introduce the cells into the microcavity and also can serve as holding reservoirs or mixing chambers for various fluids. Finally, these channels also are used to move fluids onto and off of the analysis chip. Resist stripper is used to remove the residual photoresist from the oxide or silicon nitride layer. A Bragg mirror is then grown on glass or pyrex. This reflector is generally comprised of several alternating dielectric layers such as SiO2 or AlO2. Again, the layer thicknesses are matched to the quarter wavelength of the lasing wavelength in order to provide the best reflectivity. These materials are also chosen to be transparent to the pump laser signal, which illuminates the laser gain region through the top substrate. This Bragg mirror and glass substrate is then bonded to the device ‘bottom’ by epoxy or pressure fit with a liquid-tight o-ring. This can be seen in Figure 12f. Finally, hoses for fluids can be attached to bring the sample cells into the analysis chamber. The completed device can be seen in Figure 13, and the actual device is pictured in Figure 14.

Analysis Region

Flush Channel Processing Reservoir

Outlet Channel

Staging Area

Valves

Inlet Channel

Reagent Reservoir

Processing Reservoir

1 1

2 2

Adapted from P.L. Gourley, U.S. Pat. #5793485

Laser excitation pulse

Analysis Region

Flush Channel Processing Reservoir

Outlet Channel

Staging Area

Valves

Inlet Channel

Reagent Reservoir

Processing Reservoir

1 1

2 2

Analysis Region

Flush Channel Processing Reservoir

Outlet Channel

Staging Area

Valves

Inlet Channel

Reagent Reservoir

Processing Reservoir

1 1

2 2

Adapted from P.L. Gourley, U.S. Pat. #5793485

Laser excitation pulse

Figure 13: Schematic diagram of the micro-cavity laser incorporating fluidic channels.

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Figure 14: Photograph of actual microcavity laser device. From www.sandia.gov website.

4) Technical Paper Review

a) Miniaturized imaging system The work of Descour et. al. on the development of a miniaturized imaging system for the detection of pre-cancer is of particular interest to members in the MEMS research community. The detection of pre-cancer is achieved using the miniaturized optical bench (MOB). The MOB is a miniature, optical-sectioning, fluorescence microscope and is an apt application of MEMS technology to address a pressing need of the medical community. The process flow of the manufacture of the individual optical components will be addressed in detail in later sections of the following text. The miniature microscope is made using lithographic means and is assembled using a bulk micromachined silicon microoptical table. Descour et. al. have assembled these devices using silicon spring equipped mounting slots, which will also be addressed in detail later in the text.

i) Introduction

The detection of pre-cancerous lesions is the best way to ensure patient survival. There is a pressing need for cost-effective and sensitive means to screen for pre-cancer. The means by which Descour et. al. intend to detect pre-cancer is based in the optical detection of the morphological and biochemical changes that occur. These changes may include an increase in nuclear size, an increased nuclear-to-cytoplasmic ratio, hyperchromia, pleomorphism, angiogenesis, and increased metabolic rate. These changes are currently only detectable by invasive means. This tool may be used to augment the visual-detection methods of bronchoscopy and colonoscopy, and provide information about the microscopic and biochemical processes that are occurring due to pre cancer. The MOB that is developed here may be used to develop battery-powered, pen-sized microscopes with the capability to function in multiple-modes. These multi-modal miniaturized microscopes (4M’s) could allow optical-sectioning, 3-D spectral fluorescence microscopy, and reflectance imaging. The 4Ms would allow screening for a variety of different cancers. A suggested design for the 4M is shown in Figure 15(a) below. The schematic for the design is shown in Figure 15(b) below.

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(a) (b)

Figure 15. (a) The fully incorporated pen-sized device (b) A view of the components mounted to the MOB, the device is approximately 9mm × 5mm × 3mm.

