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Harnessing Light: Optical Science and Engineering for the 21st Century 2 Optics in Health Care and the Life Sciences Optics has affected the lives of most Americans by changing the practice of medicine and offering new approaches to major health problems, such as the treatment of heart disease, cancer, kidney stones, knee injuries, and eye diseases. The use of optics and fiber optics has led to less invasive ways of treating disease by replacing open surgery with minimally invasive therapies. The basic research in biology that leads to new insights into the treatment of disease has benefited from technical advances ranging from optical methods of gene sequencing to new and more precise microscopies. This broad use of optical techniques has led to new approaches to biological research problems, new methods of medical diagnosis, and new ways to treat diseases. Tools developed for use in research have evolved into tools for patient treatment, and new and increasingly sophisticated research apparatus continues to emerge, improving our ability to study and control basic biological processes. It is the intent of this chapter to show how optics and lasers have changed the practice of medicine in ways that most readers have experienced, either directly or through a family member, and to give some view of how optical science may affect the health care of the future. In addition, the reader will have a better sense of how optics is involved in health care technologies used for applications as diverse as the determination of viral load in HIV and, potentially, the monitoring of blood glucose levels in diabetics. The material in this chapter is organized into three main topics: (1) surgery and medicine, (2) biology, and (3) biotechnology. The chapter concentrates on revolutionary developments, ones that have led to new techniques for research, diagnosis, or treatment or that could do so in the future. It concludes with some general remarks on health care
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Harnessing Light: Optical Science and Engineering for the 21st Century and the life sciences as a whole, highlighting the key challenges and opportunities that the field faces and making some recommendations to government, academia, and the private sector. Surgery and Medicine Optics has enabled laser surgery, optical diagnostic techniques, and visualization of the body's interior (see Figures 2.1 and 2.2 and Boxes 2.1 and 2.2). Although the applications of optics to surgery and medicine have increased rapidly since the invention of the laser in 1960, a number of optical techniques were used before that time. The development of rigid and flexible endoscopes—devices that allow the inside of canals (e.g., blood vessels) and hollow organs (e.g., the colon) to be viewed—is discussed in some detail elsewhere (Katzir, 1993). A number of rigid endoscopes were used in the nineteenth century, and the first flexible medical endoscope using optical fibers was demonstrated in 1959. It is worth noting that the use of microscopes by pathologists to examine tissue in order to diagnose disease was a well-established medical application of optics long before the era of the laser. The microscope is still the essential tool of the modern pathologist, although it has been made optically more advanced by the advent of computer-designed lenses and high-quality antireflective coatings. Some clinical specialties use specially modified microscopes. Ophthalmologists use a modified microscope, called a slit lamp, to project a slit-like beam of light into the eye to detect scattering objects within the cornea and lens. Advances in microscopy continue and include efforts to automate microscopy to allow initial screening for disease and infection. Arguably the most extensive use of optics in health care is in the fabrication of eyeglass frames, lenses, and contact lenses. This market was estimated at $13.2 billion in 1994 and consists of the 145 million people—55% of the total population—who wear corrective lenses (American Optometric Association, 1996). The ophthalmic market has evolved, with a variety of safe and light plastic lenses FIGURE 2.1 A flexible gastroscope, used to examine the inner surface of the stomach, together with a view of the distal (insertion) end showing working channels for tools. The image of part of a dollar bill, taken using a flexible colonoscope with a charge-coupled device (CCD) camera at the distal end, illustrates the excellent resolution available. (Courtesy of Olympus America, Inc., and N. Nishioka, Massachusetts General Hospital.)
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Harnessing Light: Optical Science and Engineering for the 21st Century FIGURE 2.2 Schematic diagram of an arthroscope, a rigid viewing scope commonly used for knee surgery. A variety of surgical tools can be passed through the working channels of the scope. (Courtesy of T Narashima, Scientist/Imagemakers.) increasingly replacing glass and with continued growth in the use of antireflection and ultraviolet (UV) blocking coatings. An additional change is the move from bifocal and trifocals with discrete zones to progressive lenses where the refractive correction varies smoothly from the bottom of the lens. Many manufacturing changes for ophthalmic optics are being implemented to enable these advanced features to be delivered to the consumer on demand (in an hour). These evolutionary developments are important but are outside the main interest of this report and are not discussed in more detail. Introduction of Lasers The medical potential of the laser has been explored almost from the invention of the ruby laser in 1960. These initial experiments were often of the ''point-and-shoot" variety, unguided by an understanding of the mechanisms by which the laser interacted with tissue or of ways to optimize these interactions. Ophthalmology was the specialty that adapted and incorporated laser techniques into clinical practice most rapidly, in large part because the interior of the eye was optically accessible (Krauss and Puliafito, 1995). By the end of the 1960s, some understanding of the mechanisms by which the laser interacts with the retina had been obtained, with both thermal and mechanical effects identified. BOX 2.1 TELEMEDICINE Telemedicine has the potential of bringing access to medical specialists to remote communities in the United States such as Indian reservations, to underserved communities in the United States, and to the entire world. The use of high-speed communications systems to transfer medical images, such as x-ray radiographs and optical micrographs of histology specimens, has been demonstrated at a number of sites. One major East Coast hospital regularly receives and reads radiographs from Saudi Arabia, returning reports within the same day. The use of teleconferencing systems to allow medical consultations involving patients who may be thousands of miles from physician consultants is also being studied in pilot projects. The technology underlying these systems is discussed in more detail in Chapter 1; it includes the development of fiber-optic communications networks and image processing and computational schemes allowing image compression. However numerous financial and legal issues must be clarified, including the malpractice aspects of teleconsultations. Several groups are developing CCD arrays for the detection of x rays used in medical imaging. When commercialized, such devices will provide x-ray image information directly in digital form, avoiding the need to scan and digitize conventional x-ray film, and will facilitate the transport and storage of radiographs.
