Abstract: Analytical reagents and mass spectrometry-based methods using these reagents for the rapid, and quantitative analysis of proteins or protein function in mixtures of proteins. The methods employ affinity labeled protein reactive reagents having three portions: an affinity label (A) covalently linked to a protein reactive group (PRG) through a linker group (L). The linker may be differentially isotopically labeled, e.g., by substitution of one or more atoms in the linker with a stable isotope thereof. These reagents allow for the selective isolation of peptide fragments or the products of reaction with a given protein (e.g., products of enzymatic reaction) from complex mixtures. The isolated peptide fragments or reaction products are characteristic of the presence of a protein or the presence of a protein function in those mixtures. Isolated peptides or reaction products are characterized by mass spectrometric (MS) techniques.

Abstract: The present invention is directed to methods for analyzing one or more fluid samples for the presence, amount or identity of one or more analytes optionally present in said samples, which uses a device having one or more round wells with a fixed diameter, the wells exposing a substrate of a specific thickness, whereby the substrate has oriented through going channels, in the area of the substrate exposed in the well is provided with at least one binding substance specific for a least one of said analytes. Using the device the sample fluid is forced to pass through the channels in the substrate from the upper side of the substrate to the lower side of the substrate and back at least one time, under conditions that are favorable to a reaction between any analyte present in the sample and the binding substances.

Abstract: Microarrays are prepared by using a separate fiber for each compound being used in the microarray. The fibers are bundled and sectioned to form a thin microarray that is glued to a backing.

Abstract: A sample lab for retaining a sample during spectrophorometric analysis includes a base plate, a sample well, and a cover plate. The sample tab may also include one, or more than one overflow opening for draining excess sample, and an overflow channel to retain the excess sample.

Abstract: A micromechanical actuator for sorting hematopoietic stem cells for use in cancer therapies. The actuator operates by diverting cells into one of a number of possible pathways fabricated in the fabrication substrate of the micromechanical actuator, when fluorescence is detected emanating from the cells. The fluorescence results from irradiating the cells with laser light, which excites a fluorescent tag attached to the cell. The micromechanical actuator thereby sorts the cells individually, with an operation rate of 3.3 kHz, however with the massively parallel 1024-fold device described herein, a throughput of 3.3 million events/second is achievable.

Abstract: An imaging system for automation of sample monitoring includes an image capture device that cooperates with a lens assembly for imaging the samples. A translator positions the lens assembly and/or a sample plate mount for acquisition of the images. Some embodiments of the system include a light source configured to provide low temperature, flash illumination of a sample plate.

Abstract: The present invention relates to a kit for determining feline blood type, wherein the kit includes a mixture comprised of a first monoclonal antibody and a second monoclonal antibody, wherein both antibodies recognize feline blood group specific A antigens. The present invention also relates to a method for determining feline blood type, wherein the method utilizes two distinct monoclonal antibodies, which recognize feline blood group specific A antigens.

Abstract: The present invention provides an apparatus and method for storing a particle-containing liquid. The storage apparatus comprises a microfluidic convoluted flow channel having a plurality of particle capture regions. The storage channel is preferably an isotropic spatially periodic channel. Sedimented particles can be resuspended following storage. This invention further provides a microfluidic analysis cartridge having a convoluted storage channel therein. The sample analysis can use optical, electrical, pressure sensitive, or flow sensitive detection. A plurality of analysis channels can be included in a single cartridge. The analysis channels can be joined to reagent inlets for diluents, indicators or lysing agents. A mixing channel can be positioned between the reagent inlet and the analysis region to allow mixing and reaction of the reagent. The cartridge can include additional valves and pumps for flow management.

Abstract: The present invention relates to a method for determining crystallization in a very small amount of a material of interest (eg a chemical or biological material of interest).

Abstract: The present invention relates to a device for use in fluid assay, in particular for assaying body fluids such as blood and urine. The device comprises a sample reservoir for a fluid sample to be assayed, the reservoir having an inlet port and an outlet port. The device comprises a fluid sample receiving zone in fluid communication with the reservoir via the inlet port and an excess fluid sample detection zone in fluid communication with the reservoir via the outlet port.

