Patent Description:
Sample separation devices, such as capillary electrophoresis devices, generally provide certain major components that include, for example, a capillary channel or array of channels, a separation medium source for providing a medium that may flow through the capillaries (e.g., a polymer fluid), a sample injection mechanism, an optical detector system or component, electrodes for producing an electric field, an anode buffer source on one end of the capillaries, and a cathode buffer source on the other end of the capillaries. Capillary electrophoresis devices generally also provide various heating components and zones to regulate the temperature of many of the aforementioned components. Regulating the temperature of many of these components can improve quality of results.

Current capillary electrophoresis devices use multiple structures to house these various components and connect or couple these structures together to provide a working capillary electrophoresis device or system. Using multiple structures has disadvantages. It is therefore desirable to provide a capillary electrophoresis apparatus with a reduced number of interconnected structures, for example, to reduce the number of necessary heating zones, reduce user handling of the structures, reduce likelihood of component failure, and reduce introduction of bubbles and other artifacts into the apparatus. <CIT> discloses a system for separating biological molecules comprising a plurality of capillaries and a capillary mount and a plurality of optical fibres corresponding to the plurality of capillaries.

Embodiments of the present invention are generally directed to systems and methods for performing sample separation assays, processes, tests, or experiments.

One aspect of the present invention involves incorporation of various components of a sample separation system into a common cartridge, cassette, or case that may be advantageously loaded into the system in a way that simplifies set up for a preforming a sample separation assay, process, test, or experiment. Another aspect involves a sample separation cartridge, cassette, or case having an optical section that, upon loading into a sample separation system or instrument, can be aligned to an optical system and/or detector in a manner that is advantageously simple, accurate, and stable. In yet another aspect of the present invention, involves a sample separation system comprising an illumination optical configuration that advantageously reduces optical noise, for example, optical noise created by Raman scattering by water molecules within a sample solution contained in one or more capillaries used during, or in preparation for, a sample separation assay, process, test, or experiment.

The following description provides embodiments of the present invention, which are generally directed to systems and methods for preparing, observing, testing, and/or analyzing biological samples.

Embodiments of the present invention may include various sample separation systems and methods including, but not limited to, capillary electrophoresis, chip based electrophoresis, lab-on-a-chip microfluidics, gel electrophoresis, electro-osmosis, chromatography, flow cytometry, and the like. Example embodiments of the present invention will be presented for capillary electrophoresis systems or instruments in order to demonstrate various aspects of the present invention that may be applicable to other separation systems, such as chip based electrophoresis and the like.

As used herein the terms "radiation" or "electromagnetic radiation" means radiant energy released by certain electromagnetic processes that may include one or more of visible light (e.g., radiant energy characterized by one or more wavelengths between <NUM> nanometers and <NUM> nanometers or between <NUM> nanometers and <NUM> nanometers) or invisible electromagnetic radiations (e.g., infrared, near infrared, ultraviolet (UV), X-ray, or gamma ray radiation).

As used herein a "radiant source" means a source of electromagnetic radiation that may be directed toward at least one sample mixture or solution in order to produce a detectable signal for determining the presence and/or quantity of one or more target sample molecules or compounds contained within the at least one sample mixture or solution. The radiant source may comprise a single source of light, for example, an incandescent lamp, a gas discharge lamp (e.g., Halogen lamp, Xenon lamp, Argon lamp, Krypton lamp, etc.), a light emitting diode (LED), an organic LED (OLED), a laser (e.g., chemical laser, excimer laser, semiconductor laser, solid state laser, Helium Neon laser, Argon laser, dye laser, diode laser, diode pumped laser, fiber laser, pulsed laser, continuous laser), or the like. Alternatively, the radiant source may comprise a plurality of individual sources (e.g., a plurality of LEDs or lasers). The radiant source may also include one or more excitation filters, such as a high-pass filter, a low-pass filter, or a band-pass filter. For example, the excitation filter comprise a colored filter and/or a dichroic filter. The radiant source may continuous or pulsed, and may comprise either a single beam or a plurality of beams that are spatially and/or temporally separated. The radiant source may be characterized by electromagnetic radiation that is primarily within the visible light range (e.g., a "light source" emitting electromagnetic radiation within a wavelength in the range of <NUM> nanometers to <NUM> nanometers or in the range of <NUM> nanometers and <NUM> nanometers), near infrared range, infrared range, ultraviolet range, or other ranges within the electromagnetic spectrum.

Referring to <FIG>, certain embodiments of the present invention comprise a system or instrument <NUM> for performing a capillary electrophoresis or similar assay, process, test, or experiment. System <NUM> comprises one or more capillaries, tubes, or channels <NUM> (four are shown in <FIG>) located on or in a capillary housing, holder, or mount <NUM>. Each capillary comprises a detection portion configured to pass electromagnetic radiation into and/or out of the capillary. In the illustrated embodiment, a capillary array <NUM> comprises four capillaries <NUM>; however, capillary array <NUM> may include more than four capillaries, for example, to provide higher throughput or shorter assay runs. Configurations of instrument <NUM> may include <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or more than <NUM> capillaries <NUM>.

