Role of precision motion control in brain cell research

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Role of precision motion control in brain cell research

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Role of Precision Motion Control in<br /> Brain Cell Research<br /> by Katerina Moloni and Mike Szulczewski<br /> <br /> The close collaboration among a neuroscientist, a<br /> laser microscopy instrumentation manufacturer,<br /> and a precision nanopositioner manufacturer has<br /> resulted in an advanced near-field confocal opti-<br /> cal microscope (NCOM) that is capable of imag-<br /> ing processes occurring within living cells in real<br /> time.1 NCOM combines the capabilities of confo-<br /> cal optical microscopy with near-field scanning<br /> optical microscopy (NSOM). The result is the<br /> ability to image, in real time, processes that occur<br /> within a living nerve cell. Multiple technologies Figure 1 Left: Principle of near-field optical microscopy using a fiber-optic<br /> are merged in the NCOM, including laser confo- probe. Right: scanning electron micrograph of a typical near-field probe tip.<br /> cal photometry, which makes high-speed (10-<br /> KHz) measurements of fluorescent signals over<br /> time. Near-field optical microscopy can attain optical Light that is<br /> resolution in the 20–50 nm range. However, it has been reflected, trans-<br /> difficult to achieve such resolution in living systems. mitted, or emit-<br /> Biological near-field microscopy has been used to image ted by the sam-<br /> actin filaments in isolated nervous system glial cells with ple is detected<br /> sub-50-nm resolution (see sidebar). Under a grant from at discrete mea-<br /> the NIH (Gaithersburg, MD), Dr. Phil Haydon, a neuro- surement<br /> scientist at the University of Pennsylvania School of points during<br /> Medicine (Philadelphia, PA), is using this technique on the scan. If the<br /> living cells to study calcium channels and single vesicles probe-to-sam-<br /> in isolated nerve terminals. ple distance is<br /> within about 5<br /> Using an NSOM for confocal microscopy nm, the NSOM<br /> actin in the brain Figure 2 Laser light at 488 nm exiting an can generate<br /> The NSOM is a scanning probe instrument that improves NSOM fiber-optic probe tip. (Photograph cour- images with a<br /> the resolution of visible-light microscopy from 500 nm to tesy of Dr. Phil Haydon.) resolution of<br /> 20–50 nm. The NSOM principle is illustrated in Figure 1. about 20–50<br /> nm. Nanopositioning and precise scanning control are<br /> An optical fiber probe tip illuminates the surface being critical for a distortion-free, high-resolution image.<br /> examined. Except at the end, the probe tip is coated<br /> with reflective aluminum and acts as an optical wave- The near-field confocal optical microscope combines the<br /> guide at the laser frequency. The uncoated end becomes NSOM technique with confocal microscopy. When<br /> a fine aperture ~20–50 nm in diameter. When a sample applied to biological samples, cells and their contents can<br /> is placed in close proximity (the near-field) to the aper- be imaged with sub-50-nm resolution. In contrast to a tra-<br /> ture, subdiffraction optical resolution can be achieved. ditional microscope, which has a single image plane, con-<br /> Figure 2 is a photomicrograph taken using a conven- focal optical microscopy is able to “optically section” the<br /> tional optical microscope and shows 488-nm light exit- sample. Figure 3 illustrates the confocal optical principle.<br /> ing from a near-field probe tip. Note that this picture is<br /> for visual demonstration purposes only and that the light The laser excitation light is directed through an optical<br /> shown at the tip of the fiber has already converted back filter (dichroic), which also acts as a beam splitter. The<br /> to the far-field light (i.e., light that can produce optical laser beam passes through the microscope objective lens<br /> resolution of no better than 500 nm). and is focused at a particular focal plane in the sample.