Abstract: A specimen collecting and testing device has a housing containing a test membrane. An elongated handle having a foam member is slidably received in the housing. Once the foam member obtains a specimen, the specimen is expelled from the foam member and delivered to a fluid chamber positioned along the test membrane. The device is then positioned horizontally and specimen that collects in the fluid chamber passes through an aperture in the fluid chamber and onto the test membrane located beneath the aperture. The test membrane may then be observed for optical changes in the case of an instant-type test, or removed for further processing. The device is particularly useful for collecting samples of urine or oral fluid for use in drug testing, pregnancy testing and the like; for testing of specimen for any of a variety of analytes such as HIV, hepatitis B, hepatitis C, etc.; and, for collecting samples of DNA for PCR testing.

Abstract: Devices and methods for utilizing dry chemistry dye indicator systems for body fluid analysis such as glucose level in whole blood are provided by incorporating a porous membrane with a skin side which enables separation of whole blood and visually reading the indicator without removing the red blood cell portion of the blood from the membrane. The devices also enable visual reading of the indicator by use of a membrane or matrix which provides separation of whole blood in a lateral flow of the blood through the matrix from the input area to a test area of the matrix. The devices also provide for microtitration of fluid samples in fixed volumetric openings containing indicator reagent. Another aspect of the device provides a determination of hematocrit level in whole blood in combination with indicator indication of analyte concentration which can be compensated for the hematocrit level. The devices provided are low cost.

Abstract: A method and apparatus for assaying a drug candidate with a biosensor having one or more sensing surface-bound biomolecules associated therewith are disclosed. The method comprises the steps of measuring the binding interaction between the drug candidate and the one or more sensing surface-bound biomolecules of the biosensor to obtain an estimate of at least one binding interaction parameter of the drug candidate, and then comparing the estimated binding interaction parameter against a mathematical expression correlated from binding interaction data associated with known drug compounds to determine an estimate of at least pharmacokinetic parameter of absorption, distribution, metabolism, or excretion (ADME) that is related to the drug candidate. The present invention allows for the simultaneous measurement of different pharmacokinetic parameters of the drug candidate, as well as an indication of the drug candidate's solubility, by use of a single analytical instrument.

Abstract: An assaying device composed of a test cartridge/cassette and cap/cover. When attached, the cap/cover seals the top of a sample well/opening on the cartridge/cassette in a fluid tight manner, to protect from contact with and contamination of the tested sample (for example, urine for drug testing), provide easier, cleaner handling of the test cartridge/cassette and prevent intermixing of another sample.

Abstract: A multi-slide assembly includes various components that alone or in combination facilitate testing of test samples with minimal manipulation of assay components. Moreover, the various device components permit centrifugation, culturing and analysis of each test sample to be performed in the same assembly. A frame retains a plurality of reaction vessel assemblies between a pair of opposing channels that are configured to slidably receive the opposing ends of each reaction vessel assembly. A strip cap seals the openings in each reaction vessel using cap members having sealing rings circumscribing the external walls thereof to form compression seals with internal walls of the reaction vessels. Furthermore, in a method of making a multi-well slide assembly, an ultrasonic welding process removably bonds a plurality of wells to a slide plate to provide an adequate seal therebetween during centrifugation, yet still enable separation thereof by an operator.

Abstract: Disclosed is an automated staining apparatus (autostainer) capable of staining sample on a microscope slide. The autostainer is capable of limiting the amount of reagent used through a process including image acquisition and processing techniques, wherein an image of the slide is acquired and used to determine a region on the slide to be stained. Also disclosed are methods of using images acquired by the autostainer in sample processing. In addition, a system including the autostainer functionally linked to an imaging system is provided.

Abstract: A method and a device for analyzing a medical fluid in a closed bag set. The bag set has an analysis device connected to integrally connected to a bag containing the medical fluid via a connection tube. The analysis device has an expandable air pocket having a reagent on an interior surface. When the medical fluid is to be tested, the air pocket is expanded and the medical fluid flows through the connection tube and into the air pocket and contacts the reagent material. The connection tube may have a valve or frangible pin or membrane preventing the medical fluid from entering the connection tube until the test procedure is started. A reaction of the reagent to the medical fluid may be detected by an optical apparatus such as a CCD or CMOS optical sensor. The air pocket may be enclosed by a cassette for exerting an under-pressure at the outer surfaces of the air pocket in order to draw medical fluid into the analysis device.

