Optics in Instruments: Applications in Biology and Medicine

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Imaging Technology

You are now ready to style your paper. The laser on biological tissue results from the conversion of light to heat, heat transfer and a tissue reaction to the temperature and the duration of the heating [34]. This interaction leads to distortion or the destruction of a tissue volume. Depending on the degree to which time is heated, and the heating time, the thermal effect of the laser produces coagulation necrosis as in the treatment of angiomas with Nd: YAG laser, or volatilization as in the treatment of skin lesions with CO 2 laser.

They are obtained with lasers emitting extremely short pulses, in the nanosecond to picosecond range on very small surfaces, which causes a destructive shock wave mainly induced by the mechanism of explosive vaporization of the target as used to treat haemangiomas [35]. In this case, the vessels of the angioma explode, which explains the vessel wall rupture, and hemorrhage.

This is also what happens during a tattoo removal when large fragments of pigment explode and give birth to smaller fragments. An effect that requires high-energy photons wavelength less than nm , with extremely short pulses 10 ns to ns. It induces a clean ablation of tissue without thermal lesions. It is used to treat corneal pathologies such as ulcers and scars, and its use in keratorefractive surgery has become a rapidly evolving field [36]. The operation is performed under local anesthesia using topical drops.

The first step of the procedure involves cutting a corneal flap surface 90 to micrometers. Until the early s, the most common way of cutting the corneal flap was the use of a microkeratome, a miniaturized and highly sophisticated mechanical device. This first very delicate cutting phase is now done by a laser, the femtosecond laser.

The cutting of the corneal flap is achieved in about ten seconds, and then the refractive sculpture is carried out using an excimer laser. Excimer lasers are powered by a chemical reaction involving an excited dimer, or excimer, which is a short-lived dimeric or heterodimeric molecule formed from two atoms, at least one of which is in an excited electronic state.

They typically produce ultraviolet light, and are used in LASIK laser in situ keratomileusis eye surgery see video: Commonly used excimer molecules include fluorine emitting at nm and noble gas compounds Argon nm, Krypton nm, etc. The femtosecond laser is the new scalpel for biologists. Since , they have gradually become familiar with this tool, which opens up great prospects in life sciences [37]. They are obtained with lasers emitting extremely short pulses, in the nanosecond to picosecond range on very small surfaces, which causes a destructive shock wave mainly induced by the mechanism of explosive vaporization of the target as used to treat haemangiomas [38].

The lasers used in biology have a wavelength located either in the infrared range or in the ultra-violet range; they operate in continuous or pulse mode. The high power density and the precise location of the laser beam suits its application to the cutting of biological tissue.

Introduction - Optics in Biology and Medicine

The high concentration of photons will destroy existing chemical bridges in the tissues. Laser-assisted microdissection LAM is a well-established technology in molecular pathology, cell biology and oncology studies, where very small tissue samples must be isolated from the surrounding material in order to perform an analysis without risk of contamination. LAM overcomes the problem of the cellular heterogeneity that characterizes tissues. It aims to recover a target cluster of cells, or a single cell precisely selected under microscope guidance, from a complex tissue section frozen or fixed by prior paraffin-embedding for subsequent molecular analysis [39] [40] Figure 1.

With the advent of PCR Polymerase Chain Reactions techniques, a technique for million-fold amplification of a single DNA molecule, microdissection enables a molecular approach of biological tissues, but with the certainty of having a highly purified cellular material for the molecular studies. Over the past 15 years, this technique for isolating specific cells from a sample responds to the need for miniaturization of analytical techniques applicable to very small cell numbers. LAM devices have gradually become more user-friendly. The development of this approach using a laser beam has greatly increased the precision and effectiveness of biological material collection Figure 2.

In this system, unlike the others, the tissue section remains stationary on the microscope and the UV laser beam moves over it when cutting. However, UV solid-state lasers nm are commonly used in each device. For the LMPC device, a gas laser operating in the ultraviolet range is used with molecular nitrogen as its gain medium, pumped by an electrical discharge.

