Abstract: A method and apparatus for autofocus on a target layer contained within a microplate well is provided. The instrument is capable of optically sensing a reference point on the underside of a microplate. This reference point is then used to focus light onto a target layer within the microplate well, the target layer having a location that is in defined relation to the reference point. The reference point is either a surface of the bottom of the microplate well or is an optically detectable mark on the underside of the microplate. In an alternate embodiment, a light position sensitive detector is used to enable deterministic autofocus for a plurality of wells on a microplate.

Abstract: An apparatus and method of use for assaying cellular motility in response to a concentration gradient of a chemotactic agent. Generally, the apparatus includes a chamber having a region for receiving a biological sample containing cells of interest and, spaced apart from such region, another region for receiving a chemotactic agent. Between these regions, a concentration gradient of chemotactic agent is established. The apparatus further includes an optical system for detecting and mapping the positions of individual cells responsive to such concentration gradient. Means for processing and analyzing the collected data are also provided. Motility determinations may be made on purified or unpurified samples. Single-site assay devices and multi-site, high-throughput assay devices are disclosed.

Abstract: A method and assay system for the displacement of background from the area surrounding target material allows more sensitive detection. The invention is for use with a system in which specific targets localized at a depth within a container are detected in a liquid containing background material that produces an optical background signal. A displacement liquid is introduced that has a density selected to form a layer encompassing the target material and displacing the material that produces the background signal. The layer of the displacement liquid is sufficient to displace background material from a depth exceeding the depth of field of the detection system. The target material is then optically detected at the depth containing the target material with diminished background allowing greater detection sensitivity.

Abstract: A method and apparatus for autofocus on a target layer contained within a microplate well is provided. The instrument is capable of optically sensing a reference point on the underside of a microplate. This reference point is then used to focus light onto a target layer within the microplate well, the target layer having a location that is in defined relation to the reference point. The reference point is either a surface of the bottom of the microplate well or is an optically detectable mark on the underside of the microplate. In an alternate embodiment, a light position sensitive detector is used to enable deterministic autofocus for a plurality of wells on a microplate.

Abstract: An energy director structure with an auxiliary weld depth limitation feature is provided. The energy director is located on a first contact shelf. A second contact shelf that is shorter, broader, forming a mesa is positioned laterally adjacent to, and below, the first contact shelf. The second contact shelf forms a mesa that limits the depth of the ultrasonic welding. Limiting the time, pressure, or temperature of the welding process additionally ensures the weld depth is limited to the height of the second contact shelf or mesa. Two plastic pieces, one of which includes an energy director and mesa structure, may be welded together to form a capillary having precise volumetric accuracy.

Abstract: A method and compound for detecting low levels of microorganisms in biological samples is disclosed. In the method, an antibiotic is conjugated to a detectable label. This antibiotic/label conjugate is then introduced into a sample containing biological material. The antibiotic binds to target microorganism where the label allows for detection of localized concentrations of the antibiotic. A compound to accomplish this method is also described. This compound is an antibiotic conjugated to a fluorescent dye. This dye has an excitation and emission wavelength that are not interfered by substances typically found in biological samples.

Abstract: A method and an apparatus for analyzing a material within a container, such as blood within a capillary in a volumetric cytometry system provides for detecting the edges of the container, counting the cells within the container, characterizing the cells within the container, and evaluating channels of data which contain information relevant to more than one of the detectable characteristics of the cells. A scanner scans a container of material including certain cells. Sampling circuitry is coupled to the scanner to generate scanned images of the material in the container. Two or more scanned images are generated based on fluorescence data from dyes that have overlapping spectra. The two scanned images are processed using a linear regression analysis among corresponding pixels in the scanned images near certain cells to characterize relative contents of two fluorescing dyes in a target cell.

Abstract: An assay and sample mixture for the enumeration of fluorescently stained target components of a whole blood sample by an imaging instrument. The sample preparation method ensures that the amount of target components per unit of volume of the whole blood sample is preserved by elimination of certain non-quantitative preparation steps while producing an even hematocrit layer within a scan capillary. Typical target components include white blood cells that express certain surface antigens, such as CD-4 and CD-8 proteins. To inhibit aggregation of the red blood cells, a reagent is added to an aliquot of whole blood sample. The aliquot of whole blood is mixed and with a preselected amount of a fluorescent dye and ligand complex which tags the target components.

Abstract: A cartridge is provided to present a biological sample for analysis by an imaging instrument. The cartridge of the invention utilizes a series of channels, capillaries, reservoirs and stop junctions to precisely move a sample, reagent and diluent through the cartridge as a function of the sum of capillary, gravitational and low centrifugal forces. The operator applies a precise amount of sample to the cartridge; therefore, the cartridge fluidics need not meter the sample prior to dilution. A practical and cost effective cartridge and assay process is disclosed which overcomes many of the limitations of the prior art. Such a cartridge is especially useful with fixed volume assays which permit low centrifugal accelerations to move the fluids within the cartridge.