Abstract: Methods, compositions and kits are disclosed. The methods are directed to determining the presence of one or more analytes in a sample suspected of containing any one of a plurality of the analytes. A combination is provided comprising in a medium (i) the sample, (ii) a binding partner for each of the analytes, (iii) for each of the analytes, a first reagent comprising a member of a signal producing system, a ligand and an analyte analog, and (iv) a second reagent comprising a binding partner for the ligand. The binding of the binding partner for the ligand is affected by the presence of an analyte and alters the amount of signal produced by the member of the signal producing system. The signal thus is modulated if one or more of the analytes are present in the sample. The amount of the signal is determined and is related to the presence of one or more of the analytes in the sample. The method may be homogeneous or heterogeneous.

Abstract: A system for detecting and analyzing patient sample quality and/or analytes, while the sample is in the metering tip used to aspirate the sample liquid from an original patient sample container, and also to dispense the liquid onto a slide test element. Spectrophotometric analysis may be done on the sample liquid while it is still in the tip which has been converted into a cuvette. One technique for such analysis is by scanning the cuvette for transmittance in a light-tight enclosure. Near-infrared and adjacent visible radiation may be used, and the absorption spectra of the liquid detected and analyzed. A possible aspect of the present invention relates to enhancing throughput of an analyzer by conducting a sample quality measurement in a process that is parallel to the main analyzer timing cycle.

Abstract: A method for measuring the hemoglobin concentration in the blood in a circuit of a dialysis machine includes directing electromagnetic waves against one section of the circuit, measuring values of the electromagnetic waves transmitted through the section, and determining values of absorption of electromagnetic waves by the blood along the section from the measured values of transmitted electromagnetic waves. The values of absorption are correlated with the values of the hemoglobin concentration. The method also includes measuring the values of at least one physical quantity of the blood, wherein the at least one physical quantity is selected from blood pressure and blood temperature, and calculating the values of hemoglobin concentration as a function of the values of absorption and the at least one physical quantity.

Abstract: A method and apparatus for screening an array of test compounds for bioactivity by contacting an array of test compounds with a detector layer capable of detecting bioactivity, and detecting a detector layer response. The detector layer is comprised of physiologically viable cells. The method and apparatus allow a large number of test compounds to be simultaneously assayed in parallel without the need for complex fluidic devices.

Abstract: An analysis device, preferably in the form of a floating buoy, is disclosed for the analysis of fluids, such as waste water. The device has a main body portion and a removable cartridge containing consumable ingredients for use in analysis.

Abstract: In one embodiment of the invention, methods and apparatus for packaging microarray suspended in a well of a well plate are provided. In another embodiment of the invention, microarrays can be successfully scanned with its target surface up or down.

Abstract: A compact sensor for detection of chemical and/or biological compounds in low concentration. The sensor comprises electromagnetic microcavities. The agent to be detected passes the microcavities, is absorbed and/or absorbed by the microcavities, and modifies the electromagnetic field inside the microcavities. After the agent has been adsorbed and/or absorbed, a probe beam is applied to the microcavities. The change of electromagnetic field is detected by the detector, and the frequency of the probe beam at which the resonance is observed, is indicative of a particular agent being present. A method for detecting chemical and/or biological compounds using the sensor.

Abstract: Apparatus and method for determining at least one parameter, e.g., concentration, of at least one analyte, e.g., urea, of a biological sample, e.g., urine. A biological sample particularly suitable for the apparatus and method of this invention is urine. In general, spectroscopic measurements can be used to quantify the concentrations of one or more analytes in a biological sample. In order to obtain concentration values of certain analytes, such as hemoglobin and bilirubin, visible light absorption spectroscopy can be used. In order to obtain concentration values of other analytes, such as urea, creatinine, glucose, ketones, and protein, infrared light absorption spectroscopy can be used. The apparatus and method of this invention utilize one or more mathematical techniques to improve the accuracy of measurement of parameters of analytes in a biological sample.

Abstract: An apparatus for holding a sensor for sensing the contents of a vessel is provided. The apparatus has an extended position where the sensor is exposed to the contents and a retracted position for cleaning the sensor. The apparatus includes a process connection configured to connect the apparatus to the wall of the vessel. The process connection defines an aperture including a first rim, a second rim angularly displaced from the first rim, and a sidewall extending between the first rim and the second rim. The sidewall is angularly displaced from the first rim by no less than 135 degrees.