Chronological succession of steps showing the laser beam cutting of a biological tissue: Blue light of a laser beam cutting a specific region, or cells of interest from a biological tissue laid after treatment on a microscope slide. YAG infrared laser with a wavelength of nm. Neoplasia is the main field of application of this technique for selecting tissue, but other fields have gradually opened up to these new methodological approaches, for instance for micro-dissecting living cells from a cell culture with the possibility of re-culturing the isolated cells [46].

De Spiegelaere W et al. Thus, from cell fragments, one of the most common applications of LAM has been the search for loss of heterozygosity LOH in malignant tumors. Cytogenetic studies, enabling analysis of the complex chromosome rearrangements that have been frequently detected in many malignancies and congenital diseases are also greatly facilitated by microdissection [50]. Proteomic analysis in tissue samples imposes certain rules in experimental protocols used for tissue processing [51]. In addition to the identification of new diagnostic and prognostic markers, this approach could help in establishing individualized treatments tailored to the molecular profile of a tumor [52].

This approach is even more powerful when it is combined with immuno-cytochemical staining using specific antibodies to label the cells of interest. The value of LAM in forensic science cannot be underestimated [53]. DNA analysis has become the prime tool for identification of the source of biological traces [54]. Most reports focus on the use of LAM to isolate a single sperm from a vaginal smear for genetic analysis in the investigation of sexual assault, the first application described by Elliott et al.

An even more challenging situation occurs when cells of a victim are much more abundant than the cells of the perpetrator. DNA analysis then becomes a crucial tool in suspect identification. Laser tweezers, often known as optical tweezers or optical traps, enable the capture and micromanipulation of microscopic particles along the three axes using the radiation pressure generated by a focused laser beam [56] [57]. The lasers used to produce optical tweezers are continuous low power lasers 0. The choice of wavelength between and nm can reduce the risk of thermal or photochemical damage.

In biological applications of optical trapping and manipulation, it is possible to remotely apply controlled forces able to catch a cell or a cellular organelle, to fix it or transport it to another cell site without inflicting optical damage [58] [59]. The optical tweezers were used to study the elasticity of the red cell membrane and to better understand how the absence or abnormality of membrane proteins could lead to a permanent deformation of these cells that could lead to promote its premature destruction [60].

One of the most useful applications has been shown to be the measure of the forces generated by kinesin and myosin, thus resolving the issue of the mechanism of kinesin walking on the submicron microtubules, or myosin on actin strands of the cytoskeleton [61]. Another field is being explored using laser ablation processes in developmental biology.

In this case, the aim is not the isolation of a part of the tissue but the alteration of the tissue itself. The idea is to alter a biological structure by mean of a laser, generally a pulsed laser. A series of high-precision laser ablation and microsurgical tissue removal experiments has been undertaken to test the functions of different parts of a biological system [ Figure 3 ]. The selective removal of cells by ablation is a powerful tool in the study of eukaryotic development-.

Ablation of two photons in drosophila embryo. The technique is called near-field scanning optical microscopy, NSOM. The concept was first suggested in in a paper that discussed the possibility of fabricating an optical aperture much smaller than the wavelength of light and positioning the aperture a distance much less than the wavelength of light from the sample. The spatial resolution is thus determined by aperture dimension rather than by diffraction, which becomes operative only in the far field.

The optical tip can take a number of forms, including specially narrowed optical fibers and hand-crafted hollow metal guides. The fabrication of tips is being aided by the use of photolithographic techniques developed in part for microelectronics applications. The tips can be spatially manipulated with atomic-scale accuracy using techniques developed for scanning tunneling microscopy STM and force microscopy. The fluorescence intensity or absorption of the sample can be measured as the tip is scanned and the signal used to generate an image in a manner similar to other scanning microscopies.

Resolutions of about 20 nm are regularly obtained with this method, which has been employed to observe single protein molecules and is being tested as a means of locating pieces of cells, such as the ribosome, that have eluded structure determination by x-ray diffraction. Localized measurements of fluorescence lifetime, described below, have also been performed using NSOM, raising the possibility of highly localized environmental probing within cells.

It should be noted, however, that the wet environment of biology makes NSOM technically more difficult to apply than with dry, solid samples. Commercialization of NSOM has begun, with at least one source of research instruments. Nonimaging microscopy is also being combined with spectroscopy. In this approach, researchers measure the phase shifts of an optical beam reflected from a sample in contact with a vibrating STM tip. The phase shift depends directly on the absorption spectrum of the molecule in the neighborhood of the tip.