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Harnessing Light: Optical Science and Engineering for the 21st Century BOX 2.2 "GETTING SCOPED" Flexible and rigid viewing scopes have changed medicine in ways many Americans have encountered. The repair of a torn meniscus in the knee is usually performed using a rigid arthroscope, through which a number of surgical tools are passed. This technique has changed knee surgery from an inpatient procedure to an outpatient one with reduced pain and convalescence. The colonoscope is used routinely to examine patients for possible colon cancer. The resulting early detection of colon cancer is often life-saving. Many gynecological procedures have become less invasive through the use of a laparoscope, which passes through the abdomen to allow access to the uterus. Laparoscopic techniques also enable numerous other procedures, such as gall bladder removal, which is discussed later in the section on minimally invasive therapy. Medical applications spread from ophthalmology into the general area of surgery, with these applications generally developing around the most readily available lasers. It is important to note that lasers can emit either short pulses of light (pulsed lasers) or a beam of light that is always on (continuous-wave, or cw, lasers) because the effects of pulsed and cw laser light can be quite different. These were primarily the pulsed ruby laser; the cw argon ion and carbon dioxide (CO2) lasers; the Nd:YAG (neodymium-doped yttrium-aluminum-garnet) laser, primarily in the cw mode; and the cw dye laser. The ability of the cw CO2 and Nd:YAG lasers to cut tissue while producing coagulation led to their use as general surgical lasers. Many companies entered the medical laser marketplace, often without a strong scientific understanding of the effects of lasers on tissue. In addition, the role of the Food and Drug Administration (FDA) in the regulation of new laser devices was not as well established as it is today, allowing the introduction of medical laser systems with unproven efficacy. Since the early 1980s a number of changes in the nature of medical laser research have occurred. There was an increasing interest in the mechanisms of laser-tissue interactions, and new clinical applications based on these interactions came into use. One of the driving forces behind this change was the initiation of the Medical Free Electron Laser (FEL) Program by the Department of Defense (DOD) in 1985. Although the program was specifically aimed at developing FEL applications, the novel pulse structure of the FEL led to an increased interest in pulsed laser effects, which in turn led to an increased understanding of laser-tissue interactions based on conventional lasers. Today, the use of optics in surgery and medicine is large and growing. For example, worldwide sales of medical laser systems reached $890 million in 1994, $1,070 million in 1995, and $1,295 million in 1996 (estimated), and they were forecast to reach $1,460 million in 1997
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Harnessing Light: Optical Science and Engineering for the 21st Century (Arons, 1997). The corresponding figures for U.S. sales were $535 million in 1994, $695 million in 1995, $830 million in 1996, and $960 million forecast for 1997. Understanding the Interaction of Light with Tissue The optical properties of tissue were studied, leading to the awareness that most tissue is an inhomogeneous substance with multiple absorbers such as melanin (the primary pigment in skin), oxyhemoglobin (a constituent of blood), and proteins. The significance of these absorbers varies with the wavelength of interest; for wavelengths greater than 1 μm, for example, water is the primary absorber. For reference, the wavelength range of visible light is about 0.4 to 0.7 µm; the wavelengths of lasers used in medicine extend to both the short (ultraviolet) and the long (infrared) side of the visible spectrum. New clinical treatments grew from increased insight into light-tissue interactions. With an understanding of the different absorption properties of various tissue components and of the depth that light penetrated into tissue came the insight that thermal effects could be confined to the optical penetration depth by using laser pulses short enough that no thermal diffusion occurred during the pulse. This led to the concept of "selective photothermolysis" in which particular sites in tissue, such as blood vessels, are targeted with minimal effect on surrounding tissue. This concept is exploited in dermatology, where the treatment of skin lesions characterized by abnormal blood vessels, such as port wine stains, is often required. New kinds of laser effects were discovered. There was increased awareness and utilization of the fact that lasers could be used to produce tissue effects other than the purely thermal ones involved in early laser surgery. The ability of pulsed lasers to cause a number of mechanical effects was recognized, studied, and used. Some of these photomechanical effects relied in turn on the ability of pulsed lasers to initiate nonlinear effects; specifically, the ability of pulsed lasers to produce optical breakdown in water was used to generate cavitation bubbles and launch stress waves. These mechanical effects found clinical use in ophthalmology, where they are employed in a procedure referred to as "photodisruption." This procedure is used to treat a side effect of cataract surgery, the formation of an opacification on the membrane that holds the opaque lens, by rupturing or tearing a portion of that membrane. Here, a simple laser procedure now avoids the need for a second, invasive surgery. The use of optical breakdown made it possible to deposit laser energy in biological media that had no linear absorption, a conceptual change. Subsequently, laser-induced mechanical effects found a second clinical application, the fragmentation of urinary tract calculi (stones) in patients, a procedure known as laser lithotripsy. This
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Harnessing Light: Optical Science and Engineering for the 21st Century technique complemented the existing method of treating urinary tract stones, which involved the use of acoustic pulses generated by a machine (the shock-wave lithotripter), that was several times more expensive than the laser system. The laser technique, which used an optical fiber to deliver light to the stone, allowed the fragmentation of stones at locations that could not be accessed by the shock-wave lithotripter because pelvic bones blocked the acoustic pulses. The mechanisms involved in this application were investigated after the effect was demonstrated and clinical trials initiated. In these studies, optical techniques from the physical sciences, such as pump-probe measurements and high-speed flash photography, played a significant role in clarifying the mechanisms of stone fragmentation. An additional nonthermal use of lasers in medicine is using light, primarily from laser sources, for cancer treatment. Drugs injected into a patient can be selectively activated by illuminating the area of interest; this can lead to the photochemical destruction of tumors. This treatment, known as photodynamic therapy (PDT), is being investigated for the treatment of a number of cancers and has recently been approved for palliation of esophageal cancer. PDT is discussed in more detail below. Today many different lasers are being used to irradiate a variety of tissue targets. Table 2.1 lists the most commonly used lasers, their wavelengths, the tissue targets, and the therapeutic interaction desired. TABLE 2.1 Common Medical Lasers and Some of Their Applications Laser Wavelength (nm) Target(s) Applications ArF Excimer 193 Tissue protein Refractive surgery Argon ion 488,514 Hemoglobin Retinal photocoagulation Nd:YAG, frequency doubled 532 Hemoglobin, tattoo pigments Tissue cutting and coagulation, tattoo removal Pulsed dye 577 Hemoglobin Removal of vascular lesions Continuous dye 630-690 Photosensitizers Photodynamic therapy Visible diode 650-690 Photosensitizers Photodynamic therapy Pulsed ruby 694 Tattoo pigments Tattoo removal Infrared diode 800 (nominal) Hemoglobin, absorbing dyes Retinal photocoagulation, tissue welding Nd:YAG 1,016 Water Tissue cutting and coagulation, many surgical applications, tattoo removal Ho:YAG 2,100 Water Tissue cutting and shrinkage Er:YAG 2,940 Water Skin resurfacing, hard and soft tissue cutting (experimental) CO2 10,600 (nominal) Water Skin resurfacing, tissue cutting and coagulation, surgery