Abstract: Analytes can be detected from among closely related substances by reacting the analyte in a test sample with a labeled binding agent which specifically binds to the analyte to form a complex. The labeled binding agent is supplied in excess, and the complex is identified through a time window relative to the detection of the excess unbound agent. The complex and labeled binding agent are isolated on a separation media and identified by the differential rate of migration.

Abstract: An energy transfer fluorescent probe for detecting a reagent is provided which includes a fluorescent reporter molecule and a quencher molecule positioned on the probe relative to the reporter molecule such that the quencher molecule quenches the fluorescence of the reporter molecule when in a first state, the quencher molecule being converted by the reagent to a second state which has a reduced ability to quench the reporter molecule. Examples of conversions of the quencher molecule from a first state to a second state include reductions, oxidations, hydrolyses, phosphate cleavages, and the conversion of amides to amines. In one embodiment, the quencher molecule is a substrate for an enzyme which converts the quencher from a first state to a second state. For example, the enzyme may be an reductase, an oxidase, hydrolytic, a peptidase or a phosphorylase. The probe is used to fluorescently detect a reagent in a sample.

Abstract: New intercalating cyanine dyes are provided in which the benzothiazole portion of the cyanine dye has been modified to produce dyes with improved properties for labelling nucleic acids. The fluorescent cyanine dyes have a positively charged substituent attached to the positively charged nitrogen on the benzothiazole portion of the cyanine dye.

Abstract: A calibration method and device for a fluorescence spectrometer which uses fluorescence from a homogeneous solid state standard as the source of calibration fluorescence and wherein the solid state standard may be built into the optical scanner and the calibration may be automatically performed as a routine step when using the optical scanner. A gold standard establishes fluorescent units, and the fluorescence spectrometer is calibrated by reference to calibration standards, such as calibration rubies, which are themselves rated against the gold standard and built into the fluorescence spectrometer to provide an unchanging reference to the gold standard and by which simultaneous calibration of two or more channels of a multi-channel fluorescence spectrometer may be accomplished, including automatically calibrating a multi-channel optical scanner when it is turned on to achieve an acceptable level of sensitivity in each channel and to adjust for any relative shift in sensitivity between the channels.

Abstract: New intercalating cyanine dyes are provided in which the benzothiazole portion of the cyanine dye has been modified to produce dyes with improved properties for labelling nucleic acids. These new intercalating cyanine dyes are cyclized fluorescent cyanine dyes represented by General Formula I ##STR1## where: n is 0, 1 or 2;Y may be either S or O;R.sub.3 and R.sub.4 may each independently be either hydrogen, C.sub.1 -C.sub.10 alkyl, C.sub.1 -C.sub.10 alkoxy or C.sub.1 -C.sub.10 alkylthio;R.sub.5 may be C.sub.1 -C.sub.50 alkyl, preferably substituted with one or more polar substituents which preferably includes one or more positively charged atoms, or a cyclized fluorescent cyanine dye of the present invention, i.e., where R.sub.5 is a linker between two cyclized fluorescent cyanine dyes;R.sub.6 and R.sub.7 may each independently be either H and C.sub.1-10 alkyl, or may be taken together to form a 5 or 6 membered ring, most preferably a 6 membered aromatic ring, optionally substituted with C.sub.1-6 alkyl or C.

Abstract: A cartridge is provided to present a biological sample for analysis by an imaging instrument. The cartridge of the invention utilizes a series of channels, capillaries, reservoirs and stop junctions to precisely move a sample, reagent and diluent through the cartridge as a function of the sum of capillary, gravitational and low centrifugal forces. The operator applies a precise amount of sample to the cartridge; therefore, the cartridge fluidics need not meter the sample prior to dilution. A practical and cost effective cartridge and assay process is disclosed which overcomes many of the limitations of the prior art. Such a cartridge is especially useful with fixed volume assays which permit low centrifugal accelerations to move the fluids within the cartridge.

Abstract: An assay and sample mixture for the enumeration of fluorescently stained target components of a whole blood sample by an imaging instrument. The sample preparation method ensures that the amount of target components per unit of volume of the whole blood sample is preserved by elimination of certain non-quantitative preparation steps while producing an even hematocrit layer within a scan capillary. Typical target components include white blood cells that express certain surface antigens, such as CD-4 and CD-8 proteins. To inhibit aggregation of the red blood cells, a reagent is added to an aliquot of whole blood sample. The aliquot of whole blood is mixed and with a preselected amount of a fluorescent dye and ligand complex which tags the target components.