System <NUM> further comprises an optical system <NUM> comprising an illumination or excitation optical system <NUM> comprising any or all of a radiant source <NUM>, a beam shaper or conditioner <NUM>, a beam divider <NUM>, and/or a beamsplitter or mirror <NUM>. Radiant source <NUM> is configured to illuminate an optical detection access or optical detection zone <NUM> of system <NUM> and/or capillaries <NUM> in which electromagnetic radiation (e.g., light, near infrared, or ultraviolet) may pass into and/or out of the detection portion of the one or more capillaries <NUM> in order to detect or measure a target, calibration, or other molecules of interest. Optical system <NUM> may further comprise a lens <NUM> and an emission optical system <NUM>. Emission optical system <NUM> may comprise lens <NUM>, a lens <NUM>, an emission filter <NUM>, and a detection system <NUM>. Radiant source <NUM> may comprise one or more of the types of radiant sources discussed above herein. In certain embodiments radiant source <NUM> comprises a diode pumped solid state (DPSS) laser having a wavelength of <NUM> nanometers.

Detection system <NUM> comprises a detector <NUM> configured to receive emissions from the optical detection zone <NUM> of capillaries <NUM>, for example to receive fluorescent emissions produced by fluorescent dyes, probes, or markers attached to target or other molecules of interest. Detector <NUM> may be an optical detector comprising one or more individual photodetectors including, but not limited to, photodiodes, photomultiplier tubes, bolometers, cryogenic detectors, quantum dots, light emitting diodes (LEDs), semiconductor detectors, HgCdTe detectors, or the like. Additionally or alternatively, detector <NUM> may be an optical detector comprising an array sensor including an array of sensors or pixels. The array sensor may comprise one or more of a complementary metal-oxide-semiconductor sensor (CMOS), a charge-coupled device (CCD) sensor, a plurality of photodiodes detectors, a plurality of photomultiplier tubes, or the like. In certain embodiments, detector <NUM> comprises two or more array sensors.

An optical system such as emission optical system <NUM> is used to collect emissions from each capillary <NUM>. In the illustrated embodiment in <FIG>, lens <NUM> is doublet lens configured to collect emission light from each of the one or more capillaries <NUM> and lens <NUM> is a doublet lens configured to reimage the emissions from each of the one or more capillaries <NUM> to a spot or focus in an image plane of emission optical system <NUM>. However, other optical configurations known in the art may be used for these purposes.

For applications in which multiple emissions at different wavelengths are produced in each of the one or more capillaries <NUM>, detection system <NUM> may further comprise one or more spectral dispersion elements <NUM> that spread the spectral content contained in different fluorescent signal to different parts (e.g., different groups of pixels) of detector <NUM>. In the illustrated embodiment shown in <FIG>, four spectral dispersion elements <NUM> are incorporated into a spectrometer <NUM> (two spectral dispersion elements <NUM> are visible in <FIG> and two more spectral dispersion elements <NUM> are located behind the two visible in <FIG>). Spectrometer <NUM> may further comprise detector <NUM>. Detection system <NUM> may be disposed within a housing or enclosure <NUM>.

Spectrometer <NUM> may be optically coupled to capillaries <NUM> and/or emission optical system <NUM> via one or more fibers or optical fibers <NUM>. In the illustrated embodiment, a first pair or bundle of optical fibers 145a is configured to receive emission light from first and second capillaries <NUM> of capillary array <NUM> and a second pair or bundle of optical fibers 145b is configured to receive emission light from third and fourth capillaries <NUM> of capillary array <NUM>. Additionally or alternatively, optical fibers <NUM> may be grouped together into a single fiber bundle or each fiber <NUM> may be separate from the remaining optical fibers <NUM>. Spectrometer <NUM> may further comprise the one or more spectral dispersion elements <NUM> and the detector <NUM>, wherein each spectral dispersion element <NUM> is configured to direct emission light from a different one of capillaries <NUM> onto a different region of detector <NUM>. Spectral dispersion elements <NUM> may comprise one or more prisms, diffractive optical elements, holographic optical elements, or the like. Spectral dispersion elements <NUM> may comprise reflective or transmissive optical elements. The use of optical fibers <NUM> have been discovered to advantageously simplify alignment and calibration of detector <NUM> for multi-fluorescent wavelength application, as discuss below herein.

In certain embodiments, optical system <NUM>, the one or more capillaries <NUM>, and capillary mount <NUM> are disposed inside of a common housing or enclosure <NUM> and spectrometer <NUM> is located outside housing <NUM> in housing <NUM>. Alternatively, spectrometer <NUM> and/or housing <NUM> may be located within housing <NUM> or directly attached to housing <NUM>. Housing <NUM> may include an opening or port to allow transfer of radiation or light from capillaries <NUM> to spectrometer <NUM>. Spectrometer <NUM> may be contained in a separate housing, as shown in <FIG>, or included inside the same instrument housing as the optical system. In contrast to the embodiment shown in <FIG>, the one or more capillaries <NUM> and/or some of associated hardware may be located outside housing <NUM>, in which case an interface with system <NUM> may be provided via an opening or port in housing <NUM>.