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Harnessing Light: Optical Science and Engineering for the 21st Century Table 2.1 includes standard surgical lasers, such as Nd:YAG and CO2, as well as some lasers whose uses are still experimental. The Er:YAG laser, for example, has been studied as a tool for dentistry (Wigdor et al., 1995) but has only recently been approved for dental use. Finally, the potential of optics and lasers for obtaining information about tissue for use either in clinical diagnosis or in providing feedback control of surgical laser systems has received increasing attention. The need for feedback control arose as situations were encountered in which the tissue response to laser irradiation depended critically on the flux and the light dose. Tissue welding, the use of lasers to join tissue by localized heating, is optimal over only a small temperature range, which makes it difficult to obtain reproducible results. A number of feedback systems, based either on tissue temperature or on changes in tissue optical properties, have been studied in control can enable tissue welding to be performed by most surgeons, an attempt to obtain reliable laser-based tissue welding. If feedback it may complement sutures for applications, such as plastic surgery, where minimal scarring is desired. A number of studies have investigated the use of laser-induced fluorescence, both from substances naturally occurring in tissue and from externally administered fluorescent marker dyes, to delineate tumors and potentially to aid in the early detection of cancer. Optical radar techniques have been applied to biological tissue, starting with the skin and soon thereafter the eye; more details are given in the discussion of optical diagnostic techniques. Minimally Invasive Therapy The growth of optical and laser techniques in medicine was in large part due to the fact that devices for delivering light to the inside of the body became available. In the 1990s, advances in such areas as CCD (charge-coupled device) camera technology and innovative new approaches by surgeons led to the development of what is now referred to as "minimally invasive therapy" (MIT; see Box 2.3). In addition to optics, the development of specialized surgical tools to allow traditional surgical manipulations such as cutting, suturing, and stapling to be performed through tiny incisions was another technology that enabled MIT. The concept of MIT is the replacement of traditional "open" surgery—with its large incisions and direct viewing of the surgical field by the physician—by several small incisions, typically punctures on the order of 5 to 10 mm in diameter, through which viewing devices and surgical tools can be passed. Optics is critical to MIT since the main concept is to use video rather than direct viewing to minimize the surgical invasiveness of the procedure. Quartz optical fibers, developed initially for fiber-optic communications, are capable of transmitting many of the laser
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Harnessing Light: Optical Science and Engineering for the 21st Century BOX 2.3 MINIMALLY INVASIVE THERAPY Advantages Shorter hospital stays Reduced patient trauma, morbidity Shorter convalescence; faster return to work Decreased expenditure on pain medication Basic Components Imaging (primarily optical; ultrasound, magnetic resonance, computerized tomography may also be used) Tissue manipulation tools Source of directed energy (electrocautery, laser, focused ultrasound) Role of Optics Video cameras Flexible endoscopes Rigid laparoscopes Laser sources Tissue characterization wavelengths of interest for therapeutic applications. An optical fiber can usually be added to an endoscope by passing it through one of the already available "channels" designed for irrigation and the passage of tools, resulting in an instrument that allows both viewing and laser irradiation. The main exception to this approach has been the CO2 laser, whose infrared wavelength could not be delivered readily using available fiber optics; in this case, rigid or flexible metallic waveguides were used as delivery channels. Despite efforts to develop an infrared fiber capable of both transmitting CO2 laser radiation and surviving in the wet environment of the human body, such fibers have not reached the point of clinical use. In addition, although the initial applications used only optical imaging to obtain information about the surgical field, the combination of x-ray, magnetic resonance, and other imaging technologies to produce fused images is envisioned by workers in MIT today. The fusion of imaging modalities may be necessary to allow internal access to solid organs such as the liver. The first extensive application of MIT was to a new procedure for gall bladder removal, the laparoscopic cholecystectomy. In this procedure, four incisions admit a viewing device, a gas infusion device to inflate the abdomen, and two surgical tools. The surgeon operates by
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Harnessing Light: Optical Science and Engineering for the 21st Century viewing a TV monitor while manipulating tools for cutting, stapling, or suturing tissue. Although a laser was initially used to stop bleeding during surgery, it was soon found that the use of a much less expensive electrocautery device was equally satisfactory, and use of the cauterizing laser in this specific MIT procedure has become minimal. A major impact of laparoscopic cholecystectomy is a drastic reduction in recovery time for the patient, with attendant savings in lost wages and lost time to employers. Hospitalization time is decreased from 4 days to 1, leading to a major cost savings. The acceptance of the procedure can be most directly appreciated by comparing the number of conventional and laparoscopic cholecystectomies between 1988 and 1994, tabulated in Table 2.2. Today, the laparoscopic procedure is the method of choice. Other examples of MIT abound; as surgeons become more skilled in the techniques involved, more complex procedures have been performed, including hernia repair and colon surgery. In orthopedics, knee and shoulder surgery is routinely performed using dedicated rigid or flexible fiber-optic viewing instruments called arthroscopes, together with dedicated miniature surgical tools for specialized operations. A word of caution is needed in considering the future of MIT techniques in the present health care environment. Since most surgery is paid for by health care providers, the acceptance of a particular MIT technique is determined by whether it reduces direct cost to the provider, not by overall societal benefits such as a decrease in time lost from work. In some cases, the direct costs of an MIT procedure can be higher than those of the older, more-invasive technique because additional tools and more sophisticated equipment are required. Thus, the introduction of new MIT techniques will require that direct costs do not increase substantially or that patient demand is such that the minimally TABLE 2.2 Growth Patterns in Minimally Invasive Surgery—Traditional and Laparoscopic Cholecystectomies Year Traditional Procedures Laparoscopic Procedures 1988 537,000 0 1989 545,000 1,000 1990 535,000 25,000 1991 410,000 125,000 1992 150,000 480,000 1993 75,000 525,000 1994 85,000 575,000 Source: W. Grundfest, Cedars Sinai Hospital.