Abstract: In order to provide an automatic analyzer which ensures accurate absorbance measurement even when ultrasonic wave intensity for agitation of the sample and reagent or the like becomes excessive, multiple reaction vessels 8 are placed as follows: As viewed from the top of the reaction disk 15, the reaction disk 15 is divided into four parts, and the side wall of the reaction vessel 8 does not intersect with two light beams 20 which intersect with each other at right angles. Herein multiple reaction vessels 8 are located at an inclined position approximately at an angle of about 45 deg. This layout allows the reaction vessel 8 to have the surface exposed to ultrasonic wave 22 intersecting to the applied ultrasonic wave appropriately at right angles, and absorbance measuring surface 21 intersecting to the applied measurement wave appropriately at right angles.

Abstract: The present invention involves an optical assay device and method of use for the detection of an analyte of interest in a sample that conveniently allows control of the flow characteristics of the sample through the device without significant user intervention. The optical assay device includes a base having an absorbent material, and a member having an optically active test stack that is rotatably coupled to the base for rotation between a lowered position and a raised position. In the lowered position, the optically active test stack contacts the absorbent material for drawing the sample through the surface. In the raised position, the optically active test stack does not contact the absorbent material.

Abstract: A method of using a fluid-transfer collection assembly includes providing a fluid-transfer collection assembly including an inlet for receiving one or more fluids, the inlet including an inlet check valve, an outlet including an outlet check valve, a test media, and a depressable, flexible bulb pump located between the inlet and the outlet and including an interior; depressing and releasing the bulb pump to draw one or more fluids into the interior of the bulb pump through the inlet check valve; and depressing the bulb pump again to impart a positive pressure in the interior of the bulb pump to cause one or more fluids in the interior of the bulb pump to exit the interior of the bulb pump through the outlet check valve and be transferred to the test media.

Abstract: A miniaturized integrated sensor (50) useful for indicating the presence of a sample analyte is disclosed. The sensor (50) has a platform (52) with an upper surface (53) and a detector (62), light source (60), waveguide (58), and reflective fixtures (60, 62) embedded in the platform (52). The light source (60) is preferably a light emitting diode and sits in a cup-shaped dimple (68) that directs light from the light source (60) toward one of the reflective fixtures (64) to uniformly distribute light across the waveguide (58). The waveguide (58) is coupled to an upper surface (53) of the sensor platform (52) and is coated with a thin film of indicator chemistry (70) which interacts with the sample analyte to produce optic signal changes that are measurable by the detector (62). A lead frame (51) in the platform (52) has pins (54, 55, 56) which provide the interface to the outside world.

Abstract: An apparatus for monitoring specific substances in a fluid is provided. The apparatus of the present invention includes a housing, a tray, transmission means, a cylindrical transparent reservoir, a cylindrical holder having a plurality of hollow frames around the surrounding thereof each of which holding a test paper, and an optical scanning assembly. The tray is disposed in the housing and rotated by the transmission means. The cylindrical transparent reservoir contains a fluid sample to be analyzed and is held on the tray so as to synchronously rotate with the tray. The cylindrical holder is disposed in the cylindrical transparent reservoir. The optical scanning assembly is fastened beside the cylindrical transparent reservoir for capturing an image of a pattern displayed on each test paper in response to at least one specific substance in the fluid sample upon rotating the cylindrical transparent reservoir and converting the image into electronic signals.

Abstract: A method for counting leukocytes which comprises liberating elastase from granulocytes contained in a specimen, adding an anti-granulocyte elastase antibody to the thus liberated elastase, measuring the antibody bonded to the elastase to thereby determine the concentration of the elastase, and then calculating therefrom the number of leukocytes contained in the specimen with the use of the ratio of the leukocyte count to the elastase concentration. This method makes it possible to conveniently and less expensively count leukocytes at a high accuracy comparable to the one established by using a conventional automatic blood cell counter, as the figure shows.

Abstract: One aspect of the present invention is a multi-well platform for fluorescence measurements, comprising a plurality of wells within a frame, wherein the multi-well platform has low fluorescence background. Another aspect of the present invention is a system for spectroscopic measurements, comprising reagents for an assay and a multi-well platform for fluorescence measurements. A further aspect of the present invention is a method for detecting the presence of an analyte in a sample contained in a multi-well platform by detecting light emitted from the sample. Another aspect of the present invention is a method from identifying a modulator of a biological process or target in a sample contained in a multi-well platform by detecting light emitted from the sample. Another aspect of the present invention is a composition identified by this method. A further aspect of the present invention is a method to identify a therapeutic.