In certain embodiments, optical fibers <NUM> are part of spectrometer <NUM>. Alternatively, optical fibers <NUM> may be separate from spectrometer <NUM>, wherein the optical fibers <NUM> are attached to spectrometer <NUM> using an optical coupler (not shown). In the illustrated embodiment, spectral dispersion elements <NUM> are advantageously configured to both disperse and focus incident emissions received from optical fiber <NUM> onto detector <NUM>.

During use, capillaries <NUM> may contain a polymer or similar solution configured to support an electric field or current. The polymer or similar solution is configured to permit the transfer or migration of one or more samples that may include one or more fluorescent dyes, probes, markers, or the like. The fluorescent dyes, probes, markers, or the like may be selected to produce a fluorescent signal during use that may be correlated to the presence or amount of one or more target molecules or sequences of molecules present at a given time within optical detection zone <NUM>. The fluorescent signal(s), light, or radiation produced within any or all of capillaries <NUM> may be directed back through lens <NUM> and the mirror so as to be received by spectrometer <NUM>.

Referring again to <FIG>, in certain embodiments, system <NUM> may comprise conditioner <NUM> and radiation from radiant source <NUM> passes through conditioner <NUM>. Conditioner <NUM> may comprise a homogenizer configured, for example, to blend different color or wavelength radiant sources and/or to provide a more even illumination cross-section of the output beam. Additionally or alternatively, system <NUM> may comprise divider <NUM>. Additionally or alternatively, emitted radiation from radiant source <NUM> may pass through beam divider <NUM> to provide a plurality of excitation, sample, illumination, or source beams <NUM>, each source beam <NUM> characterized by one or more of, one or more beam diameters, a cross-sectional shape (e.g., square, circular, or elliptical), a predetermined intensity or power profile (e.g., constant, top hat, Gaussian, etc.).

As illustrated in <FIG>, beam conditioner <NUM> and beam divider <NUM> may be configured to produce or provide source beams <NUM>, where each source beam <NUM> comprising an elliptical cross section or shape. Beam conditioner <NUM> may comprise an anamorphic beam shaper, for example, comprising one or more cylindrical lenses configured to produce beams having an elliptical cross section, wherein the beam cross section is wider in one axis than in the other perpendicular axis. Alternatively, beam conditioner <NUM> may comprise a Powell lens, for example, configured to provide a line focus and/or an elliptical beam cross section in which an intensity or power over a cross section of the beam uniform, or nearly uniform. In addition, beam conditioner <NUM> may be configured so that any diameter of the beam is greater than or less than the diameter of the beam entering beam conditioner <NUM>. In the illustrated embodiment, the beam exiting beam conditioner <NUM> is collimated. The elliptical cross section of each of source beam <NUM> may be oriented so that the long axis or dimension is oriented perpendicular or nearly perpendicular to an axis of the associated capillary <NUM>. This orientation of each source beam <NUM> and its focus has been found to advantageously reduce the sensitivity of the alignment of the capillary array <NUM> to the beams. In the illustrated embodiment shown in <FIG>, the long diameter of the beam focus is less than an inner diameter of an individual capillary <NUM>. Alternatively, as illustrated in <FIG>, the long diameter of the focused source beams <NUM> may be larger than the inner diameter of the individual capillaries <NUM>. <FIG> also illustrates the diameters and pitch of capillaries <NUM> within the array for certain embodiments. As seen in <FIG>, the inner diameter of each capillary <NUM> is <NUM> micrometers, while the focused beam has a diameter of about <NUM> micrometers.

Referring again to <FIG>, the excitation beam out of conditioner <NUM> enters beam divider <NUM>, which may be configured to produce a plurality of identical or similar source beams <NUM> from a single input beam into beam divider <NUM>. As an example, beam divider <NUM> may comprise one or more diffractive optical elements, holographic optical elements, or the like, that is configured to produce or provide four elliptical beams for illuminating each of the four capillaries <NUM>, as seen in <FIG>. The four source beams <NUM> have the same or a similar cross-section, and each beam diverges at a different angle relative to a system optical axis or general directions of light propagation. Alternatively, beam divider <NUM> may be configured to produce a plurality of beams that are parallel to one another or that converge relative to one another. In the illustrated embodiment, the beams out of beam divider <NUM> are collimated; however, some or all of the beams may alternatively be converging or diverging as they leave beam divider <NUM>. Source beams <NUM> originating from beam divider <NUM> may each be collimated as they enter lens <NUM>, but be divergent from one another. In such embodiments, lens <NUM> may be configured focus each of source beam <NUM> to a location at or near a respective capillary <NUM>, as illustrated in the magnified view of <FIG>. In addition, lens <NUM> and the source beams <NUM> out of beam divider <NUM> may be configured such that the individual beams <NUM> are each collimated relative to one another (e.g., the four beams in <FIG> may all travel parallel to one another after exiting lens <NUM>).