<br /> continued<br /> <br /> AMERICAN LABORATORY • DECEMBER 2003 19<br /> PRECISION MOTION CONTROL continued<br /> <br /> <br /> The specimen, such as a cell, is stained with an indicator<br /> dye that fluoresces when excited by the laser. The<br /> dichroic reflects the shorter excitation wavelengths and<br /> transmits the longer-wavelength fluorescence emission.<br /> The reflected emission enters the fiber-optic probe tip,<br /> which acts as a pinhole aperture. This aperture is abso-<br /> lutely critical in that it only allows the light from the focal<br /> plane to pass through it to appropriate detectors. Out-of-<br /> focus light originating from image planes slightly above or<br /> below the focal plane is blocked by the pinhole (dotted<br /> lines in Figure 3). This ability to reject light from above or<br /> below the focal plane enables the confocal microscope to<br /> perform depth discrimination and optical tomography. A<br /> true 3-D image can be created by combining a series of<br /> confocal images focused at successive planes through the<br /> depth of the specimen.<br /> <br /> Nanopositioning<br /> Researchers at Prairie Technologies, Inc. LLC (Mid-<br /> dleton, WI), developed the basic NCOM technology<br /> used by Dr. Haydon. The need then arose to precisely<br /> and reproducibly position the NSOM probe tip with<br /> respect to the cell surface (z-dimension) and feature<br /> location (x–y dimension). The cell must first be<br /> <br /> <br /> <br /> <br /> Figure 3 Principle of the confocal microscope.<br /> <br /> <br /> <br /> 20 DECEMBER 2003 • AMERICAN LABORATORY<br /> prises four elements: a piezo actuator that pro-<br /> vides motion, a mechanical translation mecha-<br /> nism (stage), a position sensor, and control elec-<br /> tronics to maintain the desired position. To<br /> realize positioning at nanometer levels of preci-<br /> sion, these four elements need to be carefully<br /> designed and optimized. The desired attributes of<br /> a nanopositioner are extremely high resolution<br /> and accuracy, stability, and quick response. Con-<br /> ventional motion-control technologies are<br /> unable to meet these requirements and still pro-<br /> vide nanometer positioning. The key is accurate<br /> position sensing and closed-loop control of the<br /> motion. nPoint nanopositioning devices have<br /> capacitance position sensors, piezo actuators, and<br /> flexure motion control integrated into a single<br /> system. Stages that control motion in one, two,<br /> or three axes are available, with virtually zero<br /> cross-talk between motion directions. The tech-<br /> Figure 4 A) Living glial cell actin filament bundles resolved by NCOM. B) nology provides a wide range of motion, com-<br /> Optical micrographs of 2-µm-diam actin filament. C) NCOM image of actin fila-<br /> bined with precise location and high scan speed.<br /> ment showing improved resolution. (Figure courtesy of Dr. Phil Haydon.)<br /> <br /> Results<br /> mapped in three dimensions, with the feature or fea- Using the technique termed biological near-field<br /> tures of interest located, and then their positions microscopy (BNFM), Dr. Haydon’s group has achieved<br /> recorded. Mapping involves step-and-repeat scanning optical resolution down to 50 nm on hydrated samples<br /> with z-motion at each point, much like a sewing where they have imaged actin filaments in isolated astro-<br /> machine needle. The z-motion locates the cell surface cyte glial cells (see sidebar). Examples of the resulting<br /> using photon-density feedback from a fluorescent dye images of actin filaments in living brain tissue glia cells<br /> marker in the cell. Using the 3-D map data, the tip can are shown in Figure 4. The researchers are now working<br /> then return to the region of interest with data recorded with this technique in living cells to study the micro-<br /> over time. Conventional stepper-motor positioning domains of calcium beneath individual channels, and to<br /> was not adequate for the precision needed. In a close resolve single vesicles in isolated nerve terminals. The re-<br /> collaboration with Prairie, engineers at nPoint, Inc. search will lead to a new understanding of how the living<br /> (Madison, WI) designed and perfected 2-axis and 3- brain develops (and degrades).