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Harnessing Light: Optical Science and Engineering for the 21st Century invasive technique will obtain reimbursement regardless of direct cost. Some studies have pointed out that evaluations of the economic benefits of MIT have produced wildly different conclusions (Cuschieri, 1995), indicating the need for care in making quantitative statements about its economic benefits. Advanced Therapeutic Applications of Lasers Currently, numerous advanced therapeutic applications of lasers are being investigated. This report illustrates the types of new clinical approaches being investigated by using a few examples that show the diversity of approaches being studied and their relation to the enhanced understanding of basic mechanisms obtained from fundamental studies. In addition, the specific optical issues involved in each of these examples are illustrated. Laser Refractive Surgery For the correction of visual defects such as nearsightedness, astigmatism, and farsightedness, laser-refractive surgery has attracted intense clinical and public interest. The basic concept is simple. Since most of the refractive power of the eye comes from the cornea, the outer surface of the eye, relatively small changes in the curvature of the cornea can correct a large number of visual defects that currently require eyeglasses or contact lenses. In a generic sense, the concept is to perform corneal "sculpting" using a laser (McDonnell, 1995; Seiler and McDonnell, 1995). Although a number of approaches to corneal sculpting have been used, the basic concept relies on the observation, made in the materials science community in the early 1980s, that UV laser radiation from pulsed excimer lasers can be used to ablate both polymers and tissue with minimal damage (typically less than 1 μm) and with high-precision and control. This basic observation served to guide the development of a number of different excimer laser systems for refractive surgery. All of these systems were designed to ablate tissue from the cornea in a controlled and predetermined manner to produce a change in the refractive power of the eye, but they differed in engineering details. In the course of development of these systems, numerous problems involving optical engineering, the safety and efficacy of the procedure, wound healing, pharmacology, and regulatory issues required solution. A powerful driving force for solving these problems was the perceived size of the market. Approximately 25% of the population of the United States suffers from myopia (nearsightedness) and constitutes potential customers for excimer laser photorefractive keratectomy (PRK). Intense effort went into developing a system that could produce the desired correction. Clinical trials were needed to determine how
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Harnessing Light: Optical Science and Engineering for the 21st Century severe a myopia could be successfully treated, as well as to find the limitations on treating other refractive defects such as hyperopia (farsightedness) and astigmatism. Long-term follow-up was necessary to determine the stability of these laser-induced changes in the cornea. After extensive clinical trials and experimentation, the FDA approved a commercial excimer laser system for PRK in October 1995. During the course of development of PRK, one of the initial problems encountered was the formation of haze in the treated cornea. Years of experimentation showed that this could be controlled by control of the laser beam profile, combined with the pharmacological treatment of some patients. A variation on PRK that shows promise in early clinical trails is LASIK (laser-assisted in situ keratomileusis) in which the anterior surface of the cornea (corneal cap) is first microtomed (cut off) to reveal the central stroma of the cornea. The stroma is appropriately ablated with an excimer laser, and the corneal cap is then replaced. The thickness profile of the cornea has been changed without affecting the anterior (front) surface of the cornea. Minimal haze is associated with this procedure, and no sutures are required. Other laser technologies are being explored for corneal sculpting. One approach relies on the use of an Ho:YAG infrared laser to heat and controllably shrink portions of the cornea outside the central visual field, avoiding the haze problem. A central issue here is biological: Will the reshaped cornea retain the new shape or relax to the original one? Cardiovascular Applications Heart disease is the leading cause of death in the United States, and the search for alternatives to expensive coronary bypass surgery has been active, with laser systems initially offering a promising approach. Cardiovascular applications provide an example of both the potential of laser techniques and the potential pitfalls in applying technological solutions to complex biological systems (Deckelbaum, 1994). The attractive feature of the laser in cardiovascular applications is its ability to deliver energy via an optical fiber to sites in very small vessels. Laser angioplasty, the use of lasers to remove blockages in arteries, is a well-known concept. Although many techniques used for angioplasty, such as laser ablation, inflatable balloons, and high-speed rotating cutters, are effective at removing blockages, a major problem shared by all of these approaches is restenosis—the vessel reclosure that occurs within 6 months in about 40% of all angioplasties. Consequently, emphasis has shifted from the development of new angioplasty techniques to the development of methods of controlling restenosis, including the insertion of metal stents and the use of photochemical therapies (see Box 2.4).
