Patent Description:
<CIT> discloses an instrument for biological analysis comprising an excitation source, an optical sensor, an excitation optical system, an emission optical system, a plurality of emission filters which are moveable into and out of the emission optical path, a rotatable filter wheel to which the plurality of emission filters are mounted, and a position source and corresponding position sensor configured to produce a position signal indicative of a position of an emission filter of the rotatable filter wheel.

Embodiments of the present invention may be better understood from the following detailed description when read in conjunction with the accompanying drawings. Such embodiments, which are for illustrative purposes only, depict novel and non-obvious aspects of the invention. The drawings include the following figures:.

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 an excitation source means a source of electromagnetic radiation that may be directed toward at least one sample containing one or more chemical compounds such that the electromagnetic radiation interacts with the at least one sample to produce emission electromagnetic radiation indicative of a condition of the at least one sample. The excitation source may comprise light source. As used herein, the term "light source" refers to a source of electromagnetic radiation comprising an electromagnetic spectrum having a peak or maximum output (e.g., power, energy, or intensity) that is within the visible wavelength band of the electromagnetic spectrum (e.g., electromagnetic radiation within a wavelength in the range of <NUM> nanometers to <NUM> nanometers or in the range of <NUM> nanometers and <NUM> nanometers). Additionally or alternatively, the excitation source may comprise electromagnetic radiation within at least a portion of the infrared (near infrared, mid infrared, and/or far infrared) or ultraviolet (near ultraviolet and/or extreme ultraviolet) portions of the electromagnetic spectrum. Additionally or alternatively, the excitation source may comprise electromagnetic radiation in other wavelength bands of the electromagnetic spectrum, for example, in the X-ray and/or radio wave portions of the electromagnetic spectrum. The excitation 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, or the like. The excitation source may comprise a plurality of individual light sources (e.g., a plurality of LEDs or lasers). The excitation 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 may include a colored filter and/or a dichroic filter. The excitation source comprise a single beam or a plurality of beams that are spatially and/or temporally separated.

As used herein, an "emission" means an electromagnetic radiation produced as the result an interaction of radiation from an excitation source with one or more samples containing, or thought to contain, one or more chemical and/or biological molecules or compounds of interest. The emission may be due to a reflection, refraction, polarization, absorption, and/or other optical effect by the a sample on radiation from the excitation source. For example, the emission may comprise a luminescence or fluorescence induced by absorption of the excitation electromagnetic radiation by one or more samples. As used herein "emission light" refers to an emission comprising an electromagnetic spectrum having a peak or maximum output (e.g., power, energy, or intensity) that is within the visible band of the electromagnetic spectrum (e.g., electromagnetic radiation within a wavelength in the range of <NUM> nanometers to <NUM> nanometers).

As used herein, a lens means an optical element configured to direct or focus incident electromagnetic radiation so as to converge or diverge such radiation, for example, to provide a real or virtual image, either at a finite distance or at an optical infinity. The lens may comprise a single optical element having an optical power provided by refraction, reflection, and/or diffraction of the incident electromagnetic radiation. Alternatively, the lens may comprise a compound system including a plurality of optical element, for example, including, but not limited to, an acromatic lens, doublet lens, triplet lens, or camera lens. The lens may be at least partially housed in or at least partially enclosed by a lens case or a lens mount.

As used herein, the term "optical power" means the ability of a lens or optic to converge or diverge light to provide a focus (real or virtual) when disposed within air. As used herein the term "focal length" means the reciprocal of the optical power. As used herein, the term "diffractive power" or "diffractive optical power" means the power of a lens or optic, or portion thereof, attributable to diffraction of incident light into one or more diffraction orders. Except where noted otherwise, the optical power of a lens, optic, or optical element is from a reference plane associated with the lens or optic (e.g., a principal plane of an optic).

As used herein, the term "biological sample" means a sample or solution containing any type of biological chemical or component and/or any target molecule of interest to a user, manufacturer, or distributor of the various embodiments of the present invention described or implied herein, as well as any sample or solution containing related chemicals or compounds used for the purpose of conducting a biological assay, experiment, or test. These biological chemicals, components, or target molecules may include, but are not limited to, DNA sequences (including cell-free DNA), RNA sequences, genes, oligonucleotides, molecules, proteins, biomarkers, cells (e.g., circulating tumor cells), or any other suitable target biomolecule. A biological sample may comprise one or more of at least one target nucleic acid sequence, at least one primer, at least one buffer, at least one nucleotide, at least one enzyme, at least one detergent, at least one blocking agent, or at least one dye, marker, and/or probe suitable for detecting a target or reference nucleic acid sequence. In various embodiments, such biological components may be used in conjunction with one or more PCR methods and systems in applications such as fetal diagnostics, multiplex dPCR, viral detection, and quantification standards, genotyping, sequencing assays, experiments, or protocols, sequencing validation, mutation detection, detection of genetically modified organisms, rare allele detection, and/or copy number variation.