Current spatial resolution is about 1 nm. This technique is at an early stage of development but demonstrates an approach that could bring optical spectroscopy truly to the level of atomic-scale spatial resolution. The future success of this endeavor will require progress in strategies for labeling specific biological sites by probe molecules and advances in nanopositioning technology. Optical microscopy studies of tissue have long relied on absorbing stains to reveal specific cellular structures of interest; without such stains, most medical histopathology would not be possible.

Traditional stains are a way of generating contrast where there is normally none; today antibody-linked fluorescent dyes are used to mark specific cell surfaces. The general strategy of many of the new optical technologies is also to tag the structure of interest with a dye molecule.

Optics in Instruments: Applications in Biology and Medicine

Much of the progress in fluorescence microscopy is linked to the development of ever more specific fluorescent probes, which may be chosen for their ability to intercalate into DNA or to label specific ions such as calcium. These fluorescent probes may be used to locate and examine specific sites by direct visualization with a microscope, or they may be sensed by a variety of optical techniques such as flow cytometry discussed below Tsien, Quantitative fluorescence-based measurement techniques are being introduced; an example is measurement of the length of long DNA molecules with fluorescence instead of pulse field electrophoresis.

Lifetime imaging is an important way to take advantage of the properties of fluorescent probes. Probe molecules can be quite sensitive to changes in their local environment; the fluorescent lifetime, fluorescence quantum efficiency, and emission wavelength of dyes can all change with environment e. The most commonly measured property is fluorescence lifetime, whose variation with the above environmental factors must be determined for each dye.

Lifetimes can be measured in the time domain using pulsed laser sources and fast detection schemes; alternatively, frequency-domain techniques can be employed to obtain equivalent information using modulated cw lasers and phase-sensitive detection. The frequency-domain approach has a significant advantage in being able to measure nanosecond-domain lifetimes using relatively simple equipment. Both techniques have been used to obtain microscopic and macroscopic images that show regions having specific lifetimes. New dye molecules with novel fluorescence properties are increasingly needed to meet the requirements of emerging technologies for visualization and other biological applications.

Ideally the dye chemist would like to control the photostability, two-photon cross section, fluorescence yield, nontoxicity, fluorescence wavelength, and lifetime of a dye. In the case of marker molecules for use with tissue in vivo, fluorescence in the wavelength region longer than about nm is desirable to.

Probes that are highly sensitive to specific chemical or physical conditions, subtle differences in pH, or the presence of chemical structures of different types are needed. For example, the systematic discovery of probes that are specific to calcium ions has allowed investigators to optically record cells responding to a variety of stimuli that induce the appearance of this ion.

Probes to target specific regions of a cell or tissue, such as the DNA of the cell nucleus or the cellular cytoplasm, are also needed. In addition to the advantage of having probes that are sensitive to chemical or physical conditions of the target, another strong driver in the development of fluorescent probes is the desire to replace traditional radioactive markers, given the rapidly rising costs of handling and disposing of radioactive tracer materials.

Every molecule undergoes some degradation when it absorbs a photon. The goal of detecting single molecules optically demands probes of even higher photostability than previously required. If a probe molecule photochemically degrades with an efficiency as low as 10 -4 , a single molecule of this probe can generate only 10 4 fluorescent photons in toto before it becomes inactive.

Combining photostability with the optical properties mentioned above provides a significant challenge. The same sophisticated new optical probes that are so useful for biological visualization also make possible the application of optical measurement and analysis methods to such biological problems as gene sorting, mapping the human genome, and investigating cellular control and communication. Flow cytometry Figure 2.

From the development of the technique in the late s to today's sophisticated research and clinical instruments, this technology has continued to make a major impact in modern-day biological research and clinical medicine. Particles to be analyzed are suspended in a liquid medium, and stains or dyes that bind to specific parts of the particle are added. Single cell suspensions can be stained for DNA content, RNA content, cell surface molecules that identify different cell types, and physiological parameters such as pH or calcium concentration.