The device shown in Figure 15 is an optical-sectioning, fluorescence microscope. The collector mirror gathers the input light, from either an onboard laser, or which may be piped in via an optical fiber. This light is then imaged upon the aperture stop of the miniaturized microscope objective. This collected light then passes through a scanning grating, a folding-flat mirror, and a dichroic beam-splitter before being projected upon the tissue that is being examined. The microscope objective is shown in the figure and consists of three lithographically processed lenses, a folding-flat mirror, and a plano-convex objective. These components are all mounted on the MOB, which is micromachined silicon and measures 9mmx5mm.

ii) Optical Design Considerations Now that the basic components of the instrument have been introduced, some of the optical design considerations that were used by the authors will be examined. The miniaturization of a microscope is in some ways very similar and in others very different from the design of a conventional instrument. A conventional optical microscope consists of two basic components, the microscope objective, and an eyepiece. The image sensor in the case of a conventional microscope is the human eye. The biggest consideration when miniaturizing a microscope is to minimize the throw, the distance between the object and the image plane of the instrument. In the 4M approach, the image will be recorded electronically rather than optically. A comfortable viewing distance is maintained e.g. 250mm. The field of view of the microscope is given as 250µm on a side, and the size of the image at the image sensor is 1000µm on a side. The authors expect that the resolution of the device, as currently configured, to be limited by the detector, rather than the diffraction-limit.

iii) Optical Design of the Miniature Microscope Objective A series of microscope-objectives were designed featuring a high numerical aperture (NA) and low transverse magnification. The low transverse magnification allows the microscope objective itself to be small, whereas the high NA yields high spatial resolution. Distances between the device and the tissue sample are 6-8mm. A schematic of the miniature microscope objective is shown in Figure 16below.

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Figure 16. The optical design of the miniaturized microscope objective with a field of view of 300µm. This is the objective used in the 4M device shown in Figure 15 above.

The design has a plano-convex lens that is followed by a folding mirror and three lithographically printed lenses. The objective is designed to operate while immersed in water. The optical elements had a pitch and yaw tolerance of ± 0.460 and a translational tolerance of ± 10µm. These are the tolerances that must be met in order for the devices resolution to be diffraction limited with regard to optical performance.

iv) Assembly of Miniature Optical Systems

The miniaturization of this optical system necessitates a means to assemble the lenses and other components onto the MOB. The MOB is a design that eliminates the need for alignment (as is the case with a macroscopic optical table) and has assembly errors that are smaller than those quoted in the previous section, which allows for very few assembly errors. The use of silicon springs and slots allow this high precision alignment to be feasible, it is the high precision of optical lithography that is the enabling technology. The cross section of each slot contains a silicon spring, as shown in Figure 17 and 18 below.

Figure 17. The MOT substrate and optical element assembly. Note the silicon spring and v-shaped channel.

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Figure 18. The silicon-spring displacement and stress in the normal direction. The silicon spring is shown in its final position. Note that the maximum stress on the spring is shown and is less

than the yield stress of silicon. The optical element is 150µm thick.

Each mounting slot contains a silicon spring, as well as a v-shaped guide slot that matches complimentary positioning features in the optical element that resides in the slot. The spring acts to press the positioning features that are present in the channel and on the optical element into alignment. The depth of insertion is limited by stops that are printed on the optical element. The authors hope to have a final alignment accuracy of ± 2µm and have a rotational accuracy of ± 0.5mrad using these alignment techniques. The optical element must be inserted into the slot and translated under the silicon spring. A top view of the test features follows in Figure 19.

(a)

(b)

Figure 19. Positioning features (a) Perspective view of a partially inserted 150µm thick glass substrate. (b) Shown in top view note the 50µm wide positioning feature, the so called v-groove.

The narrowest part of the silicon spring is 40µm wide.

The authors fabricated a variety of silicon-spring designs with widths from 30-80µm. The corresponding force range for these silicon springs was established to be 0.0175-0.116N. Figure 19 shows the experimental results for test elements that had been coated with a hybrid glass and printed with the

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alignment features using a binary-photomask. The positioning features have a 35x35µm cross-section and are 50µm wide. The rectangular alignment features fit into the v-shaped groove. This results in positioning accuracy as shown in table 1 below.

Table 1: The measured positioning accuracies of the test elements shown in Figure 19 above.