Source beams <NUM> out of beam divider <NUM> in <FIG> may be reflected by a mirror <NUM> and directed toward capillaries <NUM>. Additional mirrors and/or diffractive elements may be included as desired to direct the four beams toward capillaries <NUM>, for example, to meet packaging constraints. The beams from beam divider <NUM> continue to diverge after reflection off the mirror until they are received by lens <NUM>. Mirror <NUM> may be a dichroic mirror, or the like, which may be configured to reflect light at a predetermined wavelength or light over a predetermined wavelength range, while transmitting light or other electromagnetic radiation that is outside the predetermined wavelength or wavelength range. In some embodiments, mirror <NUM> comprises a dichroic mirror having more than one predetermined wavelength or wavelength range, for example, when the radiant source comprises more than one distinct wavelength or wavelength range. In the illustrated embodiment, the source beams <NUM> from beam divider <NUM> are reflected by mirror <NUM>, while emitted radiation from optical detection zone <NUM> is transmitted or largely transmitted by mirror <NUM>. Alternatively, the location of capillaries <NUM> may be located along the optical axis of beam divider <NUM> and mirror <NUM> may be configured to transmit, or largely transmit, the excitation beams, while reflecting emissions from the optical detection zone <NUM>.

Emission filter <NUM> may be located between lenses <NUM>, <NUM> and may be configured block or attenuate light from the radiant source, thereby eliminating or reducing the about of light from the radiant source that is receive by spectrometer <NUM>. In certain embodiments, the focal length of lenses <NUM>, <NUM> are selected to produce a magnification of capillaries <NUM>, or of emission radiation from capillaries <NUM>, that is different than one (e.g., to produce a magnified or demagnified image). For example, lens <NUM> may be selected to have a numerical aperture (NA) that is twice the NA of the lens <NUM>, resulting in a system magnification of two. In certain embodiments, lens <NUM>, <NUM> has an NA of <NUM> and lens <NUM> has an NA of <NUM>. In some embodiments, the focal length or NA of lenses <NUM>, <NUM> may be selected to (<NUM>) provide a focal spot, or focal point, at or near capillary array <NUM> that has a predetermined size or diameter and (<NUM>) simultaneously providing an NA that is matched to the NA of spectrometer <NUM> and/or the NA of the optical fiber system used to transfer light into spectrometer <NUM>.

Source beams <NUM> are configured to illuminate samples within optical detection zone <NUM> of each of the capillaries <NUM> to produce respective emissions, for example fluorescent emissions produced by fluorescent dyes, probes, or markers attached to the target molecules or molecules of interest. The emissions may be configured to indicate the presence or amount of target molecules or molecules of interest. The emissions may be focused or re-image onto a plane using lenses <NUM>, <NUM> or some other suitable emission optical system. Emission filter <NUM> may be configured to filter out unwanted radiation, such as excitation light produced by radiant source <NUM>. Alternatively, as shown illustrated in <FIG>, emission light from capillaries <NUM> may be focused or re-image onto to input or receiving ends of optical fibers <NUM>, then propagated by optical fibers <NUM> into spectrometer <NUM>. Each fiber <NUM> may be associated with (e.g., receive radiation from) a corresponding one of capillaries <NUM>. Using optical fibers <NUM>, radiation from capillaries <NUM> is then transferred into spectrometer <NUM>, where it is dispersed by wavelength onto a detector <NUM>. In the illustrated embodiment, emission radiation from optical fibers 145a enter on one side of spectrometer <NUM> and radiation from optical fibers 145b enter on another side of spectrometer <NUM>. In this manner, the spectrum from each of fiber <NUM> (or capillaries <NUM>) is directed onto a different portion of detector <NUM>. This configuration has been found to advantageously allow the spectrum from each of multiple capillaries <NUM> to be produced and detected simultaneously on a single or reduced number of array detectors <NUM>. Detector <NUM> may be configured to receive the emissions from the samples contained in capillaries <NUM> and to produce emission signal that may be further processed. For example, spectrometer <NUM> may be configured to separate the signals created by different fluorescent dyes, probes, or markers, for example, created by dyes, or probes, markers corresponding to different DNA or RNA bases (e.g., adenine, thymine (or uracil), cytosine, and guanine).

System <NUM> may further comprise a computer or processing system <NUM> including a data processing system, a computer program product <NUM> configured to program processing system <NUM>, and display or other output device <NUM>. Processing system <NUM> may be used to control or obtain data from system <NUM>, for example, to monitor and/or control one or more electrical parameters (e.g., radiant source power, detector supply power, cathode/anode voltage, or current through one or more of each capillary <NUM> or a group of the capillaries <NUM>) or to measure or control various run or process parameters such as temperature or pressure (e.g., system or capillary <NUM> temperature, pressure of a pump or syringe for filling capillaries <NUM> with a polymer solution or the like). Processing system <NUM> may be coupled to detection system <NUM>, for example to provide read detected fluorescence signals. In certain embodiments, detection system <NUM> passes a signal to processing system <NUM> corresponding to the intensity of emissions received at various wavelengths scanned by detection system <NUM>. Computer program product <NUM> may be used to configure processing system <NUM> to process received spectral data from detection system <NUM> that may be used during runtime of instrument <NUM> to calibrate instrument <NUM> or to correct for spectral error, for example, as disclosed in <CIT>. Display or other output device <NUM> is coupled to processing system <NUM> and may be used to display or report data related to an assay, process, test, or experiment such as run parameter values, spectral data, run condition data, run quality data, warning flags, and the like, for example, as disclosed in <CIT>.