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Harnessing Light: Optical Science and Engineering for the 21st Century BOX 2.6 IMMUNE SYSTEM MONITORING FOR HIV The AIDS epidemic is an excellent example of a critical medical problem that is being studied using optical biomedical instrumentation. AIDS currently affects more than 100 million people worldwide and is the leading cause of death among young adult males in the United States. Our understanding of this terrible disease has grown out of intense scientific research that has occurred over the past 15 years. A large portion of the research has focused on the impact of the AIDS virus on the human immune system. The primary tool used in this research has been the flow cytometer. For example, using flow cytometry, immunologists were able to determine the precise subgroup of white blood cells, the CD4 cell, that is attacked by the virus. The flow cytometer has evolved from the primary scientific tool used to understand the impact of the AIDS virus on the immune system into the principal clinical diagnostic instrument that is now the standard of care for monitoring CD4 levels in infected individuals. Flow cytometry data on CD4 concentrations in peripheral blood are used to guide physicians in choosing the antiviral and antibiotic drug therapies appropriate at various stages of the disease. Another class of optical instrumentation that is of critical importance in the battle against AIDS is the automated genetic sequencer. Using this instrument, which typically incorporates a scanning laser fluorimeter, scientists have been able to sequence the complete genome of the AIDS virus. This information has provided insight into the structure of the surface proteins of the virus and has helped lead to effective methods for sensitive detection of viral proteins in peripheral blood. Detecting viral protein in a peripheral blood sample is currently the accepted diagnostic method for verifying HIV infection. Gene sequencing instruments are also used to monitor genetic changes in the virus that signal the evolution of viral mutants resistant to drug therapies and mutants that might elude the current generation of tests used to ensure the safety of the U.S. blood supply. It is interesting to note that flow cytometers and automated gene sequencing instruments were developed in the late 1970s and early 1980s, precisely the time when the AIDS epidemic began. This timing was quite fortunate since without these instruments, our knowledge of the AIDS virus, its common modes of transmission, and possible strategies for combating it would have been severely affected and the epidemic would most definitely be significantly worse. The next generation of AIDS diagnostic techniques will focus on determining the concentration of free HIV in peripheral blood, the viral load. This diagnostic measurement has proven to be of great importance for developing promising new anti-HIV drugs, the protease inhibitors, and for determining effective therapies involving combinations of these antiviral drugs. Several different techniques have been developed using DNA chemistry for viral recognition and optical detection for quantification, for example, quantitative competitive polymerase chain reaction (PCR) and branch DNA. Both of these techniques are usually performed in sophisticated molecular biology laboratories and are not yet suitable for a typical hospital clinical laboratory.
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Harnessing Light: Optical Science and Engineering for the 21st Century The impact of flow cytometry on modern biomedical research is large. The total annual market for flow cytometry instruments and reagents is estimated to be $300 million worldwide, of which U.S. manufacturers control on the order of 90%. Presently, clinical applications account for two-thirds of the total market, or about $200 million annually. One measure of the impact of flow cytometry is that, on the average, three out of four issues of Science contain an article with flow cytometric data. The technology is used worldwide, even in developing countries with limited funds for high-technology instrumentation. In these countries the major application is the analysis of white blood cell subpopulations in AIDS patients. In the future, flow cytometers will be easier to use, more compact, and located in smaller hospitals or even doctor's offices. An integrated system with a flow cytometer on a chip that contains excitation source, detection, and fluidics is a realistic goal. On the research side, sensitive flow cytometry techniques orders of magnitude faster and more sensitive than currently used methods are being developed to analyze the size of DNA fragments and to sequence DNA. A variety of technological improvements are needed for this to occur. New compact light sources that emit light in the blue and ultraviolet will be needed to match the dyes currently in routine use. Detection and light filtration systems that are compact, efficient, and easy to use are also necessary. One of the unique aspects of flow cytometry, whether in a clinical or a research laboratory, is that competent cytometrists must be well founded in a variety of disciplines from computer science to biology to optical sciences. Currently, there is no interdisciplinary degree program that adequately prepares either users or developers of the technology for the breadth of information and understanding that they need. Bioengineered Fluorescent Indicators A number of novel fluorescent indicators based on molecular biology have become available that serve as indicators of processes going on within living cells. For example, the green fluorescent protein (GFP) from a luminescent jellyfish is a protein that spontaneously modifies itself to generate a strongly fluorescent internal chromophore. Two mutants of different colors can engage in fluorescence resonance energy transfer, which can then be spectroscopically studied to monitor the presence or absence of protein-protein interaction inside living cells. Optical readouts of membrane potential, protein phosphorylation, and proteolysis are also under development. Even more recently, techniques have been developed to incorporate the gene for a bioluminescent molecule into bacteria and other molecules (Contag et al., 1995). This has allowed tracking of the spread of bacteria, as well as the action of antibiotics, throughout the body of small animals. The same
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Harnessing Light: Optical Science and Engineering for the 21st Century approach may be useful in signaling successful gene therapy. More broadly, it appears we can now alter the optical properties of living organisms in order to monitor the spread and control of disease in living animals and eventually humans. Micromanipulation Techniques A new application of optics in biology is the use of light to actively manipulate the molecules, mechanisms, and structures that determine biological function. Laser beams can be used, with proper handling, to create optical traps or "tweezers" that capture and manipulate cells and even subcellular organelles. Optical tweezers are even being used to determine the forces involved in the locomotion of single biological molecules. The force that light can exert was predicted by James Clerk Maxwell in his theory of electromagnetism of 1873 but was not demonstrated experimentally until the turn of the century. One reason for the delay is that radiation pressure is extraordinarily feeble. Milliwatts of power (corresponding to very bright light) impinging on an object