The pitch rotation given in Table 1 matches the tolerance calculated by the authors for the microscope objective shown in Figure 19 above. The measured yaw rotations are small compared to the values given at the end of the preceding section. The translation was measured using high magnification optical microscope images, and the authors state that improvements in positioning accuracy may be made by using the intended circular cross-section positioning features rather than the rectangular ones used by the authors in this study. The authors need to further examine the assembly issues addressed here, although a zero-alignment procedure for the assembly of the 4M device is feasible. Figure 20 below shows an assembled and fabricated device, as shown in the design schematic of Figure 15 above.

(a) (b) (c)

Figure 20. A miniature microscope objective. (a) Schematic to scale for NA=0.4, m=4 microscope objective. (b) Silicon MOT micromachined substrate with objective lens aperture, silicon springs,

and mounting slots. (c) Partially assembled miniaturized microscope objective.

v) Fabrication of Micro-Optics The micro-optical, and opto-mechanical components are fabricated based upon the simultaneous printing using lithography and the hybrid sol-gel method. This method has the advantage over conventional fabrication techniques, which have multiple steps, as the method used by the authors is single step. The hybrid sol-gel technique also has the potential to improve the surface quality of the elements and speeds up the fabrication of thick structures.

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The authors demonstrated the fabrication of micro-lens arrays with thicknesses up to 100µm thick. Recent experiments using binary tone photomasks demonstrate structures with thicknesses of greater than 100µm with root mean square surface roughness of 10-20nm as shown in Figure 21 below.

vi) Patterning With Binary Photomasks When using a binary photomask with a mercury UV lamp to expose the fabrication of binary optical elements may be achieved. Figure 21below shows an early version of the diffractive lens elements and show the alignment features already discussed above.

Figure 21. Lithographically patterned opto-mechanical structures patterned using binary photomasks.

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Figure 22. Lithographically patterned optical elements. (a) A micro-optical element patterned in hybrid glass material on a 150µm thick glass substrate. The hybrid material is 17.8µm thick. (b) A more recent test element with the more recent protruding positioning features and with a hybrid

material thickness of 34µm.

Figure 22 shows current using binary photomask patterning of hybrid glass. Thicknesses of greater than 100µm have been achieved. Figure 22(a) shows a 1000µm diameter cylindrical test structure patterned to an average height of 110µm, whereas the authors demonstrate another structure in Figure 22(b) with an average height of 118µm. The plot in Figure 23(a) is the height profile of Figure 23(b) along the line shown.

vii) Patterning With Greyscale Photomasks

The fabrication of lenslets with 3D structure requires that one use greyscale photomasks, which is what the authors used to decrease the degree of polymerization of the hybrid glass as one moves outward from the center of the lenslet. Figure 23, which follows, shows the topography of a segment of a lenslet array.

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Figure 23. Segment of a lenslet array patterned in hybrid glass using a greyscale photomask (a) Lenslet in isometric view. (b) A diagonal profile of the surface shown in (a).

This means of fabricating convex lenses shows a patterned depth of 59µm. The authors measured the rms roughness of the lenslet in Figure 23 to be 10-45nm. The use of greyscale photomasks allows freedom in the design of optical surfaces that need not be spherical.

viii) Hybrid Glass Material The hybrid glass material used by the authors in this study is an inorganic base matrix that is prepared by hydrolosis and condensation of alkoxysilanes. The inorganic base matrix is provided with side chains containing terminal carbon double bonds that allow the material to photopolymerize. Acrylate monomers may be added to the material to increase crosslinking density. The viscosity of the prepolymer needs to be high to make structures with high aspect ratios. Also, the material needs to adhere well to the substrate. An additional consideration is that the polymer should not shrink excessively during the baking step or adhesion problems will result. The hybrid glass material has an index of refraction of 1.53 at 632.8nm and a rms roughness value of 10-45nm. The transmission of a 150µm thick film of this hybrid glass material deposited on a 1.1mm thick glass substrate is greater than 97% at wavelengths of 450-1600nm.

b) MEMS Microcavity Laser Gourley et. al. have a different approach to a very similar problem. This group of researchers approaches the problem of cell differentiation (which is one of the means to detect pre-cancerous lesions) by making a MEMS microcavity laser. The microcavity laser can analyze living or fixed cells from humans, and is