The particles are then introduced into a fluid flow and passed through a nozzle that produces a stream of droplets containing individual particles. These particles pass through a region in which focused visible. Marrone, Los Alamos National Laboratory. Multiple sensors are used to detect fluorescence signals, which are recorded.

The sensors may also be used to detect light scattering by the particles. In some cases, signals from the sensors are used to activate an additional cell sorting process based on deflecting previously charged droplets by charged deflection plates. The development of optically based measurement techniques and new probes occurred in parallel with the application of this technology to basic biological studies and routine clinical assays. Routine clinical applications of flow cytometry fall predominantly into two categories: Immunophenotyping, the identification and enumeration of white blood cells by analysis of surface molecules with fluorescent-dye-labeled antibodies, is used in various medical applications including monitoring AIDS progression Box 2.

DNA content measurements provide clinicians with information about the number of proliferating cells in a population and the normality of the cellular DNA content. Such measurements have application in grading cancer cells and determining disease prognosis. These applications of flow cytometry in the clinical arena are used worldwide. Sorting capabilities of flow cytometers are used to physically separate large numbers of human chromosomes.

Chromosome-specific libraries have been generated for each of the human chromosomes. Such libraries are an important component of genetic engineering, a technique that allows specific genes to be inserted into cells and organisms.

Introduction - Optics in Biology and Medicine | Biophotonics Imaging Laboratory

The availability of these materials has also played an important role in the establishment and rapid progress of the Human Genome Project, which promises new understanding of the genetic basis of disease. 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 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.

It is interesting to note that flow cytometers and automated gene sequencing instruments were developed in the late s and early s, 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 typica l hospital clinical laboratory. The impact of flow cytometry on modern biomedical research is large. 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. 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.

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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. This has allowed tracking of the spread of bacteria, as well as the action of antibiotics, throughout the body of small animals.

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. 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 but was not demonstrated experimentally until the turn of the century. One reason for the delay is that radiation pressure is extraordinarily feeble. The advent of lasers in the s 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, 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. 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. 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. 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. 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 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. Oligonucleotides are small polymers made up of nucleotides, which are subunits of DNA Lipshutz et al. 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.

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. Courtesy of Affymetrix, Inc. Affymetrix and GeneChip are registered trademarks used by Affymetrix, Inc. 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 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. 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. 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. 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.

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. 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 CO 2 laser, the high-repetition-rate, frequency-doubled Nd: Lasers and optics have made possible noninvasive treatment of many diseases of the eye and have become essential to the practice of.

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.

Description

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 CO 2 and YAG surgical lasers. YAG laser offers compatibility with existing quartz fiber optics and may replace CO 2 in some cases.

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-. 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. 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.

References and links

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. NSF should increase its efforts in biomedical optics and pursue opportunities in this area aggressively.

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. Caring for the Eyes of America.

Medical laser market hits new high. Photonic detection of bacterial pathogens in living hosts. Whither minimal access surgery: Cardiovascular applications of laser technology. Optical spectroscopy of tissue-like phantoms using photon density waves. Lasers and Optical Fibers in Medicine.

Using oligonucleotide probe arrays to access genetic diversity. Excimer laser corneal surgery: New strategies and old enemies. MR imaging with hyperpolarized 3 He gas. Handbook of Biological Confocal Microscopy , 2nd ed. Excimer laser photorefractive keratectomy. Practical Flow Cytometry , 3rd ed.

Biological applications of optical forces. Technique tracks messenger molecules in living cells. News , July 18,: Fluorescence lifetime imaging microscopy FLIM: Optical instrumentation for bio-imaging is an important component of biological and medical science progress. On the other hand, biomedical applications are critical drivers for technology development in optics and photonics.

The development of optics in biological and biomedical sciences i. The range of applications is very broad as well, from medical diagnostics and treatment to basic research to understand how living organisms behave and work. The connection between application and technology development is a great challenge but also a great opportunity. In this feature issue of Biomedical Optics Express, we have collected several papers that represent some of the technologies discussed at the Bio-Optics: These papers highlight advances in diagnostic microscopy [ 1 — 5 ], optical coherence tomography [ 6 ], multimodal imaging [ 7 , 8 ] and selected applications [ 9 , 10 ].

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