According to embodiments of the present invention, one or more samples or solutions containing at least one biological targets of interest may be contained in, distributed between, or divided between a plurality of a small sample volumes or reaction regions (e.g., volumes or regions of less than or equal to <NUM> nanoliters, less than or equal to <NUM> nanoliter, or less than or equal to <NUM> picoliters). The reaction regions disclosed herein are generally illustrated as being contained in wells located in a substrate material; however, other forms of reaction regions according to embodiments of the present invention may include reaction regions located within through-holes or indentations formed in a substrate, spots of solution distributed on the surface a substrate, samples or solutions located within test sites or volumes of a capillary or microfluidic system, or within or on a plurality of microbeads or microspheres.

While devices, instruments, systems, and methods according to embodiments are generally directed to dPCR and qPCR, embodiments of the present invention may be applicable to any PCR processes, experiment, assays, or protocols where a large number of reaction regions are processed, observed, and/or measured. In a dPCR assay or experiment according to embodiments, a dilute solution containing at least one target polynucleotide or nucleotide sequence is subdivided into a plurality of reaction regions , such that at least some of these reaction regions contain either one molecule of the target nucleotide sequence or none of the target nucleotide sequence. When the reaction regions are subsequently thermally cycled in a PCR protocol, procedure, assay, process, or experiment, the reaction regions containing the one or more molecules of the target nucleotide sequence are greatly amplified and produce a positive, detectable detection signal, while those containing none of the target(s) nucleotide sequence are not amplified and do not produce a detection signal, or a produce a signal that is below a predetermined threshold or noise level. Using Poisson statistics, the number of target nucleotide sequences in an original solution distributed between the reaction regions may be correlated to the number of reaction regions producing a positive detection signal. In some embodiments, the detected signal may be used to determine a number, or number range, of target molecules contained in the original solution. For example, a detection system may be configured to distinguish between reaction regions containing one target molecule and reaction regions containing two or at least two target molecules. Additionally or alternatively, the detection system may be configured to distinguish between reaction regions containing a number of target molecules that is at or below a predetermined amount and reaction regions containing more than the predetermined amount. In certain embodiments, both qPCR and dPCR processes, assays, or protocols are conducted using a single the same devices, instruments, or systems, and methods.

Referring to <FIG>, a system, apparatus, or instrument <NUM> for biological analysis comprises one or more of an electronic processor, computer, or controller <NUM>, a base, mount, or sample block assembly <NUM> configured to receive and/or processes a biological or biochemical sample, and/or an optical system, apparatus, or instrument <NUM>. Without limiting the scope of the present invention, system <NUM> may comprise a sequencing instrument, a polymerase chain reaction (PCR) instrument (e.g., a real-time PCR (qPCR) instrument and/or digital PCR (dPCR) instrument), capillary electrophoresis instrument, an instrument for providing genotyping information, or the like.

Electronic processor <NUM> is configured to control, monitor, and/or receive data from optical system <NUM> and/or base <NUM>. Electronic processor <NUM> may be physically integrated into optical system <NUM> and/or base <NUM>. Additionally or alternatively, electronic processor <NUM> may be separate from optical system <NUM> and base <NUM>, for example, an external desktop computer, laptop computer, notepad computer, tablet computer, or the like. Communication between electronic processor <NUM> and optical system <NUM> and/or base <NUM> may be accomplished directly via a physical connection, such as a USB cable or the like, and/or indirectly via a wireless or network connection (e.g., via Wi-Fi connection, a local area network, internet connection, cloud connection, or the like). Electronic processor <NUM> may include electronic memory storage containing instructions, routines, algorithms, test and/or configuration parameter, test and/or experimental data, or the like. Electronic processor <NUM> may be configured, for example, to operate various components of optical system <NUM> or to obtain and/or process data provided by base <NUM>. For example, electronic processor <NUM> may be used to obtain and/or process optical data provided by one or more photodetectors of optical system <NUM>.