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smaller, less expensive, has a higher diagnostic yield, and has the potential to offer better point-of-care delivery to the patient. Cells are used in this device as internal optical guides in a semiconductor microcavity.

i) Introduction Gourley et. al. use a microcavity laser that operates at resonant frequencies that are based upon the dielectric properties of the cell. Cells may be gathered by scraping, from body fluids, or by any of a number of other means. The cell acts as a component of the system and superimposes information about it’s biochemical state, as well as it’s structure, onto the emitted laser light, this light may then be piped, via fiber optic, to a high speed computer for analysis. A schematic of the device follows in Figure 24. High-resolution spectrometry allows the authors to resolve the emitted light into narrow spectral peaks. The spacing and intensity of these peaks are characteristic fingerprints for each cell, which the authors state could readily detect sickle-cell enemia. A picture of this device follows in Figure 25. This rapid and accurate means of differentiating between different cells places his technology in competition with the 4M device described in the previous sections, which I will comparatively discuss in the closing conclusion and summary section.

Figure 24. A schematic of the device in the upper left hand corner, and a white blood cell, surrounded by red blood cells in the lower right hand corner.

Conventional means of examining cell morphology, like cell staining, are slow, labor intensive, and are often incorrect. The biocavity laser has won a number of government awards and the concept of the biocavity laser is being currently developed into a “nanolaser biochip” that evaluates tumor cells by quantifying their protein content. The authors report that, initially at least, only a few hundred cells are require to detect abnormal growth. This ability to detect cancer with such a small tissue sample is crucial for resecting a tumor margin or grading highly localized tumor malignancy.

Figure 25. Compact spectrometer reads microlaser output.

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Additional applications for this device include the monitoring of cells during drug testing, in real time. Also, important is the high-speed detection and/or eradication of rare cells within a population. Additionally, higher cell identification rates may be achieved by this device. Gourley et. al. have shown that the biocavity laser has the ability to probe the human immune system, characterize genetic disorders, and distinguish between normal and cancerous cells. Taking advantage of the lasers speed and efficiency rare events, such as dying or infected cells may be used as a part cellular analysis. Precancerous cells, as well as blood cancers may be detected. The authors also state that they can mass produce their devices and provide a 10-100 times decrease in cost, which is another major advantage of MEMS over conventional technology.

c) Conclusion and Discussion Both of the MEMS based devices, developed by Gourley and Descour, have the potential to drastically change the way we as patients interact with doctors. Screening for pre-cancer, cancer and a host of other medical maladies could involve a relatively inexpensive, fast, and painless way to interact with the medical community. Both of these devices are relatively compact and, when mass-produced, promise to be less expensive than current means of medical screening. Descour’s device seems particularly well suited to look for cervical, colonic, and oral cancers. Essentially, Descour’s device, the 4M is particularly suited to looking at cells on surfaces, and determining their health. The multiple modality of Descours device could lend it an advantage, inasmuch as it is a more flexible tool, allowing both image and spectral information to be gathered and analyzed. On the other hand, Gourley’s device is particularly effective for looking at cells in a liquid, for example, blood, or any other body fluid. Each of these devices seem to me, at least while in the developmental stages, to have a medium within which they are particularly effective. The possibility exists that these seemingly complimentary technologies could be in direct market competition, although I suspect that the strengths of each will restrict them to effective pre-cancerous screening of different types.

5) Future Work The Bio-Cavity laser concept is fairly robust, however there are several distinct areas that need further research before this device can go into commercial production. One of the largest challenges is interfacing the Bio-MEMS device with the outside world. Commercially viable ways to reliably route the fluids into and out of the chip do not exist at this time. The micro-fluidics is the most complicated part of this device functionally. Further problems arise from biocompatibility issues. Certain enzymes may be able to actually attack the Bio-Cavity laser. This can reduce the reflectivity of the mirrors and lead to reduced finesse of the optical cavity, thereby hindering the sensitivity of the device. It is also possible that certain cells may react when exposed the semiconductor active region and dielectric mirrors. The Bio-Cavity laser is designed to image cells sequentially as they travel through the microfluidic channels. If the cells clot this can clog the mechanism. Similarly, if the cells rupture, the interpretation of the results can become more difficult. At this stage the device needs to be tested with real cell collected during an actual surgery. Currently the device has shown good reliability in differentiating between cancerous and non-cancerous cultured cells. However during an actual procedure a great variety of cells will be sent to the device and detection algorithms need to be developed for real-world cellular samples. The easiest way to achieve this would be through collaboration with practicing surgeons. However, it may be necessary to start with a cancer model in another species.