Referring to <FIG>, computer or processing system <NUM> may be configured to execute instruction codes contained in a computer program product <NUM>. Computer program product <NUM> may comprise executable code in an electronically readable medium that may instruct one or more computers such as computer or processing system <NUM> to perform processing that accomplishes the exemplary method steps performed by the embodiments discussed herein. The electronically readable medium may be any non-transitory medium that stores information electronically and may be accessed locally or remotely, for example via a network connection. In alternative embodiments, the medium may be transitory. The medium may include a plurality of geographically dispersed media each configured to store different parts of the executable code at different locations and/or at different times. The executable instruction code in an electronically readable medium directs the illustrated computer or processing system <NUM> to carry out various exemplary tasks described herein. The executable code for directing the carrying out of tasks described herein would be typically realized in software or firmware. However, it will be appreciated by those skilled in the art that computers or other electronic devices might utilize code realized in hardware to perform many or all the identified tasks without departing from the present invention. Those skilled in the art will understand that many variations on executable code may be found that implement exemplary methods within the spirit and the scope of the present invention.

The code or a copy of the code contained in computer program product <NUM> may reside in one or more storage persistent media (not separately shown) communicatively coupled to computer or processing system <NUM> for loading and storage in persistent storage device <NUM> and/or memory <NUM> for execution by a processor <NUM>. Computer or processing system <NUM> also includes I/O subsystem <NUM> and peripheral devices <NUM> (e.g., display or output device <NUM>). I/O subsystem <NUM>, peripheral devices <NUM>, processor <NUM>, memory <NUM>, and persistent storage device <NUM> may be coupled via a common bus <NUM>. Like persistent storage device <NUM> and any other persistent storage that might contain computer program product <NUM>, memory <NUM> may a non-transitory media (even if implemented as a typical volatile computer memory device). Moreover, those skilled in the art will appreciate that in addition to storing computer program product <NUM> for carrying out processing described herein, memory <NUM> and/or persistent storage device <NUM> may be configured to store various data elements disclosed or referenced and illustrated herein.

Those skilled in the art will appreciate computer or processing system <NUM> illustrates just one example of a system in which a computer program product in accordance with embodiments of the present invention may be implemented. To cite but one example of an alternative embodiment, execution of instructions contained in a computer program product in accordance with an embodiment of the present invention may be distributed over multiple computers, such as, for example, over the computers of a distributed computing network.

Referring to <FIG>, in certain embodiments, a sample separation system or instrument <NUM>, such as a capillary electrophoresis (CE) instrument, is configured for separating biological molecules, for example, for separating sample nucleotide molecules or sample amino acid molecule according to length of the different molecules. Where possible, embodiments of system <NUM>, as well as methods, elements, and/or parameter values associated with system <NUM>, may be incorporated into embodiments of system <NUM> and into methods, elements, and/or parameter values associated with system <NUM>. Conversely, where possible, embodiments of system <NUM>, as well as methods, elements, and/or parameter values associated with system <NUM>, may be incorporated into embodiments of system <NUM> and into methods, elements, and/or parameter values associated with system <NUM>.

System <NUM> comprises one or more capillaries <NUM>, an electronic or voltage supply <NUM>, one or more cathodes <NUM>, one or more anodes <NUM>, a sample source container <NUM>, a sample destination container <NUM>, radiant source <NUM>, detection system <NUM>, and processing system <NUM> including a data processing system configured by computer program product <NUM>, and display or output device <NUM>. Instrument <NUM> may include multiple capillaries <NUM> (e.g., four capillaries <NUM>, as shown in <FIG>); however, only one capillary <NUM> is illustrated in <FIG> for simplicity. Configurations of instrument <NUM> may include <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or more than <NUM> capillaries. Sample separation could also be performed by other means including using gel electrophoresis and microfluidics, such as on a lab-on-a-chip.

System <NUM> may be used to perform a capillary electrophoresis or other sample separation assay, experiment, or process. A sample mixture or solution <NUM> containing various samples or sample molecules 515a is first prepared in or delivered into sample source container <NUM>. At least a portion of sample mixture <NUM> is subsequently loaded into cathode <NUM> end of capillary <NUM>, for example using a pump or syringe, or by applying a charge or electric field to capillary <NUM>. Once loaded into the anode end of capillary <NUM>, voltage supply <NUM> creates a voltage difference between cathode <NUM> and anode <NUM>. The voltage difference causes negatively charged, dye-labeled samples 515a to move from sample source container <NUM> to sample destination container <NUM>. During the assay, process, test, or experiment, various samples (e.g., nucleotides or amino acid molecules) pass through an optical detection zone <NUM> and are illuminated by radiant source <NUM> to produce respective emissions, for example fluorescent emissions produced by fluorescent dyes, probes, or markers attached to the target molecules or molecules of interest. The emissions may be configured to indicate the presence or amount of target molecules or molecules of interest. Longer and/or less charged dye-labeled samples 515a move at a slower rate through capillary <NUM> than do shorter and/or higher charged dye-labeled samples, thereby creating some separation between samples of varying lengths and charges. As each of samples 515a passes through an excitation beam generated by radiant source <NUM>, a dye on a leading element (a leading element might, e.g., be a nucleotide) of a sample 515a exhibits fluorescence that is detected by detection system <NUM>. Detection system <NUM> may be coupled to provide signals to processing system <NUM> in response to detected fluorescence. In particular, detection system <NUM> passes a signal to processing system <NUM> corresponding to the intensity of emissions received at various wavelengths scanned by detection system <NUM>. Computer program product <NUM> configures data processing system <NUM> to process the received spectral data and may, for example during runtime of instrument <NUM>, calibrate instrument <NUM> to correct for spectral error, for example, as disclosed in <CIT>. A display or other output device <NUM> is coupled to processing system <NUM> and may be used to display or report data related to the assay, process, test, or experiment such as run parameter values, spectral data, run condition data, run quality data, warning flags, and the like, for example, as disclosed in <CIT>.