produce piconewtons of force (1 pN = 10-12 N). The advent of lasers in the 1960s finally enabled researchers to study radiation pressure through the use of intense, collimated sources of light. By focusing laser light into narrow beams, researchers demonstrated that tiny particles, such as polystyrene spheres a few micrometers in diameter, could be displaced and even levitated against gravity using the force of radiation pressure. Under the right conditions, the intense light gradient near the focal region can achieve stable three-dimensional trapping of dielectric objects. Optical traps can be used to capture and remotely manipulate a wide range of larger particles, varying in size from several nanometers to tens of micrometers (Svoboda and Block, 1994). Subsequently, it was shown that these "optical tweezers" could manipulate living things such as viruses, yeasts, bacteria, and protozoa. Experiments during the past few years have begun to explore the rich possibilities afforded by optical trapping in biology. Although still in their infancy, laser-based optical traps have already had significant impact. Tweezers afford an unprecedented means for manipulation on the microscopic scale. Optical forces are minuscule on the scale of larger organisms, but they can be significant on the scale of macromolecules, organelles, and even whole cells. A force of 10 piconewtons, equal to 1 microdyne, can tow a bacterium through water faster than it can swim, halt a swimming sperm cell in its track, or arrest the transport of an intracellular vesicle. A force of this magnitude can also stretch, bend, or otherwise distort single macromolecules, such as DNA and RNA, or macromolecular assemblies, including cytoskeletal components such as microtubules and actin filaments. Proteins such as
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Harnessing Light: Optical Science and Engineering for the 21st Century myosin, kinesin, and dynein produce forces in the piconewton range. Optical traps are therefore especially well suited to studying mechanics or dynamics at the cellular and subcellular levels. The possibilities for further development and use of optical tweezers in biology and medicine are extraordinary. There are many areas in which optical tweezers can be expected to provide visual images or better understanding of biological processes that involve motion. For example, the micromechanics of DNA-modifying enzymes (such as DNA and RNA polymerases) can be observed and protein synthesis manipulated at the most basic level; receptor-ligand interactions can be manipulated by physically constraining the reactants; small structures such as biosensors and microtubules could be constructed; mechanical properties of filaments can be measured directly; and forces allowing cells to crawl or chromosomes to move from place to place can be determined. The National Science Foundation (NSF) should increase its efforts in biomedical optics and pursue opportunities in this area aggressively. This will require a broader interpretation of the NSF charter regarding health care in order to support promising technologies that bridge the NIH and NSF missions. Biotechnology Just as optics is playing an important enabling role in the development of new research techniques for fundamental biology, it is also becoming increasingly important in the biotechnology industry. Many of the devices and techniques discussed above in the context of biological research, such as flow cytometry and fluorescent molecular probes, play similarly important roles in biotechnology applications. In a general sense, biotechnology involves measurement, manipulation, and manufacture of large biologically significant molecules such as proteins and DNA. Among the applications for which optical methods are most important are genetic sequencing and pharmaceutical development. DNA Analysis The development of new instrumentation for DNA sequencing has been driven by the Human Genome Project, which is the largest government-funded project in the health sciences. The general strategy of all such instruments involves tagging the four distinct bases that occur in DNA with fluorescent dyes that have different emission wavelengths. Currently an argon ion laser is used to excite fluorescence. Sequence information is obtained by monitoring the multicolored fluorescent emission from large (50 cm x 70 cm) electrophoretic gels.
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Harnessing Light: Optical Science and Engineering for the 21st Century High-efficiency confocal laser scanning systems, which are commercially available, currently provide the fastest method for gene sequencing. Although they represent a major improvement over first-generation instruments, these devices are still considered approximately 100 times too slow to meet the goals of the Human Genome Project. The next generation of instruments, currently under development, incorporates integrated optics, hollow fibers for capillary electrophoresis, and red and infrared dyes for better spectral separation of the fluorescent indicators. The polymerase chain reaction (PCR) used for DNA amplification is pervasive in biology today, being used for detection of viruses in blood, monitoring of viral loads in AIDS patients, detection of inherited disease tendencies, and forensics. Although current PCR systems are of laboratory bench-top size, the availability of miniaturized optics allows the development of miniaturized versions. These micro-PCR systems will allow quantitative detection of the nucleic acids formed and will use microspectrometers to monitor fluorescent tags in real-time. The ultimate goal is to combine these optical monitors with control and analysis software that will determine the thermal cycling used in the PCR process. It is interesting to note that the problem of miniaturizing the liquid handling aspects of such systems presents formidable technical challenges whose solutions have yet to be found. Oligonucleotide probe arrays, sometimes referred to as DNA chips (Figure 2.8), combine both optical and chemical techniques to obtain genetic information. Oligonucleotides are small polymers made up of nucleotides, which are subunits of DNA (Lipshutz et al., 1995). The basic goal of these chips is to make possible the performance of a large number of operations probing the sequence of DNA in parallel. The chips are made by light-directed chemical synthesis, which is in turn based on photolithographic techniques developed for the semiconductor industry and on solid-phase chemical synthesis. The photolithographic techniques are used to "deprotect" or activate small synthesis sites consisting of hydroxyls on a solid substrate. The sites are selected using photolithographic masks. The activated region can then be reacted with a chemical building block to produce a new compound. By combining many of these activation steps with multiple cycles of photo-protection and chemical reaction, a chip with a high-density checkerboard array of oligonucleotides can be produced. For example, if the resolution of the chemical process is 100 µm, 104 sites can be produced per square centimeter. These sites are essentially probes for specific DNA sequences. The target or unknown sequence is labeled with a fluorescent dye and exposed to the chip. It binds most strongly to sites that match a portion of its DNA sequence, resulting in localized patches of high fluorescence. Laser scanning confocal microscopy, described previously, is