In certain embodiments, electronic processor <NUM> may integrated into optical system <NUM> and/or base <NUM>. Electronic processor <NUM> may communicate with external computer and/or transmit data to an external computer for further processing, for example, using a hardwire connection, a local area network, an internet connection, cloud computing system, or the like. The external computer may be physical computer, such as a desktop computer, laptop computer, notepad computer, tablet computer, or the like, that is located in or near system <NUM>. Additionally or alternatively, either or both the external computer and electronic processor <NUM> may comprise a virtual device or system, such as a cloud computing or storage system. Data may be transferred between the two via a wireless connection, a cloud storage or computing system, or the like. Additionally or alternatively, data from electronic processor <NUM> (e.g., from optical system <NUM> and/or base <NUM>) may be transferred to an external memory storage device, for example, an external hard drive, a USB memory module, a cloud storage system, or the like.

In certain embodiments, base <NUM> is configured to receive a sample holder or sample carrier <NUM>. Sample holder <NUM> may comprise a plurality or array of spatially separated reaction regions, sites, or locations <NUM> for containing a corresponding plurality or array of biological or biochemical samples <NUM>. Reaction regions <NUM> may comprise any plurality of volumes or locations isolating, or configured to isolate, the plurality of biological or biochemical samples <NUM>. For example, reaction regions <NUM> may comprise a plurality of through-hole or well in a substrate or assembly (e.g., sample wells in a standard microtiter plate), a plurality of sample beads, microbeads, or microspheres in a channel, capillary, or chamber, a plurality of distinct locations in a flow cell, a plurality of sample spots on a substrate surface, or a plurality of wells or openings configured to receive a sample holder (e.g., the cavities in a sample block assembly configured to receive a microtiter plate).

Base <NUM> may comprise a sample block assembly configured to control the temperature of sample holder <NUM> and/or biological samples <NUM>. Sample block assembly <NUM> may comprise one or more of a sample block, a Peltier device or other apparatus for controlling or cycling temperature, and/or a heat sink (e.g., for aiding in stabilizing a temperature). Base <NUM> may comprise a thermal controller or thermal cycler, for example, to provide or perform a PCR assay.

Reaction apparatus <NUM> may include sample holder <NUM>. At least some of the reaction regions <NUM> may include the one or more biological samples <NUM>. Biological or biochemical samples <NUM> may include one or more of at least one target nucleic acid sequence, at least one primer, at least one buffer, at least one nucleotide, at least one enzyme, at least one detergent, at least one blocking agent, or at least one dye, marker, and/or probe suitable for detecting a target or reference nucleic acid sequence. Sample holder <NUM> may be configured to perform at least one of a PCR assay, a sequencing assay, or a capillary electrophoresis assay, a blot assay. In certain embodiments, sample holder <NUM> may comprise one or more of a microtiter plate, substrate comprising a plurality of wells or through-holes, a substrate comprising a one or more channels or capillaries, or a chamber comprising plurality of beads or spheres containing the one or more biological samples. Reaction regions <NUM> may comprise one or more of a plurality of wells, a plurality of through-holes in substrate, a plurality of distinct locations on a substrate or within a channel or capillary, a plurality of microbeads or microspheres within a reaction volume, or the like. Sample holder <NUM> may comprise a microtiter plate, for example, wherein reaction regions <NUM> may comprise at least <NUM> well, at least <NUM>, or at least <NUM> wells.

In certain embodiments, sample holder <NUM> may comprise a substrate including a first surface, an opposing second surface, and a plurality of through-holes disposed between the surfaces, the plurality of through-holes configured to contain the one or more biological samples, for example as discussed in Patent Application Publication Numbers <CIT> and <CIT>. In such embodiments, the substrate may comprise at least <NUM> through-holes or at least <NUM>,<NUM> through-holes. In certain embodiments, sample holder <NUM> may comprise an array of capillaries configured to pass one or more target molecules or sequence of molecules.

In certain embodiments, system <NUM> may optionally include a heated or temperature controlled cover <NUM> that may be disposed above sample holder <NUM> and/or base <NUM>. Heated cover <NUM> may be used, for example, to prevent condensation above the samples contained in sample holder <NUM>, which can help to maintain optical access to biological samples <NUM>.

In certain embodiments, optical system <NUM> comprises an excitation source, illumination source, radiation source, or light source <NUM> that produces at least a first excitation beam 405a characterized by a first wavelength and a second excitation beam 405b characterized by a second wavelength that is different from the first wavelength. Optical system <NUM> also comprises an optical sensor or optical detector <NUM> configured to receive emissions or radiation from one or more biological samples in response to excitation source <NUM> and/or to one or more of excitation beams 405a, 405b. Optical system <NUM> additionally comprises an excitation optical system <NUM> disposed along an excitation optical path <NUM> between excitation source <NUM> and one or more biological samples to be illuminated. Optical system <NUM> further comprises an emission optical system <NUM> disposed along an emission optical path <NUM> between the illuminated sample(s) and optical sensor <NUM>. In certain embodiments, optical system <NUM> may comprise a beamsplitter <NUM>. Optical system <NUM> may optionally include a beam dump or radiation baffle <NUM> configured reduce or prevent reflection of radiation into emission optical path <NUM> from excitation source <NUM> that impinges on beamsplitter <NUM>.