In certain embodiments, system <NUM> comprises a delivery system <NUM> comprising a polymer reservoir <NUM> containing a polymer or polymer solution <NUM>, a polymer valve <NUM>, and a pump <NUM> (e.g., a syringe) configured to receive or draw polymer <NUM> from polymer reservoir <NUM> and to pump or load polymer <NUM> into capillary <NUM>. Delivery system <NUM> further comprises a buffer reservoir <NUM> containing a buffer solution <NUM> and a buffer valve <NUM>. In the illustrated embodiment, buffer reservoir contains the one or more anodes <NUM>. In certain embodiments, all or some of components of delivery system <NUM> are part of a cassette or cartridge <NUM> that may further comprise capillaries <NUM>, cartridge <NUM> may also comprise the one or more cathodes <NUM> (e.g., one cathode <NUM> for each of a plurality of capillaries <NUM>). Examples of cassette or cartridges suitable for use with embodiments of the present invention are disclosed in <CIT>.

In certain embodiments, the sample separation assay, process, test, or experiment comprises the following activities:.

Referring to <FIG>, in certain embodiments a system or instrument <NUM>, such as a capillary electrophoresis (CE) instrument, is configured separating biological molecules, for example, for separating sample nucleotide molecules or sample amino acid molecule according to length of the different molecules. Where possible, embodiments of system <NUM>, as well as methods, elements, and/or parameter values associated with systems <NUM>, <NUM>, may be incorporated into embodiments of systems <NUM>, <NUM> and into methods, elements, and/or parameter values associated with systems <NUM>, <NUM>. Conversely, where possible, embodiments of systems <NUM>, <NUM>, as well as methods, elements, and/or parameter values associated with system <NUM>, may be incorporated into embodiments of system <NUM> and into methods, elements, and/or parameter values associated with system <NUM>.

System <NUM> comprises a housing or enclosure <NUM> and detection system <NUM> shown in <FIG> that may be disposed within housing <NUM>. Detection system <NUM> comprises a plurality of optical fibers <NUM>, the receiving ends of which are coupled, mounted, or attached to an optical fiber mount <NUM>. The receiving ends of optical fibers <NUM> are configured to receive emissions from optical detection zone <NUM> of respective ones of capillaries <NUM>. System <NUM> also comprises computer processing system <NUM>, computer program product <NUM>, and display or other output device <NUM>. System <NUM> further comprises a plurality of capillaries <NUM> comprising optical detection zone <NUM>, which are coupled, mounted, or attached to a capillary mount <NUM>. In certain embodiments, capillary mount <NUM> may be held or supported by a support structure <NUM> that in turn is mounted or attached to a base <NUM>.

System <NUM> further comprises emission optical system <NUM> and an excitation optical system <NUM> comprising any or all of a radiant source <NUM>. Emission optical system <NUM> comprises lenses <NUM>, <NUM> that are disposed along an optical axis or path <NUM> between capillaries <NUM> and the entrance end of optical fibers <NUM>. Lens <NUM> is configured to collect emission light from each of the capillaries <NUM> and lens <NUM> is configured to reimage the emissions from each of the one or more capillaries <NUM> to a spot or focus in image plane of emission optical system <NUM> that is at or near the input or receiving ends of optical fibers <NUM>; however, other optical configurations known in the art may be used for these purposes.

With further reference to <FIG>, capillaries <NUM> and capillary mount <NUM> may be part of a cartridge or cassette <NUM> that may also include support structure <NUM> and base <NUM>. Base <NUM> may be mounted or attached to cartridge <NUM>. Cartridge <NUM> may be removed from system <NUM> and replaced by another cartridge <NUM>' (not shown) that is configure the same or similar to cartridge <NUM> shown in <FIG> and <FIG>. In certain embodiments, cartridge <NUM>' (not shown) may have the same or similar form, but contain modified or different elements than cartridge <NUM>. For example, cartridge <NUM>' (not shown) may have more or fewer capillaries <NUM> than the four capillaries <NUM> of cartridge <NUM>, for example, <NUM>, <NUM>, or <NUM> capillaries <NUM>.

Capillaries <NUM> are coupled, mounted, or attached to capillary mount <NUM> such that portions of capillaries within optical detection zone <NUM> are fixedly located relative to one another. In similar fashion to capillaries <NUM>, optical fibers <NUM> are coupled, mounted, or attached to optical fiber mount <NUM> such that the input or receiving ends of optical fibers <NUM> are fixedly located relative to one another. It has been discovered that fixedly mounting capillaries <NUM> and the receiving ends of optical fibers <NUM> advantageously simplifies alignment between of optical fibers <NUM> with respective capillaries <NUM>. This arrangement also has been found to improve the accuracy and durability of the alignment between optical fiber <NUM> and capillaries <NUM>.

