In response to a broad-based need for point-of-care multiplex diagnostic capability, we have developed a novel hybrid platform to analyze optically encoded microspheres arranged on a 2-dimensional planar array. The microspheres which we have initially selected are developed by Luminex Inc. as substrates for sandwich-type fluorescent immunoassays and are typically used in conjunction with a customized flow analyzer. CCD-based optics are the essential feature which enables the development of a rugged diagnostic instrument which can be scaled for point-of-care applications. We have characterized the Multiplex Immunoassay Diagnostic System (MIDS) using a benchtop prototype built around a conventional 12-bit CCD. This system is capable of resolving up to 6 discrete classes of fluorescent microbeads, and measuring their corresponding reporter signal. The MIDS sensitivity to the phycoerythrin (PE) reporter compared favorably to that of the reference Luminex flow system, and is capable of identifying viral, bacterial, and protein simulants in laboratory samples, at concentrations less than 1μg/ml. The ability to
resolve small differences in the average PE fluorescence is a direct function of CCD performance, and may be a necessary trade-off for developing a portable and economical detection system. However, we are
confident that the MIDS platform can easily be scaled to meet the nominal requirements of any given point-of-care or screening application, and furthermore provide much-needed diagnostic functionality in this particular environment.
Researchers at Lawrence Livermore National Laboratory are developing means to collect and identify fluid-based biological pathogens in the forms of proteins, viruses, and bacteria. To support detection instruments, we are developing a flexible fluidic sample preparation unit. The overall goal of this Microfluidic Module is to input a fluid sample, containing background particulates and potentially target compounds, and deliver a processed sample for detection. We are developing techniques for sample purification, mixing, and filtration that would be useful to many applications including immunologic and nucleic acid assays. Many of these fluidic functions are accomplished with acoustic radiation pressure or dielectrophoresis. We are integrating these technologies into packaged systems with pumps and valves to control fluid flow through the fluidic circuit.
Optically generated acoustic waves have been used to temporarily permeate biological cells. This technique may be useful for enhancing transfection of DNA into cells or enhancing the absorption of locally delivered drugs. A diode- pumped frequency-doubled Nd:YAG laser operating a kHz repetition rates was used to produce a series of acoustic pulses. An acoustic wave was formed via thermoelastic expansion by depositing laser radiation into an absorbing dye. Generated pressures were measured with a PVDF hydrophone. The acoustic waves were transmitted to culture and plated cells. The cell media contained a selection of normally-impermeable fluorescent-labeled dextran dyes. Following treatment with the opto-acoustic technique, cellular incorporation of dyes, up to 40,000 Molecular Weight, was noted. Control cells that did not receive opto-acoustic treatment had unremarkable dye incorporation. Uptake of dye was quantified via fluorescent microscopic analysis. Trypan Blue membrane exclusion assays and fluorescent labeling assays confirmed the vitality of cells following treatment. This method of enhanced drug delivery has the potential to dramatically reduce required drug dosages and associated side effects and enable revolutionary therapies.
Stress waves generated at the end of optical fibers through thermoelastic expansion have been used for several purposes, including the destruction of blood clots, the destruction of kidney and gallstones, and the induction of cell permeability changes for drug delivery. We have undertaken the modeling of stress wave propagation in an effort to optimize the stress wave effects in these therapies. In particular, we have studied techniques to focus the stress wave in order to increase the pressure magnitude for a given pulse energy and to alter the compressive/tensile pressure ratio. This study includes the effects of acoustic wavelength and focusing fiber tip shape.
Laser induced stress waves are being used in a variety of medical applications, including drug delivery and targeted tissue disruption. Stress waves can also be an undesirable side effect in laser procedures such as ophthalmology and angioplasty. Thus, a study of the effects of stress waves on a cellular level is useful. Thermoplastic stress waves were produced using a Q-switched frequency-doubled Nd:YAG laser (lambda equals 532 nm) with a pulse duration of 4 ns. The laser radiation was delivered to an absorbing media. A thermoplastic stress wave was produced in the absorbing media and propagated into plated cells. The energy per pulse delivered to a sample and the spot size were varied. Stress waves were quantified. We assayed for cell viability and damage using two methods. The threshold laser parameters for cell damage were defined for three cell lines.