In the illustrated embodiment shown in <FIG>, as well as other embodiments of the invention disclosed herein, excitation source <NUM> comprises a radiation source <NUM>. Radiation source <NUM> may comprise one or more of at least one an incandescent lamp, at least one gas discharge lamp, at least one light emitting diode (LED), at least one organic light emitting diode, and/or at least one laser. For example, radiation source <NUM> may comprise at least one Halogen lamp, Xenon lamp, Argon lamp, Krypton lamp, diode laser, Argon laser, Xenon laser, excimer laser, solid-state laser, Helium-Neon laser, dye laser, or combinations thereof. Radiation source <NUM> may comprise a light source characterized by a maximum or central wavelength in the visible band of the electromagnetic spectrum. Additionally or alternatively, radiation source <NUM> may comprise an ultraviolet, infrared, or near-infrared source with a corresponding maximum or central wavelength within on one of those wavelength bands of the electromagnetic spectrum. Radiation source <NUM> may be a broadband source, for example, having a spectral bandwidth of at least <NUM> nanometers, at least <NUM> nanometers, or at least <NUM> nanometers, where the bandwidth is defined as a range over which the intensity, energy, or power output is greater than a predetermined amount (e.g., where the predetermined amount is at or about <NUM>%, <NUM>%, or <NUM>% of a maximum or central wavelength of the radiation source). Excitation source <NUM> may additionally comprise a source lens <NUM> configured to condition emissions from radiation source <NUM>, for example, to increase the amount of excitation radiation received at sample holder <NUM> and/or into biological samples <NUM>. Source lens <NUM> may comprise a simple lens or may be a compound lens including two or more elements.

In certain embodiments, excitation source <NUM> further comprises two or more excitation filters <NUM> moveable into and out of excitation optical path <NUM>, for instance, used in combination with a broadband excitation source <NUM>. In such embodiments, different excitation filters <NUM> may be used to select different wavelength ranges or excitation channels suitable for inducing fluorescence from a respective dye or marker within biological samples <NUM>. One or more of excitation filters <NUM> may have a wavelength bandwidth that is at least ±<NUM> nanometers or at least ±<NUM> nanometers. Excitation filters <NUM> may comprise a plurality of filters that together provide a plurality of band passes suitable for fluorescing one or more of a SYBR® dye or probe, a FAM™ dye or probe, a VIC® dye or probe, a ROX™ dye or probe, or a TAMRA™ dye or probe. Excitation filters <NUM> may be arrange in a rotatable filter wheel (not shown) or other suitable device or apparatus providing different excitation channels using excitation source <NUM>. In certain embodiments, excitation filters <NUM> comprise at least <NUM> filter or at least <NUM> filter.

In certain embodiments, excitation source <NUM> may comprise a plurality of individual excitation sources that may be combined using one more beamsplitters or beam combiners, such that radiation from each individual excitation source is transmitted along a common optical path, for example, along excitation optical path <NUM> shown in <FIG>. Alternatively, at least some of the individual excitation sources may be arranged to provided excitation beams that propagate along different, non-overlapping optical paths, for example, to illuminate different reaction regions of the plurality of reaction regions <NUM>. Each of the individual excitation sources may be addressed, activated, or selected to illuminate reaction regions <NUM>, for example, either individually or in groups or all simultaneously. In certain embodiments, the individual excitation sources may be arrange in a one-dimensional or two-dimensional array, where one or more of the individual excitation sources is characterized by a maximum or central wavelength that is different than that of at least one of the other individual excitation sources in the array.

In certain embodiments, first excitation beam 405a comprises a first wavelength range over which an intensity, power, or energy of first excitation beam 405a is above a first predetermined value and second excitation beam 405b comprises a second wavelength range over which an intensity, power, or energy of second excitation beam 405b is above a second predetermined value. The characteristic wavelength of the excitation beams 405a, 405b may be a central wavelength of the corresponding wavelength range or a wavelength of maximum electromagnetic intensity, power, or energy over the corresponding wavelength range. The central wavelengths of at least one of the excitation beams <NUM> may be an average wavelength over the corresponding wavelength range. For each excitation beam <NUM> (e.g., excitation beams 405a, 405b), the predetermined value may be less than <NUM>% of the corresponding maximum intensity, power, or energy; less than <NUM>% of the corresponding maximum intensity, power, or energy; less than <NUM>% of the corresponding maximum intensity, power, or energy; or less than <NUM>% of the corresponding maximum intensity, power, or energy. The predetermined values may be the same for all excitation beams <NUM> (e.g., for both excitation beams 405a, 405b) or the predetermined values may be different from one another. In certain embodiments, the wavelength ranges of the first and second excitation beams 405a, 405b do not overlap, while in other embodiments at least one of the wavelength ranges at least partially overlaps that of the other. In certain embodiments, the first and second central wavelengths are separated by at least <NUM> nanometer. In certain embodiments, at least one of the first and second wavelength ranges has a value of at least <NUM> nanometer or at least <NUM> nanometers.