<br /> axis piezo-driven closed-loop nanopositioners. Prairie<br /> specified the size, speed, and accuracy requirements Reference<br /> and nPoint developed and customized a positioner that 1. Doyle RT, Szulczewski MJ, Haydon PG. Extraction of near-field<br /> was reduced by 80% in size. The end result is a more fluorescence from composite signals to provide high resolution images<br /> refined positioner that can be adapted to any inverted- of glial cells. Biophys J May 2001; 80:2477–8.<br /> stage instrument and therefore can be used for applica-<br /> tions other than NSOM.<br /> <br /> A nanopositioner is basically a mechanical stage that<br /> consists of a moveable component inside a rigid frame.<br /> The moveable and static portions are electrodischarge<br /> machined from a monolithic block and connected by<br /> flexure “hinges.” The moveable component can move in Dr. Moloni is Vice President of Marketing, nPoint, Inc., 1617 Sher-<br /> any or all of three translational axes, as well as in angular man Ave., Madison, WI 53705, U.S.A.; tel.: 608-204-8756; fax:<br /> axes such as rotation or tilt. The flexure mounting ensures 608-310-8774; e-mail: Mr. Szulczewski<br /> that motion in one dimension does not cause motion in is President, Prairie Technologies, Inc., Middleton, WI, U.S.A. This<br /> any other dimension. work was a direct result of the National Institutes of Mental Health SBIR<br /> Phase I and Phase II Funding on the grant entitled “Near-Field Optical<br /> The NCOM motion control or positioning system com- Point Measurements on Living Cells,” ref. no. 5R44 MH57612-03.<br /> <br /> <br /> <br /> AMERICAN LABORATORY • DECEMBER 2003 21<br /> PRECISION MOTION CONTROL continued<br /> <br /> <br /> <br /> Role of glial cells and actin in the brain<br /> Tiny glial cells make up about half of the weight of the brain and<br /> outnumber neurons (the major brain cell of study) more than 10<br /> to 1. Recently, it has been shown that these star-shaped astro-<br /> cytes or brain glial cells provide nutrition, housekeeping, and<br /> structural support for neurons. They clean up brain debris by<br /> digesting parts of dead neurons, transport nutrients to neurons,<br /> hold neurons in place, and regulate the content of extracellular<br /> space. Over the past five years, researchers have found that glia<br /> may also be essential for the correct wiring of the brain. Experi-<br /> ments have shown that by themselves, neurons connect together<br /> poorly, but a combination of neurons and glia results in strong<br /> connections, or synapses, between neurons. In the brain, such<br /> connections allow nerve cells to pass along messages about our<br /> every sensation, thought, and movement. A weakening of these<br /> The larger objects are neurons; the smaller objects are the glial cells (opti- connections could be responsible for memory loss and other<br /> cal micrograph). symptoms of strokes and Alzheimer’s disease. When glia are<br /> prodded, they can release calcium, which may then influence<br /> communication between neurons. The calcium ions emitted by one glial cell can trigger surrounding glia to release calcium as well,<br /> spreading a signal outward like the ripples caused by throwing a stone into a pond. The calcium wave releases glutamate from the glial<br /> cells and has a direct impact on the firing of the neurons.<br /> <br /> Glutamate is a neurotransmitter, a chemical used by neurons to communicate with each other. Actin is a giant globular protein<br /> molecule that resides in every cell. Actin polymerizes to form bundles or networks of filaments. By polymerizing and depolymeriz-<br /> ing—a calcium-dependent process—actin controls cell mobility and the flow of material into and out of the cell.<br /> <br /> Glia are almost certainly needed for the formation of strong synapses as the brain develops, but the importance of this effect in the<br /> intact brain has yet to be fully understood. It is also possible that glia control the strength of synapses in the fully developed brain,<br /> intensifying some circuits and weakening others .<br /> <br /> <br /> <br /> <br /> 22 DECEMBER 2003 • AMERICAN LABORATORY<br />



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