The generation of vapor-driven bubbles is common in many emerging laser-medical therapies involving soft tissues. To successfully apply such bubbles to processes such as tissue break-up and removal, it is critical to understand their physical characteristics. To complement previous experimental and computational studies, an analytic mathematical model for bubble creation and evolution is presented. In this model, the bubble is assumed to be spherically symmetric, and the laser pulse length is taken to be either very short or very long compared to the bubble expansion timescale. The model is based on the Rayleigh cavitation bubble model. In this description, the exterior medium is assumed to be an infinite incompressible fluid, while the bubble interior consists of a mixed liquid-gas medium which is initially heated by the laser. The heated interior provides the driving pressure which expands the bubble. The interior region is assumed to be adiabatic and is described by the standard water equation-of- state, available in either tabular, or analytic forms. Specifically, we use adiabats from the equation-of-state to describe the evolution of the interior pressure with bubble volume. Analytic scaling laws are presented for the maximum size and duration of bubbles as functions of the laser energy and initially heated volume.
Peter Amendt, Richard London, Moshe Strauss, Michael Glinsky, Duncan Maitland, Peter Celliers, Steven Visuri, David Bailey, David Young, Darwin Ho, Charles Lin, Michael Kelly
Formation of vapor bubbles is characteristic of many applications of short-pulse lasers in medicine. An understanding of the dynamics of vapor bubble generation is useful for developing and optimizing laser-based medical therapies. To this end, experiments in vapor bubble generation with laser light deposited in an aqueous dye solution near a fiber-optic tip have been performed. Numerical hydrodynamic simulations have been developed to understand and extrapolate results from these experiments. Comparison of two-dimensional simulations with the experiment shows excellent agreement in tracking the bubble evolution. Another regime of vapor bubble generation is short-pulse laser interactions with melanosomes. Strong shock generation and vapor bubble generation are common physical features of this interaction. A novel effect of discrete absorption by melanin granules within a melanosome is studied as a possible role in previously reported high Mach number shocks [Lin and Kelly, SPIE 2391, 294 (1995)].
The formation and evolution of acoustic waves and vapor bubbles as a result of laser irradiation have received considerable attention, particularly with respect to angioplasty, thrombolysis, and ophthalmic laser applications. Pressure waves and bubbles have been implicated in undesirable tissue damage yet they can be beneficially utilized while limiting their negative impact. Either planar or spherical pressure waves can be produced through manipulation of irradiation parameters and geometry. An OPO laser emitting approximately 5 ns pulses of visible radiation was delivered through an optical fiber to a cuvette containing dye dissolved in either water or glycerin. Absorption was varied by altering the dye concentration and wavelength of the OPO laser and the spot size was varied by employing multiple sizes of optical fiber. A nitrogen-pumped dye laser with a pulse duration of approximately 5 ns was used as an illumination source. A Mach-Zehnder interferometer technique enabled visualization and quantification of the pressure waves; bubble evolution was monitored with shadowgrams. A comparison was made between experimental and theoretical results for water and glycerin.
In various pulsed-laser medical applications, strong stress transients can be generated in advance of vapor bubble formation. To better understand the evolution of stress transients and subsequent formation of vapor bubbles, 2D simulations are presented in channel or cylindrical geometry with the LATIS computer code. Differences with 1D modeling are explored, and simulated experimental conditions for vapor bubble generation are presented and compared with data.