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6) References Cited P.L. Gourley, J.D. Cox, J.K. Hendricks, A.E. McDonald G.C. Copeland, D.Y. Sasaki, M. Curry, and S.L. Skirboll, “Semiconductor Microcavity Laser Spectroscopy of Intracellular Protein in Human Cancer Cells” Proc. SPIE, 4265, 113-124 (2001). T. French, P.L. Gourley, and A.E. McDonald, “Optical properties of fluids in microfabricated channels” Proc. SPIE, 2978, 123-128 (1997). P.L. Gourley and A.E. McDonald, “Semiconductor microlasers with intracavity microfluidics for biomedical applications” Proc. SPIE, 2978, 186-196 (1997). M.F. Gourley and P.L. Gourley, “Integration of Electro-Optical Mechanical Systems and Medicine: Where are we and Where can we go?” Proc. SPIE, 2978, 197-204 (1997). Paul L. Gourley, “Resonant-cavity apparatus for cytometry or particle analysis” U.S. Patent No. 5793485, 36 pp. (1998). American Cancer Society. Facts and Figures 2002 NASA, Cancer Detection Device, SpinOff (1998) (http://www.sti.nasa.gov/tto/index.html) S.O. Kasap, Optoelectronics and Photonics: Principles and Practices, Prentice Hall, Upper Saddle River, NJ, 2001 K. E. Meissner, P. L. Gourley, T. M. Brennan, B. E. Hammons, and A. E. McDonald, “Intracavity spectroscopy in vertical cavity surface-emitting lasers for micro-optical-mechanical systems,” Applied Physics Letters, vol 69 (11), 9 Sept. 1996

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7) Biographical Sketches

a) Ryan McClintock i) Education Undergraduate: B.S E.E. Northwestern University (2001) Graduate: Anticipated Ph. D. Northwestern University (200?)

ii) Appointments Research Assistant, Center for Quantum Devices, Northwestern University March 1999 – Present National Defense Science and Engineering Graduate Fellow, August 2001 - present

iii) Publications Relevant: None Other Significant: A. Yasan, R. McClintock, K. Mayes, S. R. Darvish, H. Zhang, P. Kung and M. Razeghi. Comparison of ultraviolet light-emitting diodes with peak emission at 340 nm grown on GaN substrate and sapphire. Applied Physics Letters, 81 (12) 16 September 2002, p. 2151-2153 A. Yasan, R. McClintock, K. Mayes, S.R. Darvish, P. Kung, and M. Razeghi. Top-emission ultraviolet light-emitting diodes with peak emission at 280 nm. Applied Physics Letters, 81 (5), 29 July 2002, p. 801-802 A. Yasan, R. McClintock, S. R. Darvish, Z. Lin, K. Mi, P. Kung, and M. Razeghi. Characteristics of high-quality p-type AlxGa1-xN/GaN superlattices. Applied Physics Letters, 80 (12), 25 March 2001, p. 2108-2110 P. Sandvik, K. Mi, F. Shahedipour, R. McClintock, A. Yasan., P. Kung, M. Razeghi. AlxGa1-xN for solar-blind UV detectors. Journal of Crystal Growth, 231 (3), Oct. 2001, p. 366-370 R. McClintock, P. Sandvik, K. Mi, F. Shahedipour, A. Yasan, C. Jelen, P. Kung, M. Razeghi. AlxGa1-xN materials and device technology for solar blind ultraviolet photodetector applications. Proceedings of SPIE, 4288, June 2001 , p. 219-229

iv) Synergistic Activities Worked closely with several undergraduate students to provide them with hand-on experience conducting academic research at the Center for Quantum Devices. Collaborated with Northwestern University Professors to provide students taking introductory solid-state courses with hands-on demonstrations of semiconductor characterization equipment.