Abstract: The present invention provides a method of detecting human antibodies in a sera solution. The invention also provides a method of quantitating anti-glycolipid antibody levels in solutions. The invention provides a method of diagnosing disease states, including neurological diseases, by quantitating a subject's antibody levels.

Abstract: Systems and methods for establishing and/or maintaining the accuracy of a multivariate calibration model designed for quantitative optical spectroscopic measurement of attributes or analytes in bodily tissues, bodily fluids or other biological samples, which are particularly useful when the spectral absorbance of the attribute or analyte is small relative to the background. The present invention provides an optically similar reference sample to reduce the effect of instrument or environment variation on the measurement capability of the model. The optically similar reference can be a gel composition having scattering particles suspended therein. The reference gel can be directly applied to a spectroscopic instrument sampler, or can be in a container specifically designed for optimal coupling to a spectroscopic instrument.

Abstract: There is disclosed a measuring system that is for use with an automatic chemical analyzer and accomplishes accurate measurement reproducibility and low cost. The system has a turntable on which plural reaction cells are arranged circumferentially. A light source and a spectroscopic detector are located on opposite sides of the reaction cell in a detection position. The amount of light transmitted through the cell is measured. A motor having an encoder is connected to the turntable. The output signal from the detector corresponding to each reaction cell is accepted in response to the output signal from the encoder.

Abstract: The measurement of the wavelength shifts in the reflectometric interference spectra of a porous semiconductor substrate such as silicon, make possible the highly sensitive detection, identification and quantification of small analyte molecules. The sensor of the subject invention is effective in detecting multiple layers of biomolecular interactions, termed “cascade sensing”, including sensitive detection of small molecule recognition events that take place relatively far from the semiconductor surface.

Abstract: A blood cell detector includes an orifice section having a single orifice, a first supplying section for supplying a first blood specimen into the orifice section, a second supplying section for supplying a second blood specimen into the orifice section, and first and second electrodes provided on opposite sides of the orifice for detecting a change in impedance of each of the first and second blood specimens when the first and second blood specimens are selectively caused to pass through the orifice.

Abstract: The invention discloses an apparatus and a method for determining the volume of individual red blood cells or other particles that are suspended in liquids. The sample is disposed into an optical cuvette suitable for microscopic analysis. An absorbing dye is added that does not leak into the cells, and that is able to absorb light at wavelengths that are only weakly absorbed by the cells. The cell volume is determined using transmitted light intensity values measured (i) in a first area comprising a single cell, (ii) in a second area close to that cell, and (iii) in said second area, after changing the cuvette thickness by a known amount.

Abstract: The invention discloses a method and apparatus for determining the volume of single red blood cells or other particles that are suspended in liquids. The sample is disposed into an optical cuvette suitable for microscopic analysis. A fluorescent dye is added that does not leak into the cells, and that is able to absorb excitation light and emit fluorescence light at wavelengths that are only weakly absorbed by the cells. The cell volume is determined using fluorescence intensity values measured (i) in a first area comprising a single cell, (ii) in a second area close to that cell, and (iii) in said second area, after changing the cuvette thickness by a known amount.

Abstract: A weathering test method comprising making active oxygen and light simultaneously act on a test piece, a weathering test method comprising successive and/or alternate steps of making active oxygen and light simultaneously act on a test piece and making at least one of light, oxygen, and water to act on the test piece, and apparatus for carrying out the methods are disclosed. In evaluating weatherability of organic materials, articles made of organic materials or articles coated with organic materials, the methods and apparatus achieve acceleration of deterioration of test pieces to greatly reduce the testing time.

Abstract: A blood test container and a blood test method are provided which facilitate the procedures starting from blood collection and ending with measurement of various components present in the blood while eliminating a risk for a tester to come into contact with the blood.

Abstract: A diagnostic device for use in performing clinical tests on a fluid, including a light transmitting plate and a panel adhered to and substantially covering the plate. The panel has an opening therein formed and a strip of pH testing material, substantially smaller than the panel, is fixed between the plate and the panel to fill substantially the opening, wherein the pH testing material produces a color indicative of fluid pH upon contact with the fluid. A window formed in the panel exposes a portion of the plate wherein the panel forms a wall on the perimeter of the window to confine fluid applied to the plate, within an area defined by the window.