Excitation optical system <NUM> is configured to direct excitation beams 405a, 405b to the one or more biological samples. Where applicable, references herein to excitation beams 405a, 405b may be applied to embodiment comprising more than two excitation beams <NUM>. For example, excitation source <NUM> may be configured to direct at least five or six excitation beams <NUM>. Excitation beams 405a, 405b may be produced or provided simultaneously, may be temporally separated, and/or may be spatially separated (e.g., wherein excitation beams 405a is directed to one reaction region <NUM> and excitation beams 405b is directed to a different reaction region <NUM>). The excitation beams <NUM> may be produced sequentially, for example, by sequentially turning on and off different-colored individual radiation source <NUM> that are characterized by different wavelengths or by sequentially placing different color filters in front of a single radiation source <NUM>. Alternatively, excitation beams 405a, 405b may be produced simultaneously, for example, by using a multi-wavelength band filter, beamsplitter, or mirror, or by coupling together different individual radiation source <NUM>, such as two different-colored light emitting diodes (LEDs). In some embodiments, excitation source <NUM> produces more than two excitation beams <NUM>, wherein excitation optical system <NUM> directs each of the excitation beams to one or more biological samples <NUM>.

Referring to <FIG>, excitation source <NUM> may comprise at least <NUM> individual radiant sources 425a, 425b, 425c, 425d, 425e that are combined to transmit along a common excitation optical path <NUM>. Excitation source <NUM> may also comprise corresponding sources lenses 428a, 428b, 428c, 428d, 428e. Radiation from radiant sources 425a, 425b, 425c, 425d, 425e may be combined using a plurality of combiner optical elements 429a, 429b, 429c. Combiner optical elements 429a, 429b, 429c may comprise one or more of a neutral density filter, a <NUM>/<NUM> beamsplitter, a dichroic filter or mirror, a cube beamsplitter, or the like. Combiner optical elements 429a, 429b, 429c are one example of how to combine various individual sources <NUM> and it will be appreciated that other combinations and geometrical arrangements of individual radiant sources <NUM> and combiner optical elements <NUM> are within the scope of embodiments of the present invention. One or more of individual radiant sources 425a, 425b, 425c, 425d, 425e may be characterized by a central wavelength and/or wavelength range that is differ from that of the other individual radiant sources 425a, 425b, 425c, 425d, 425e.

Referring to <FIG>, the spectral distribution of radiation source <NUM> may be selected in a non-obvious manner to enable at least five excitation beams <NUM> of different colors or excitation channels to be used with one common beamsplitter <NUM>, while simultaneously maintaining acceptable or predetermined data throughput for all excitation channels, for example, during each cycle of the qPCR assay. As used herein, the term "excitation channel" means each of several, distinct electromagnetic wavelength bands providing by an excitation source (e.g., excitation source <NUM>) that are configured to illuminate one or more biological samples (e.g., biological samples <NUM>). As used herein, the term "emission channel" means each of several, distinct emission wavelength bands over which electromagnetic radiation is allowed to pass onto an optical sensor or detector (e.g., optical sensor <NUM>).

<FIG> shows the relative energy over the wavelength spectrum for three different radiation sources. The dashed line plot is the spectrum of a Halogen lamp (herein referred to as "Source <NUM>") characterized by relatively low energy levels in the blue wavelength range of the visible spectrum and increasing energy until a peak at about <NUM> nanometers. The dash-dot spectrum plot is that of a commercially available LED light source (herein referred to as "Source <NUM>"), which has peak energy at around <NUM> nanometers and a lower peak from about <NUM> nanometers to about <NUM> nanometers, then steadily decreasing energy into the red wavelength range of the visible spectrum. The solid line plot is the spectrum of another LED light source (herein referred to as "Source <NUM>") according to an embodiment of the present invention (e.g., an exemplary spectrum for excitation source <NUM>). <FIG> shows integrated energy over various excitation channels for each of the three sources shown in <FIG>, where the spectrums for these channels are those of typical excitation filter used in the field of qPCR. The wavelength ranges and excitation filter designations are shown below in Table <NUM>, where X1 is excitation channel <NUM>, X2 is excitation channel <NUM>, and so forth.