v) Collaborators & other Affiliations Collaborators Aaron Gin Kathryn Mayes William McBride Graduate and Postdoctoral Advisors Manijeh Razeghi

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b) Kathryn Mayes i) Education Undergraduate: B.S E.E. Northwestern University (2002) Graduate: Anticipated Ph. D. Northwestern University (200?)

ii) Appointments Research Assistant, Center for Quantum Devices, Northwestern University April 2002 – Present Walter P. Murphy Graduate Fellow, August 2002 - present

iii) Publications

Relevant: None Other Significant: A. Yasan, R. McClintock, K. Mayes, S. R. Darvish, H. Zhang, P. Kung and M. Razeghi. Comparison of ultraviolet light-emitting diodes with peak emission at 340 nm grown on GaN substrate and sapphire. Applied Physics Letters, 81 (12) 16 September 2002, p. 2151-2153 A. Yasan, R. McClintock, K. Mayes, S.R. Darvish, P. Kung, and M. Razeghi. Top-emission ultraviolet light-emitting diodes with peak emission at 280 nm. Applied Physics Letters, 81 (5), 29 July 2002, p. 801-802

iv) Collaborators & other Affiliations Collaborators Aaron Gin Ryan McClintock William McBride Graduate and Postdoctoral Advisors Manijeh Razeghi

c) Aaron Gin i) Education Undergraduate: B.S E.E. Valparaiso University (1999) Graduate: M.S.E.E. Stanford University (2001) Graduate: Anticipated Ph. D. Northwestern University (200?)

ii) Appointments

Research Assistant, Microstructures Group, under Dr. Fabian Pease and Dr. Calvin Quate, Stanford University 1999-2001 Research Assistant, Center for Quantum Devices, Northwestern University 2001 – Present Tau Beta Pi Graduate Fellow 1999-2000 National Science Foundation Graduate Fellow, 2000 - present

iii) Publications

Relevant: None Other Significant: Y. Wei, A. Gin, M. Razeghi, G.J. Brown, “Type II InAs/GaSb superlattice photovoltaic detectors with cutoff wavelength approaching 30 µm”, Appl. Phys. Lett. 81, 3675 (2002).

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M. Razeghi, Y. Wei, A. Gin, G. J. Brown, D. K. Johnstone, “Type-II InAs/GaSb superlattices and detectors with ?c>18µm”, Proc. SPIE 4650, 111 (2002). M. Razeghi, Y. Wei, A. Gin, G. J. Brown, “Quantum dots of InAs/GaSb type II superlattice for infrared sensing”, Mat. Res. Soc. Symp. Proc. 692, 99 (2002). Y. Wei, A. Gin, M. Razeghi, G. J. Brown, “Advanced InAs/GaSb superlattice photovoltaic detectors for very long wavelength infrared applications”, Appl. Phys. Lett. 80, 3262 (2002). A. Gin, P. D. Tougaw, S. Williams, “An alternative geometry for quantum-dot cellular automata”, J. Appl. Phys. 85, 8281 (1999). A. Gin, S. Williams, H. Meng, P. D. Tougaw, “Hierarchical design of quantum-dot cellular automata devices”, J. Appl. Phys. 85, 3713 (1999).

iv) Collaborators & other Affiliations Collaborators Kathryn Mayes Ryan McClintock William McBride Graduate and Postdoctoral Advisors Manijeh Razeghi

d) William McBride i) Education Undergraduate: B.S. Physics The College of William and Mary (2000) Undergraduate: B.S. Chemistry The College of William and Mary (2000) Graduate: Anticipated Ph.D. Northwestern University (200?)

ii) Appointments Research Assistant, Nanoscience and Technology Lab, Northwestern University August 2002 – Present Walter P. Murphy Graduate Fellow, August 2001 – August 2002

iii) Publications

Relevant: None Other Significant: None

iv) Collaborators & other Affiliations

Collaborators Aaron Gin Ryan McClintock Kathryn Mayes Graduate and Postdoctoral Advisors Rod Ruoff