Abstract: A diagnostic kit for use in performing clinical tests on a fluid specimen includes slides with attached slipcovers, tests solutions for facilitating microscopic analysis procedures, a differential diagnosis chart to provide an immediate visual indication of any microscopy including syndrome identifiers and diagnostic criteria, specimen obtaining sterile applicators, and test result cards. Each individual slide is a transparent plate covered by a panel overlay that is sufficiently thick for defining a plurality of test site wells. Indicia indicators disposed on the panel overlay adjacent to each individual test site well, provides technical assistance in the performance of diagnostic analysis on a fluid specimen deposited within each test site well. Each test site well is sufficiently deep to confine the fluid specimen as well as a measured drop quantity of one or more of the test solutions.

Abstract: Electrokinetic devices having a computer for correcting for electrokinetic effects are provided. Methods of correcting for electrokinetic effects by establishing the velocity of reactants and products in a reaction in electrokinetic microfluidic devices are also provided. These microfluidic devices can have substrates with channels, depressions, and/or wells for moving, mixing and monitoring precise amounts of analyte fluids.

Abstract: A system and method for on-site drug testing rapidly and objectively performs a drug screen on a urine sample at a collection center. The system includes a plurality of collection centers, wherein each collection center includes a test station for performing the drug testing. The test station includes collection cups for holding urine samples, test kits which couple to the collection cups in a fixed position, and an automatic reader which holds the collection cups and test kits in a fixed position under a predetermined level of illumination and produces an electronic image of the test kit. The test station also includes a computer workstation and a bar-code reader. The computer workstation generates electronic test order forms and receives image data from the automatic imaging system to evaluate the test results as indicated on the test kits. The bar-code reader is used to enter bar-code information which associate the collection cups and the test kits with the respective people being tested.

Abstract: A process, a test kit, calibration standards, and the method of preparing such standards as useful for the qualitative and/or quantitative determination of total solid phase- or microparticle-immobilized, amine-containing reactants such as proteins is provided. The operating principle of the invention is distinct from a classical immunoassay based on antibody-antigen immune interaction or from standard calorimetric proteins assays of soluble proteins and allows the detection of as low as attogram (10−18 gm) levels of total analyte per microparticle.

Abstract: A surface plasmon resonance apparatus for detecting a soluble analyte (e.g. a protein) or a particulate analyte (e.g. a cell), the apparatus comprising: (a) a sensor block adapted to receive a sensor, said sensor, for example a sensor slide, having a metallized sensor surface capable of binding the analyte; (b) a light source capable of generating an evanescent wave at the sensor surface of a sensor slide on the sensor block; (c) a first detector capable of detecting light from the light source which is internally reflected from the sensor surface; and (d) a second detector (e.g. a video camera) capable of detecting light scattered or emitted from an analyte bound thereto. Optionally the apparatus further comprises a second light source for increasing the intensity of the light scattered or emitted from an analyte bound to the sensor surface, preferably, this is sited to such as to minimize the amount of light transmitted therefrom which is detected by the first detector.

Abstract: In a test strip measuring system, such as for measuring the concentration of certain substances in body fluids, the system consists of a test strip onto which the fluid to be investigated is applied, and a measuring device into which the test strip is inserted for making the desired measurements. The system further includes a code carrier with a memory storing information concerning the manufacturing of the test strip and/or the measurement to be carried out in using the test strip. Still further, the system includes an adapter through which the code carrier is connectable with a data processing unit for the measuring device. On one hand, the adapter allows one and the same code carrier to be used with different measuring device and on the other hand allows different code carriers to be used with one and the same measuring devices.

Abstract: A device for identifying analytes in a biological sample, including a substrate having a surface lying substantially in a first plane, a plurality of targets, each having a wall lying substantially in a second plane offset from the first plane, and a receptor coating applied to one of the surface and the target walls for binding analytes present in the biological sample when the biological sample is applied to the substrate. A laser beam is sequentially directed onto each of the plurality of target, the laser being positioned relative to the substrate such that when the beam is directed onto a target, a first half of the beam is reflected back to the laser from the wall of the target and a second half of the beam is reflected back to the laser from the surface of the substrate adjacent the target.