In the field of qPCR, one important performance parameter is the total time to obtain emission data for samples containing multiple target dyes. For example, in some cases it is desirable to obtain emission data from multiple dyes or probes over one or more emission channels, designated M1-M6, for each excitation channel used to illuminate the sample(s) (e.g., M1-M6 with X1, M2-M6 with X2, M3-M6 with X3, M4-M6 with X4, M5-M6 with X5, and/or M6 with X6). The inventors have found that when Source <NUM> is used in a system having a single, broadband beamsplitter for five or six excitation/emission filter channels (e.g., excitation channels X1-X6 with combinations emission channels M1-M6), the amount of time to obtain data for excitation channel <NUM> and/or excitation channel <NUM> could be unacceptably long for certain applications. To remedy this situation, it is possible to use one or more narrow band, dichroic beamsplitters for excitation channels <NUM> and/or <NUM> to increase the amount of excitation light receive by the sample(s), and the amount of emission light received by the sensor (so that the overall optical efficiency is increased by using dichroic beam splitter, in this case). However, this precludes the use of a single beamsplitter arrangement, as shown in <FIG> and, therefore, the corresponding advantages of a single beamsplitter configuration (e.g., reduced size, cost, complexity) are lost. A better solution has been discovered in which a light source, such as Source <NUM>, is used in combination with a single beamsplitter (e.g., a broadband beamsplitter such as a <NUM>/<NUM> beamsplitter), such as beamsplitter <NUM>. It has been found that the relative energy in excitation channels X1, X5, and/or X6 may be used to identify an excitation source <NUM> suitable for use with a single beamsplitter embodiment to provide acceptable total integration time for collecting emission data over five or six excitation channels. Using LED Source <NUM> and LED Source <NUM> as examples, the following data shown in Table <NUM> below may be derived for the data shown in <FIG>.

Based on such data, the inventors have found that, in certain embodiments, improved performance (e.g., in terms of shorter Channel <NUM> integration time) may be obtain when X1/X2 is greater than <NUM> (e.g., greater than or equal to <NUM>). Additionally or alternatively, in other embodiments, improved performance (e.g., in terms of shorter Channel <NUM> integration time) may be obtain when X5/X2 is greater than <NUM> (for example, greater than or equal to <NUM>) and/or when X6/X2 is greater than <NUM> (for example, greater than or equal to <NUM>). For the criteria set forth here, "X1" means an excitation channel that has a spectral output characterized by a maximum power, energy, or intensity within the wavelength band including <NUM>-<NUM> nanometers; "X2" means an excitation channel that has a spectral output characterized by a maximum power, energy, or intensity within the wavelength band including <NUM>-<NUM> nanometers; "X5" means an excitation channel that has a spectral output characterized by a maximum power, energy, or intensity within the wavelength band including <NUM>-<NUM> nanometers; "X6" means an excitation channel that has a spectral output characterized by a maximum power, energy, or intensity within the wavelength band including <NUM>-<NUM> nanometers.

Referring again to <FIG>, excitation beams <NUM> are directed along excitation optical path <NUM> during operation toward sample processing base <NUM>, for example, toward reaction regions <NUM> when sample holder <NUM> is present. When present, source lens <NUM> is configure to condition excitation beams <NUM>, for example, to capture and direct a large portion of the emitted radiation from excitation source <NUM>. In certain embodiments, one or more mirrors <NUM> (e.g., fold mirrors) may be incorporated along excitation optical path <NUM>, for example, to make optical system <NUM> more compact and/or to provide predetermined package dimensions. <FIG> illustrated one mirror <NUM>; however, addition mirrors may be used, for example to meet packaging design constraints. As discussed in greater detail below herein, additional lenses may be disposed near sample holder <NUM>, for example, in order to further condition the excitation beams <NUM> and/or corresponding emissions from biological samples contained in one or more reaction regions.

Emission optical system <NUM> is configured to direct emissions from the one or more biological samples to optical sensor <NUM>. At least some of the emissions may comprise a fluorescent emission from at least some of the biological samples in response to at least one of the excitation beams <NUM>. Additionally or alternatively, at least some of the emissions comprise radiation from at least one of the excitation beams <NUM> that is reflected, refracted, diffracted, scattered, or polarized by at least some of the biological samples. In certain embodiments, emission optical system <NUM> comprise one or more emission filters <NUM> configured, for example, to block excitation radiation reflected or scattered into emission optical path <NUM>. In certain embodiments, there is a corresponding emission filter <NUM> for each excitation filter <NUM>. Referring to <FIG>, in certain embodiments, the excitation filter <NUM> are arranged in an excitation filter wheel <NUM> and/or the emission filters <NUM> are arranged in an emission filter wheel <NUM>.