We present a study of the short-timescale fluid dynamic response of water to a fiber-delivered laser pulse of variable energy and spatial profile. The laser pulse was deposited on a stress confinement timescale. The spatial profile was determined by the fiber core radius, r, and the water absorption coefficient, (mu) a. Considering 2D cylindrical symmetry, the combination of fiber radius and absorption coefficient parameters can be characterized as near planar, symmetric, and side-directed. The spatial profile study shows how the stress wave varies as a function of geometry. For example, relatively small absorption coefficients can result in side-propagating shear and tensile fields.
Time-resolved infrared (IR) radiometry was used to measure surface temperatures during pulsed Er:YSGG
(l=2.79 mm) and Er:YAG (l=2.94 mm) laser irradiation of dental enamel. Scanning electron microscopy (SEM)
was used to determine the melting and vaporization thresholds and to characterize other changes in the
surface morphology. The magnitude and temporal evolution of the surface temperature during multiplepulse irradiation of the tissue was dependent on the wavelength, fluence, and pre-exposure to laser pulses. Radiometry and SEM micrographs indicate that ablation is initiated at temperatures well below the melting and vaporization temperatures of the carbonated hydroxyapatite mineral component (1200 °C). Ablation occurred at lower surface temperatures and at lower fluences for Er:YAG than for Er:YSGG laser irradiation: 400 °C vs. 800 °C and above 7 J/cm2 vs. 18 J/cm2, respectively. However, the measured surface temperatures were higher at l=2.79 mm than at l=2.94 mm during low fluence irradiation (<7 J/cm2). Spatially dependent absorption in the enamel matrix is proposed to explain this apparent contradiction.
We review the basic laser ablation processes of dental hard tissue for wavelengths ranging from the IR to the UV. The underlying tissue removal mechanisms extend from water- mediated explosive, to thermomechanical, to plasma-mediated processes. This discussion is based on a literature review of the current state of hard tissue removal under various irradiation conditions combined with some new data using surface temperature measurements. The most effective tissue removal mechanism is the water-mediated explosive process in the IR at wavelengths between 3 and 10 micrometers . Highly controlled tissue removal at low ablation rates can be obtained in the near IR (around 1 micrometers ) using plasma-mediated ablation, provided the irradiation parameters are chosen appropriately. Similarly small ablation rates combined with good tissue specificity characterize the ablation in the UV region of the spectrum. The ablation mechanism in the UV is largely dominated by photothermal processes, although photochemical and thermomechanical processes may contribute.
Dental hard tissues can be ablated efficiency by (lambda) equals 3 micrometers laser irradiation with minimal subsurface thermal damage. However, the potential of lasers operating in the region of the infrared for caries preventive treatments has not been investigated. In this study, the caries inhibition potential of Er:YAG ((lambda) equals 2.94 micrometers ) and Er:YSGG ((lambda) equals 2.79 micrometers ) laser radiation on dental enamel was evaluated at various irradiation intensities. Pulsed IR radiometry and scanning electron microscopy (SEM) were used to measure the time-resolved surface temperatures during laser irradiation and to detect changes in the surface morphology. The magnitude and temporal evolution of the surface temperature during multiple pulse irradiation of the tissue was dependent on the wavelength, irradiation intensity, and the number of laser pulses. Radiometry and SEM micrographs indicated that ablation was initiated at temperatures of approximately 300 degree(s)C for Er:YAG and 800 degree(s)C for Er:YSGG laser irradiation, well below the melting and vaporization temperatures of the carbonated hydroxyapatite mineral component (m.p. equals 1200 degree(s)C). Nevertheless, there was marked caries inhibition for irradiation intensities below those temperature thresholds, notably 60% and 40% inhibition was achieved after Er:YSGG and Er:YAG laser irradiation, respectively. These results indicate that the Er:YSGG laser can be used effectively for both preventive dental treatments and for hard tissue removal.
Recently there has been much interest in lasers and their potential use to replace the dental drill. The research has been directed towards vital dental tissues. It must be understood that any laser to be used in dentistry which will replace the dental drill must also ablate and remove existing dental materials. Some concern exists about the ablation products when the Er:YAG laser is used to ablate dental materials. It is incumbent on the professionals using these lasers to understand the materials being produced by these lasers and protect themselves and their patients from possible toxic products. It is the intent of this paper to evaluate the products produced by the ablation of both dental amalgam and composite dental restorative materials and compare them with those produced by the traditional dental handpiece (drill).
