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Removable sorting cuvette and nozzle

Removable sorting cuvette and nozzle description/claims

The present application claims priority to U.S. application Ser. No. 11/711,320, filed Feb. 26, 2007, which is continuation application of U.S. application Ser. No. 10/259,332, filed Sep. 27, 2002, now issued as U.S. Pat. No. 7,201,875, both of which are incorporated herein by reference in its entirety.

The present invention relates to flow cytometry.

Flow analysis has proven to be an important technology for the analysis of discrete targets. The applications of this technology include cellular assay to investigate a variety of cellular features including DNA content, specific nucleic acid sequences, chromatic structure, RNA content, specific antigens, surface receptors, cell morphology, DNA degredation and other assay targets. The targets of a flow cytometer may be multicellular organisms (e.g. microfilaria), cellular aggregates, viable cells, dead cells, cell fragments, organelles, large molecules (e.g. DNA), particles such as beads, viral particles or other discrete targets of this size range. The term “cells”, as used throughout, is used to refer to such discrete targets. This technology has a number of different applications, including diagnostic, clinical and research applications.

Flow cytometry measures targets flowing through an analytical region in a flow cell. In the flow cell a core stream is injected into the center of a sheath flow stream flowing at a constant flow rate. The core stream is a liquid sample, which may be injected from a sample tube. Injection generally requires insertion of an aspiration tube into the sample tube and pressurization of the head above the liquid in the sample tube such that sample liquid is pressure driven from the sample tube into the injection tube.

The flow stream is directed into a tapered portion of the flow cell body and through an analytical region. In one design, the stream is directed through a nozzle and analyzed in air. In a second design, the stream is directed through a channel for analysis.

Analysis takes place by optical interrogation of particles as each particle passes a detection region. In most systems, one or more laser beams are directed by steering mirrors and illumination lenses through the analytical region. If more than one laser are used, a dichroic stack may be used to combine the beams and direct the beams through the stream to be analyzed.

Some of the light passing through the analytical region will be scattered by particles. Detectors measure the intensity of forward and side scatter. In addition, the illumination beam will excite fluorescence from target particles in the flow stream that have been labeled with a fluorescent dye. Emitted fluorescence is collected by a collection lens and transmitted to detection optics. The detection optics separate the collected light (e.g. using filters and dichroic mirrors) into light at specific wavelengths. Light at specific wavelengths, or within specific wavelength ranges, are detected by individual light detection devices (e.g. photomultiplier tubes). The signal from the various detectors is sent to a data processor and memory to record and characterize detection events.

In addition to analysis of particles, flow cytometer systems may also be designed to sort particles. After leaving the optical analysis region, the flow stream may be separated into droplets. One common method of droplet generation is to vibrate the nozzle from which the flow stream emerges. This may be done by vibration of the nozzle alone, or vibration of the entire flow cell. The resultant separated droplets adopt a spacing which is a function of the stream velocity and the vibration wavelength. Droplets containing the target of interest are charged by a charging device such as a charging collar. The charged droplets are directed between two charged deflection plates, which angularly deflect charged droplets. The deflected droplets are then collected in containers positioned in the path of falling deflected particles.

Known flow cytometry similar to the type described above are described, for example in U.S. Pat. Nos. 3,960,449; 4,347,935; 4,667,830; 5,464,581; 5,483,469; 5,602,039; 5,643,796 and 5,700,692. All references noted are hereby expressly incorporated by reference. Commercial flow cytometer products include FACSort™, FACSVantage™, FACSCount™, FACScan™, and FACSCalibur™ systems all manufactured by BD Biosciences, the assignee of the instant invention.

The described system presents a number of advantages for the analysis of particles (e.g. cells), allowing rapid analysis and sorting. However a number of limitations to the system exist.

The system requires precise alignment of various elements to function properly. The lasers must be precisely positioned to properly direct light to the objective. To aid in this positioning, the laser or other illumination source is commonly mounted on an x-y-z stage, allowing three-dimensional positioning of the laser. The steering mirrors for the laser beams must be precisely positioned to properly direct the illumination beam to the objective. This generally requires that the mirrors be mounted to allow for angular adjustment. The illumination lens system must be exactly positioned such that the illumination lens focuses the illumination light onto the target area. This lens is also generally mounted such that it can be repositioned along the x-y-z axes.

The flow cell must be positioned such that the angle at which the illumination beam impinges the flow stream and the distance from the flow stream to the illumination lens does not change. Commonly the flow cell is mounted on a stage, which allows x-y-z positioning of the flow cell. In addition the stage holding the flow cell may also allow for angular repositioning of the flow cell (e.g. α and θ positioning). This angular adjustment is critical for sorting, which requires precise prediction of the sort stream direction. In addition, the optics used for detection of scattered light and fluorescence also must be properly aligned.

The stream in air jet must also be aligned, to ensure that the stream in air is directed in the intended direction. This alignment is effected by angular rotation of the flow cell. This alignment is additionally important if the optical interrogation of the stream takes place in a stream-in-air. The alignment procedure for a stream in air system requires first locating the stream-in-air with respect to both the illumination and the light collection optics and then focusing each of these components on a location within the stream in air.

Alignment requires user time and considerable user expertise. At times it is difficult to determine which element requires adjustment. Set up of the instrument generally requires a diagnostic of alignment with elements realigned by repositioning as needed. This occurs at least once a day, more frequently if an element is replaced or removed. Realignment necessitates both instrument down time and user time and expertise. The time required to perform the alignment procedure is highly dependent on both the condition of the system and the skill of the operator. In addition, the need for constant realignment reduces the repeatability of system performance.

A few attempts have been made to address the problem of the need for repeated alignment of some elements of a flow system. U.S. Pat. Nos. 5,973,842 and 6,042,249 to Spangenberg disclose an optical illumination assembly for use with an analytical instrument. This assembly may include an illumination source (e.g. a laser), a spatial filter, a beam shaping aperture and a focus lens. All elements are illumination optical elements, not the flow cell or light collection elements. Each component is mounted on a plate, frame or mounting cylinder, which in turn are mounted on a platform. Each of the plates or frames is movable along two axes by micrometer adjustments using adjusters with opposing spring plungers. Following an initial adjustment, the plates or frames are secured into a fixed location using screws or other devices to fix the plates or frames into place. The adjusters or springs are removed once the frames or plates are secured. The focus lens would be mounted such that it would be moved along 3 axes (x-y-z movement) and subsequently also be fixed into a location. This allows fixation of the light generation and illumination optics. However, the cuvette would still be adjusted to be positioned at the focal spot of the illumination. This would be required on a routine basis.

U.S. Pat. No. 4,660,971 discloses an illumination configuration in which a focus lens is in contact with a flow cell. A spring biases the lens against a housing, positioning the lens at a selected focal length from the flow cell. This maintains a relative axial position between the lens and the flow cell.

These references, while providing a method in which some of the issues relating to the alignment of the illumination optics are addressed, do not provide a method in which the flow cell and the light collection optics may also be fixed. Fixing all of these elements significantly further simplifies the alignment of the instrument.

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