In certain embodiments, emission optical system <NUM> comprises a sensor lens <NUM> configured to direct emissions from at least some of the biological samples onto optical sensor <NUM>. Optical sensor <NUM> may comprise a single sensor element, for example, a photodiode detector or a photomultiplier tube, or the like. Additionally or alternatively, optical sensor <NUM> may comprise an array sensor including an array of sensors or pixels. Array sensor <NUM> 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. Sensor lens <NUM> may be configured to from an image from the emissions from one or more of the plurality of biological samples <NUM>. In certain embodiments, optical sensor <NUM> comprises two or more array sensors <NUM>, for example, where two or more images are formed from the emissions from one or more of the plurality of biological samples <NUM>. In such embodiments, emissions from one or more of the plurality of biological samples <NUM> may be split to provide two signals of the one or more of the plurality of biological samples <NUM>. In certain embodiments, the optical sensor comprises at least two array sensors.

Beamsplitter <NUM> is disposed along both excitation and emission optical paths <NUM>, <NUM> and is configured to receive both first and second excitation beams 405a, 405b during operation. In the illustrated embodiment shown in <FIG>, beamsplitter <NUM> is configured to transmit the excitation beams <NUM> and to reflect emissions from the biological samples <NUM>. Alternatively, beamsplitter <NUM> may be configured to reflect the excitation beams and to transmit emissions from the biological samples <NUM>. In certain embodiments, beamsplitter <NUM> comprises a broadband beamsplitter having the same, or approximately the same, reflectance for all or most of the excitation beams <NUM> provided by excitation source <NUM> and directed to the reaction regions <NUM> (e.g., excitation beams 405a, 405b in the illustrated embodiment). For example, beamsplitter <NUM> may be a broadband beamsplitter characterized by a reflectance that is constant, or about constant, over a wavelength band of at least <NUM> nanometers, over a wavelength band of at least <NUM> nanometers, or over the visible wavelength band of the electromagnetic spectrum, over the visible and near IR wavelength bands of the electromagnetic spectrum, or over a wavelength band from <NUM> nanometers to <NUM> nanometers. In certain embodiments, beamsplitter <NUM> is a neutral density filter, for example, a filter having a reflectance of, or about, <NUM>%, <NUM>%, or <NUM>% over visible wavelength band of the electromagnetic spectrum. In certain embodiments, beamsplitter <NUM> is a dichroic beamsplitter that is transmissive or reflective over one or more selected wavelength ranges, for example, a multi-wavelength band beamsplitter that is transmissive and/or reflective over more than one band of wavelengths centers at or near a peak wavelength of excitation beams <NUM>.

In certain embodiments, beamsplitter <NUM> is a single beamsplitter configure to receive some or all of the plurality of excitation beams <NUM> (e.g., excitation beams 405a, 405b), either alone or in combination with a single beam dump <NUM>. Each excitation beam may be referred to as an excitation channel, which may be used alone or in combination to excite different fluorescent dyes or probe molecule in one or more of the biological samples <NUM>. By contrast many prior art systems and instruments, for example, in the field of qPCR, provide a plurality of excitation beams by using a separate beamsplitter and/or beam dump for each excitation channel and/or each emission channel of the system or instrument. In such prior art systems and instruments, chromatically selective dichroic filters are typically used in at least some of the excitation channels to increase the amount of radiation received at the samples. Disadvantages of systems and instruments using different beamsplitters and/or beam dumps for each channel include an increase in size, cost, complexity, and response time (e.g., dues to increased mass that must be moved or rotated when changing between excitation and/or emission channels). The inventors have discovered that it is possible to replace these plural beamsplitters and/or beam dumps with the single beamsplitter <NUM> and/or single beam dump <NUM>, while still providing an acceptable or predetermined system or instrument performance, for example, by proper selection of spectral distribution of excitation source <NUM> and/or by configuring the systems or instruments to reduce the amount of stray or unwanted radiation received by optical sensor <NUM> (as discuss further herein). Thus, embodiments of the present invention may be used to provide systems and instruments that have reduced size, cost, complexity, and response time as compared to prior art systems and instruments.