Referring to <FIG> and <FIG>, in certain embodiments, each capillary <NUM> comprises capillary core <NUM> made of a core material and an outer coating or layer <NUM> surrounding capillary core <NUM>. For example, capillary core <NUM> may comprise fused silica and outer layer <NUM> may comprise a polyimide coating. The central portion of capillary core <NUM> comprises a channel <NUM> through which sample solution and molecules are contained. In such embodiments, for example, when outer layer <NUM> comprises a material that is optically opaque or translucent, optical access to material located in channel <NUM> may be provided by removing outer layer <NUM> along the portion of capillary <NUM> within optical zone <NUM>. As illustrated in <FIG> and the magnified view of <FIG>, in certain embodiments, capillaries <NUM> are mounted to capillary mount <NUM> so that out layers <NUM> of adjacent capillaries <NUM> touch or contact one another. In this way, it has been discovered that the spacing between channels can be easily and accurately provided and maintained. Alternatively, spacers of predetermined thickness may be place between at least two adjacent capillaries on each side of optical detection zone <NUM>. For example, spacer of differing thickness may be placed between different sets of adjacent capillaries to increase the accuracy of the spacing between adjacent capillaries <NUM> and/or to provide a predetermined spacing between adjacent capillaries <NUM>. In other embodiments, capillaries <NUM> may be place in a fixture, such as a V-block, to provide a predetermined spacing between adjacent capillaries <NUM>.

The outer diameter of capillaries <NUM> may be equal to or about <NUM> micrometers, for example, <NUM> ±<NUM> micrometers. In certain embodiments, the outer diameter of capillaries <NUM> is from <NUM> micrometers to <NUM> micrometer, for example, from <NUM> micrometers to <NUM> micrometers. In such embodiments, the diameter of channel <NUM> may be from <NUM> micrometers to <NUM> micrometers, for example, from <NUM> micrometers to <NUM> micrometers. In certain embodiments, the thickness of outer layer <NUM> is from <NUM> micrometers to <NUM> micrometers, for example, from <NUM> micrometers to <NUM> micrometers. In certain embodiments, the outer diameter of each capillary <NUM> is <NUM> ±<NUM> micrometers, the diameter of channel <NUM> is <NUM> ±<NUM> micrometers, and the thickness of outer layer <NUM> is <NUM> micrometers.

In certain embodiments, optical fiber mount <NUM> is coupled, mounted, or attached to a motion or translation stage <NUM>. In use, capillaries <NUM> may be easily aligned using an alignment method comprising:.

In certain embodiments, the alignment signal comprises a measured signal from detector <NUM> based on emissions from a single one of the capillaries <NUM>. Additionally or alternatively, the alignment signal comprises a measured signal from detector <NUM> based on emissions from a more than one of the capillaries <NUM>, for example, based on an average emission from all or some of the capillaries <NUM>.

It has been discovered that this alignment method advantageously allows all the capillaries to be simultaneously aligned to the respective optical fibers <NUM> and, as a consequence, to be simultaneously aligned to the same corresponding areas on detector <NUM> each time the alignment method is performed. The reason emissions from each capillary <NUM> illuminate the same corresponding areas on detector <NUM> each time is because the output (or emitting or distal) ends of each optical fiber <NUM> are in a fixed position relative to detector <NUM>. Therefore, emitted emissions from the output end of optical fibers <NUM> will travel the same path each time to detector <NUM>. When capillaries <NUM> need to be replaced by a new set of capillaries <NUM> and the alignment method rerun, the new capillaries <NUM> will have the same or nearly the same spacing between capillaries as the old set of capillaries <NUM>. Thus, when the disclosed alignment method is performed again, the only emissions from capillaries <NUM> received at detector <NUM> are those emission that pass from the same output ends of optical fibers <NUM>. In prior art systems that directly reimage capillary emissions (i.e., systems that do not use the optical fiber arrangement disclosed herein), slight changes in a new, replacement set of capillaries will cause emissions from the new set of capillaries to be reimaged onto slightly different portions of the detector. Because of this, the detector itself in non-optical fiber based systems must be recalibrated each time, since different areas or, for example, pixels of a CCD or CMOS array detector, have different sensitivities. Therefore, because of the inventive use of optical fibers <NUM> in combination with the fixed mounting configurations of capillaries <NUM> and optical fibers <NUM>, no recalibration of detector <NUM> is necessary when a replacement set of capillaries <NUM> is used.

In the illustrated embodiment shown in <FIG>, translation stage <NUM> is used to translate or move the input ends of optical fibers <NUM> in a transverse direction during the above alignment method. Additionally or alternatively, capillary mount <NUM> may be attached to a motion or translation stage and move instead of, or in addition to, translation stage <NUM>. In other embodiments, relative motion between capillaries <NUM> and optical fibers <NUM> may be accomplished during the alignment method above by making changes to emission optical system <NUM>. For example, a turning mirror or an additional refractive element may be place in the optical path from capillaries <NUM> and optical fibers <NUM>. Adjusting the turning mirror or additional refractive element can then be used to move the reimaged emissions from capillaries <NUM> and so align the reimaged emissions to the receiving ends of optical fibers <NUM>. In other embodiments, the alignment method can be implemented using longitudinal motion in place of or in addition to the transverse movement discussed above with translation stage <NUM>, for example, in order to move the reimaged emissions toward or away from the input ends of optical fibers <NUM>, thereby increasing the amount of emission entering optical fibers <NUM>. In yet other embodiments, emission optical system <NUM> comprises a zoom lens or other optical elements configured to change the magnification of the reimaged emissions from capillaries <NUM>, for example, to accommodate slight changes in spacing between different sets of capillaries <NUM> used in system <NUM>.