The erbium:YAG laser coupled with a cooling stream of water appears to be an effective means of removing dental hard tissues. However, before the procedure is deemed clinically viable, there are several important issues of safety and efficacy that need to be explored. In this study we investigated the surface that remains following laser ablation of dentin and compared the results to the use of a dental handpiece. Specifically, we studied the effect the laser radiation had on the bonding of composite to dentin. The crowns of extracted human molars were removed revealing the underlying dentin. An additional thickness of material was removed with either a dental handpiece or an Er:YAG laser by raster scanning the samples under a fixed handpiece or laser. Comparable surface roughnesses were achieved. A cylinder of composite was bonded onto the prepared surfaces following the manufacturer's directions. The dentin-composite bond was then shear stressed to failure on a universal testing apparatus and the maximum load recorded. Preliminary results indicated that laser irradiated samples had improved bond strengths. SEM photographs of the surfaces were also taken to compare the two methods of tooth preparation.
Any laser that will be used by dentist to replace the dental drill (handpiece) must remove dental hard tissues safely. These lasers must also have the ability to ablate the restorative dental materials which are present in the teeth being treated. Prior to any laser being used to treat humans a thorough knowledge of the effects of the laser treatment on dental materials must be understood. Cores of dental amalgam were created and sliced into thin wafers for this experiment. Ablation efficiency and thermal changes were evaluated with and without water. It appears as if the Er:YAG laser can effectively ablate amalgam dental material with and without water. The water prevents the temperature from increasing much above baseline and does not reduce efficiency of ablation.
It is understood that if a laser is to replace the dental high speed handpiece it must be able to ablate dental materials which are present in teeth being treated with the laser. It is the intent of this paper to evaluate the effects of the Er:YAG laser on dental composite restorative material concentrating on the etch rate with and without waterspray. Composite dental material is used to form plugs of known thickness and the etch rate of the Er:YAG laser on this material is determined. The results are compared with those obtained from studies of the Er:YAG on dentin and enamel. In these studies the water reduced the efficiency of the Er:YAG laser 15 - 20% on these tissues.
Erbium lasers can effectively cut hard biological tissues despite the limited water content of these materials. However, deposition of thermal energy into the tissue can be a concern for tissue such as teeth. It has been demonstrated in animal models that permanent damage can occur with a pulpal temperature rise of 5 degree(s)C. Therefore, it is necessary to cool such materials during the ablation process to avoid a buildup of thermal energy. an Er:YAG laser was used to ablate hard dental materials, such as dentin and enamel, in vitro. Temperature measurements were made by inserting a thermocouple probe at various locations within teeth. a fine stream of water flowing over the irradiation site was an effective means of cooling the teeth and did not limit the ablation rate significantly over the range of flow rates tested. Temperature rises near the ablation site were limited to less than 5 degree(s)C with the water spray; temperature rises of greater than 20 degree(s)C were seen with no water spray. Moderate variation in the water flow rate had a minimal effect on the temperature rise; most of the thermal energy was convected away with water flow rates as low as 5 ml/min.
The object of this paper is to evaluate the thermal effect of the Er:YAG laser on human teeth and discuss the preliminary studies of the effects of water on the ablation efficiency of the Er:YAG laser. Human extracted teeth were sectioned into varying thicknesses in a horizontal plane and placed in a holder and aligned in a position where the laser was in focus on the surface of the tooth. First the laser was set at a known energy and the number of pulses needed to ablate through the known thickness of tooth were counted and a proportion of energy vs. ablation depth (etch rate) was determined. After etch rate was determined a thermocouple was placed on the back surface of the tooth section in line with the laser. The laser was then used to cut into the tooth just above the thermocouple and the temperature determined with and without water.
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