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Harnessing Light: Optical Science and Engineering for the 21st Century FIGURE 2.8 Part of a ''DNA chip," showing fluorescently labelled DNA bound to an 8,000-site GeneChip® probe array. (Courtesy of Affymetrix, Inc., Santa Clara, Calif. Copyright ©Affymetrix, Inc. All rights reserved. Affymetrix and GeneChip are registered trademarks used by Affymetrix, Inc.) used to produce a map of fluorescence intensity versus site on the chip. Since the chemical composition at each site is known from the synthesis procedure, the unknown sequence can be deduced. Applications envisioned for these probe arrays include rapid sequencing of DNA as well as the detection of mutations associated with resistance to antiviral drugs used in the treatment of AIDS. Although the commercial success of the DNA chip will depend on many factors, including the development of competing technologies, it illustrates the way sophisticated optical techniques, developed in part for the semiconductor industry, are being used for biotechnology. Pharmaceutical Screening Pharmaceutical screening to find drugs that have optimal biological activity for a particular clinical application is a good example of the potential impact of advanced fluorescent indicators on biotechnology. These applications, now in the early stages of development, would allow the screening of very large numbers of potential pharmaceuticals using only minute quantities of the candidate drug and small groups of cells. The pharmaceutical industry has developed very large libraries of semirandomly generated candidate compounds for drug discovery. The libraries contain thousands to millions of different chemicals, usually synthesized by combinatorial sequences of reaction steps. The libraries now encompasses a wide variety of chemical families, including many that could be suitable for orally active drugs to treat major diseases. However, screening these huge libraries to find which members possess optimal biological activity is a tremendous challenge. Only picomole quantities of each candidate are available, so most traditional pharmaceutical assays are too insensitive. Thus, there is a great need for bioassays that can be miniaturized to microliter or smaller assay volumes and performed at the rate of thousands to millions per day. Such bioassays have to be easily adaptable both to known drug receptors and to the thousands of new potential macromolecular targets being found by human genome sequencing. Optically based methods to accomplish this are being investigated. The basic concept is to combine recent improvements in microscopic
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Harnessing Light: Optical Science and Engineering for the 21st Century imaging with new fluorescent indicators of intracellular signaling to allow bioassays on single cells or small groups of cells. Cells can now be genetically engineered to be responsive to signaling pathways of interest or to mimic target disease processes. They are then grown by tissue culture in billions or trillions as required. Zeptomole (1 zmol = 10-21 mol) to attomole (1 amol = 10-18 mol) quantities of compound suffice to activate or inhibit individual cells, which can be imaged in microscopic volumes. The best known intracellular fluorescent indicators report calcium signals and are already in use for drug screening at the cellular level. However, gene expression is a more universal and stable readout, which can be monitored by introducing an optically easy-to-detect enzyme for the protein that the cell would normally express. For example, reporter enzymes, such as ß-lactamase, together with carefully designed, membrane-permeant fluorogenic substrates can disrupt fluorescence energy transfer in the substrate and change the emission color from green to blue. This color change is so dramatic that it can easily be seen by the unaided eye and is precisely quantifiable by two-color flow cytometry or standard ratio image processing. Flow cytometry should enable selection and cloning of cell lines whose ß-lactamase expression is optimally sensitive to known drugs, hormones, or disease-mimicking alterations. The same enzyme system provides a nondisruptive optical readout to measure the effect of novel drug candidates on single cells or small clusters of cells. In this way the cumulative activity of nearly any specific signal transduction pathway of choice may be monitored optically. The practical challenge is now to integrate the techniques of molecular biology, cell culture, optical signal transduction, organic synthesis, microscale liquid handling, high-performance optical imaging, and automated data analysis into a coherent, robust, and economically viable system. Summary and Recommendations Surgery and Medicine Optics has enabled the development of rigid and flexible viewing scopes that allow minimally invasive diagnosis and treatment of numerous sites inside the body, such as the colon, the knee, and the uterus. Lasers have become accepted and commonly used tools for a variety of surgical applications. These include the CO2 laser, the high-repetition-rate, frequency-doubled Nd:YAG laser (KTP 532), and the Nd:YAG laser. Lasers and optics have made possible noninvasive treatment of many diseases of the eye and have become essential to the practice of
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Harnessing Light: Optical Science and Engineering for the 21st Century ophthalmology. Inpatient procedures have often become outpatient ones as a result. Lasers are now used extensively in dermatology for the treatment of pigmented lesions, tattoos, wrinkles, and other problems. This use has become widespread because research has led to an understanding of how to target specific tissue sites by the proper choice of laser wavelength and pulse width. Biological response, rather than the sophistication of a particular optical technique, is often the critical issue in clinical applications. Close cooperation between physical scientists and physicians is necessary to successfully address clinical problems. One example is laser angioplasty. New infrared solid-state lasers are being used to complement the more established CO2 and YAG surgical lasers. The Ho:YAG laser offers compatibility with existing quartz fiber optics and may replace CO2 in some cases. The Er:YAG laser is unique in its ability to cut bone with minimal thermal damage. Photomechanical effects have been recognized as clinically significant and often useful; they are used commonly in ophthalmology and urology. Light-activated drugs are being used to treat both cancer and noncancer diseases by photodynamic therapy. These photochemical treatments are able to affect not only cells and tissue, but also specific growth factors and signaling processes in tissue. Noninvasive monitoring of basic body chemistries, such as glucose concentration, remains a major challenge for optics. The basic science required for the development of such monitoring techniques is often missing or incomplete. As laser medicine and surgery have moved from being almost entirely empirical arts to having a solid basis in the underlying physics and chemistry of laser-tissue interaction, new and less painful laser treatments for numerous diseases have been developed. The disease-oriented structure of NIH does not encourage the funding of biomedical optical technology programs. Lasers and fiber-based instrumentation have enabled many new minimally invasive therapies that reduce total (direct plus lost time) health care costs. Optically based diagnostic methods are less developed than therapeutic ones, but they offer potentially improved techniques for the medical laboratory (more accurate blood tests), the clinic (techniques to complement x-ray mammography), and home care (noninvasive glucose monitoring). New laser technologies and effects are now quickly assimilated by the medical care community. However, the FDA regulatory process makes commercialization of new technologies costly. Close cooperation among optical scientists, physicians, and FDA personnel may improve the process. Optics and lasers will continue to facilitate the development of new medical systems. Visible diode lasers, diode-pumped solid-state lasers, light-emitting diodes, and compact optical parametric oscillators are some of the devices on which such systems will be built. Feedback control will attract increasing attention as opti-
