The Optics of Life: A Biologists Guide to Light in Nature

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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.

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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.

A quick guide to light microscopy in cell biology

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.

Proteins such as. 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.

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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.

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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.

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Laser scanning confocal microscopy, described previously, is. Courtesy of Affymetrix, Inc. All rights reserved. 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.

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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. 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. 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.