In certain embodiments, the alignment signal used in the above alignment method is produced due to Raman scattering of water molecules within one or more of the channels <NUM> of capillaries <NUM>, for example, water molecules contained in a polymer solution used to conduct a capillary electrophoresis assay, process, test, or experiment. The use of Raman scattering from water molecules, which is typically a source of noise, has been unexpected discovered to be suitable for the above alignment method because this signal remains constant over time and, for example, between different filling of capillaries <NUM> with the polymer solution use in capillary electrophoresis. Because of the stability of this signal source, Raman scattering can also be used to calibrate detector <NUM>, as well as provide alignment between capillaries <NUM> and optical fibers <NUM>. In such embodiments, the signal produced by Raman scatter may be measured during or after the alignment method and the detector may then be calibrated based on the value of the measured signal from detector <NUM>. In addition, the use of Raman scatter from water molecules allows the alignment method to be conducted before or after a sample has been introduced into the capillaries <NUM> for a capillary electrophoresis run or other sample separation assay, process, test, or experiment using system <NUM>. In other embodiments, the alignment method may be conducted during a sample separation assay, process, test, or experiment. In such embodiments, emissions from one or more of capillaries <NUM> may be used to adjust alignment during the assay, process, test, or experiment.

Referring to <FIG>, <FIG>, and <FIG>, in certain embodiments, system <NUM> further comprises an optical interface, cover, or snout <NUM> that is configured to engage, interface, or mate with capillary mount <NUM> and/or support structure <NUM>. As seen in <FIG>, base <NUM> may comprise a spring <NUM>, whereby capillary mount <NUM> and/or support structure <NUM> may be held against, mounted to, or engaged with optical interface <NUM> by a contact force that is determined by the amount of compression of spring <NUM> as cartridge <NUM> is placed or aligned within system <NUM>. Optical interface <NUM> may comprise turning mirror <NUM> and/or turning mirror <NUM>, which are part of excitation optical system <NUM>.

Mirrors <NUM>, <NUM> may be configured to a guide a source, source, illumination, or excitation beam <NUM> from radiant source <NUM>, through capillaries <NUM>, and into a beam dump <NUM>. Excitation optical system <NUM> may further comprise other optical elements not shown in <FIG>, for example, lenses, prisms, polarizers, additional mirrors, and the like. For example, one or more lenses may be place along the optical path between radiant source <NUM> and capillaries <NUM> to condition source beam <NUM> to provide a predetermined illumination characteristic as it passes through the plurality of capillaries <NUM>.

It has been discovered that mounting turning mirror <NUM> with optical interface <NUM> advantageously provides a more stable alignment of source beam <NUM> to capillaries <NUM>, since any expansion or contraction along optical axis <NUM> of capillary mount <NUM> and/or support structure <NUM> due to temperature variations over time is compensated for the same or approximately the same movement of turning mirror <NUM> in the direction of optical axis <NUM>. Thus, the position of source beam <NUM> through capillaries <NUM> remains constant or very stable with movement of the of the capillaries due to temperature change. If, for example, source beam <NUM> traveled directly from radiant source <NUM> to capillaries <NUM> (i.e., without first reflecting off turning mirror <NUM>), the position of source beam <NUM> through capillaries <NUM> in the direction parallel to optical axis <NUM> would change as the location of capillaries <NUM> changed due to temperature variation in capillary mount <NUM> and/or support structure <NUM>.

Claim 1:
A system (<NUM>) for separating biological molecules, the system comprising:
a plurality of capillaries (<NUM>) configured to separate biological molecules in a sample, each capillary comprising a detection portion (<NUM>) configured to pass electromagnetic radiation into the capillary;
a capillary mount (<NUM>), the plurality of capillaries coupled to the capillary mount such that the detection portions are fixedly located relative to one another;
a plurality of optical fibers (<NUM>) corresponding to the plurality of capillaries, each optical fiber comprising a receiving end configured to receive emissions from a respective one of the detection portions;
a fiber mount (<NUM>), the optical fibers being coupled to the fiber mount such that the receiving ends of the optical fibers are fixedly located relative to one another;
an emission optical system (<NUM>) configured to direct emissions from the detection portions into the receiving ends of the optical fibers;
an optical detector (<NUM>) configured to produce an alignment signal when emissions from at least one of the plurality of capillaries is transmitted through a respective at least one of the optical fibers and onto the optical detector; and
a motion stage (<NUM>) configured to move to a plurality of locations, one or more of the fiber mount, or at least a portion of the emission optical system;
wherein the motion stage is configured to align the receiving ends of the optical fibers to the detection portions based on values of the alignment signal at the plurality of locations.