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Harnessing Light: Optical Science and Engineering for the 21st Century cal and magnetic resonance imaging systems are coupled with laser-based treatment systems. Mechanisms should be developed for encouraging increased public and private investment in noninvasive optical monitoring of basic body chemistries. Clearer separation of the roles of the public sector—basic science and proof of principle—and the private sector—device development—is needed. Better understanding of how light interacts with tissues will continue to be important for the development of optical techniques for treatment and diagnosis. NIH should establish a study section for RO1 grants devoted to biomedical applications of light and optical technology. An initiative to identify the human optical properties suitable for noninvasive monitoring should also be established. Tools for Biology Confocal laser scanning microscopy and computed microscopy have enabled depth-resolved microscopic imaging that allows three-dimensional information to be acquired. Two-photon techniques have not only enhanced the capabilities of fluorescence microscopy but also opened up new possibilities for performing spatially localized photochemistry within cells. The potential of these techniques is relatively unexplored. Near-field microscopy, a nonimaging technique, allows microscopy with resolutions of tens of nanometers, far less than the diffraction limit for light. Fluorescent markers have replaced many of the radioactive tags used to mark the presence of specific molecules, such as proteins, and in DNA sequencing, thus eliminating the complications associated with handling and disposing of radioactive materials. Flow cytometry, which is based on laser and optical technology, has become both a standard clinical assay and a frequently used research tool. Optical micromanipulation techniques (optical tweezers) have found uses in the study of the forces involved in molecular locomotion and in the manipulation of cells and molecules within them. The use of fluorescence techniques as quantitative assays will grow as more quantitative measurement techniques are introduced. New microscopies (confocal, two-photon, near-field) are extending the capabilities of traditional microscopy by enhanced resolution and the ability to image in depth. Lasers and optical methods have become an integral tool for many essential biological technologies and methods. The continual development of new, specific, and inexpensive molecular probes is necessary for optimal utilization of fluorescence-based techniques. The development of instrumentation that solves significant biological problems requires interdisciplinary teams that are aware of both available technology and biological questions. The advances in technology that are now being applied build upon long-term investments in basic research. Examples are the understanding of two-photon
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Harnessing Light: Optical Science and Engineering for the 21st Century absorption, which builds on basic quantum mechanical calculations that are more than 60 years old, and the development of optical tweezers, which grew out of studies of optical levitation. NSF should increase its efforts in biomedical optics and pursue opportunities in this area aggressively. This will require a broader interpretation of the NSF charter regarding health care in order to support promising technologies that bridge the NIH and NSF missions. Biotechnology Lasers have become essential parts of all systems used for DNA sequencing, ranging from those that are commercially available to more experimental capillary electrophoresis systems. Optics is being employed in a number of biotechnology applications, from sophisticated systems using DNA chips to simpler systems using transmission probes. Scientists, engineers, and technicians with cross-disciplinary training will enhance the transfer of optical science into biology and medicine. References American Optometric Association. 1996. Caring for the Eyes of America. St. Louis, Mo.: American Optometric Association. Arons, I. 1997. Medical laser market hits new high. Med. Laser Rep. 11:1-2. Contag, C.H., P.R. Contag, J.I. Mullins, S. Spilman, D.K. Stevenson, and D.A. Benaron. 1995. Photonic detection of bacterial pathogens in living hosts. Mol. Microbiol. 18:593. Cuschieri, A. 1995. Whither minimal access surgery: Tribulations and expectations. Am. J. Surg. 169:9-19. Deckelbaum, L.I. 1994. Cardiovascular applications of laser technology. Lasers Surg. Med. 15:315-341. Gratton, E., and J.B. Fishkin. 1995. Optical spectroscopy of tissue-like phantoms using photon density waves. Comments Mol. Cell Biophys. 8:307-357. Huang, D., E.A. Swanson, C.P. Lin, J.S. Schuman, W.G. Stinson, W. Chang, M.R. Hee, T. Flotte, K. Gregory, C.A. Puliafito, and J.G. Fujimoto. 1991. Optical coherence tomography. Science 254:1178-1181. Katzir, A. 1993. Lasers and Optical Fibers in Medicine. San Diego, Calif.: Academic Press. Krauss, J.M., and C.A. Puliafito. 1995. Lasers in ophthalmology. Lasers Surg. Med. 17:102-159.
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Harnessing Light: Optical Science and Engineering for the 21st Century Lipshutz, R.J., D. Morris, M. Chee, E. Hubbell, M.J. Kozal, N. Shah, N. Shen, R. Yang, and S.P.A. Fodor. 1995. Using oligonucleotide probe arrays to access genetic diversity. BioTechniques 19:442-447. McDonnell, P.J. 1995. Excimer laser corneal surgery: New strategies and old enemies. Invest. Ophthalmol. Vis. Sci. 36:4-8. Middleton, H., R.D. Black, B. Saam, G.D. Cates, G.P. Cofer, R. Guenther, W. Happer, L.W. Hedlund, G.A. Johnson, K. Juvan, and J. Swartz. 1995. MR imaging with hyperpolarized 3He gas. Magn. Reson. Med. 33:271-275. Pawley, J.B. 1995. Handbook of Biological Confocal Microscopy, 2nd ed. New York: Plenum Press. Seiler, T., and P.J. McDonnell. 1995. Excimer laser photorefractive keratectomy. Surv. Opthalmol. 40:89-118. Shapiro, H.M. 1995. Practical Flow Cytometry, 3rd ed. New York: Wiley-Liss. Svoboda, K., and S.M. Block. 1994. Biological applications of optical forces. Ann. Rev. Biophys. Biomol. Struct. 23:247-285. Tsien, R.Y. 1994. Fluorescent imaging: Technique tracks messenger molecules in living cells . Chem. Eng. News, July 18,: pp. 34-44. Wang, X.F., A. Periasamy, and B. Herman. 1992. Fluorescence lifetime imaging microscopy (FLIM): Instrumentation and applications. Crit. Rev. Anal. Chem. 23:1-26. Wigdor, H.A., J.T. Walsh, S.R. Visuri, D. Fried, and J.L. Waldvogel. 1995. Lasers in dentistry. Lasers Surg. Med. 16:103-133. Williams, R.M., D.W. Piston, and W.W. Webb. 1994. Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry. FASEB J. 8:804-813. Yodh, A., and B. Chance. 1995. Spectroscopy and imaging with diffusing light. Phys. Today 48:34-40.
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