Referring to <FIG>, in certain embodiments, system <NUM> comprises an instrument housing <NUM> and sample holder drawer <NUM> comprising base <NUM> and configured during use to receive, hold, or contain sample holder <NUM> and to position sample holder <NUM> to provide optical coupling thereof with optical system <NUM>. With drawer <NUM> closed (<FIG>), housing <NUM> may be configured to contain or enclose sample processing system <NUM> and optical system <NUM>. In certain embodiments, housing <NUM> may contain or enclose all or portions of electronic processor <NUM>.

Referring to <FIG>, in certain embodiments, optical system <NUM> may further comprise a lens <NUM> and/or a lens array <NUM>, which may comprise a plurality of lenses corresponding to each of the reaction regions <NUM> of sample holder <NUM>. Lens <NUM> may comprises a field lens, which may be configured to provide a telecentric optical system for a least one of sample holder <NUM>, reaction regions <NUM>, lens array <NUM>, or optical sensor <NUM>. As shown in illustrated embodiment in <FIG> and <FIG>, lens <NUM> may comprise a Fresnel lens.

Referring again to <FIG> and <FIG>, in certain embodiments, base <NUM> comprises a sample block assembly <NUM> comprising a sample block <NUM>, temperature controller <NUM>, such as a Peltier device <NUM>, and a heat sink <NUM>. Sample block assembly <NUM> may be configured to provide a thermal controller or thermal cycling (e.g., provide a PCR assay or temperature profile), maintain a temperature of sample holder <NUM> or biological sample(s) <NUM>, and/or otherwise maintain, control, adjust, or cycle heat flow or temperature of sample holder <NUM> or biological sample(s) <NUM>.

With additional reference to <FIG>, in certain embodiments, optical system <NUM> includes an imaging unit <NUM> comprising an optical sensor circuit board <NUM>, sensor lens <NUM> (which may be a compound lens, as illustrated in <FIG>), an inner lens mount <NUM>, an outer lens mount <NUM>, a threaded housing <NUM>, and a focusing gear <NUM>. Optical sensor circuit board <NUM>, threaded housing <NUM>, and sensor lens <NUM> together may form a cavity <NUM> that encloses or contains optical sensor <NUM> and may be configured to block any external light from impinging optical sensor <NUM> that does not enter through sensor lens <NUM>. Outer lens mount <NUM> comprises an outer surface containing gear teeth <NUM> that may be moveably or slideably engaged with the teeth of focusing gear <NUM> via a resilient element (not shown), such as a spring. In certain embodiments, focusing gear <NUM> moves or slide along a slot <NUM> of a plate <NUM>, as illustrated in <FIG>. Inner lens mount <NUM> comprises a threaded portion <NUM> that engages or mates with a threaded portion of threaded housing <NUM>.

Inner lens mount <NUM> may be fixedly mounted to outer lens mount <NUM>, while threaded housing <NUM> is fixedly mounted relative to optical sensor circuit board <NUM>. Inner lens mount <NUM> is moveably or rotatably mounted to threaded housing <NUM>. Thus, focusing gear <NUM> and outer lens mount <NUM> may be engaged such that a rotation of focusing gear <NUM> also rotates outer lens mount <NUM>. This, in turn, causes inner lens mount <NUM> and sensor lens <NUM> to move along an optical axis of sensor lens <NUM> via the threads in inner lens mount <NUM> and threaded housing <NUM>. In this manner, the focus of sensor lens <NUM> may be adjusted without directly engaging sensor lens <NUM> or its associated mounts <NUM>, <NUM>, which are buried within a very compact optical system <NUM>. Engagement with focusing gear <NUM> may be either by hand or automated, for example using a motor (not shown), such as a stepper motor or DC motor.

Referring to <FIG> and <FIG>, in certain embodiments, imaging unit <NUM> further comprises a locking device or mechanism <NUM>. Locking device <NUM> comprises an edge or tooth <NUM> that may be slideably engaged between two teeth of focusing gear <NUM> (see <FIG>). As illustrated in <FIG>, locking device <NUM> may have a first position (<FIG>) in which focusing gear <NUM> is free to rotate and adjust the focus of sensor lens <NUM> and a second position (<FIG>) is which focusing gear <NUM> is locked in position and impeded or prevented from rotating. In this manner, the focus of sensor lens <NUM> may be locked while advantageously avoiding direct locking contact or engagement with threads <NUM> of inner lens mount <NUM>, which could damage the threads and prevent subsequent refocusing of sensor lens <NUM> after being locked into position. Operation of locking device <NUM> may be either manually or in an automated manner. In certain embodiments, locking mechanism <NUM> further comprises a resilient element such as a spring (not shown), wherein rotation of focusing gear <NUM> may be accomplished by overcoming a threshold force produced by the resilient element.

Referring to <FIG>, optical system <NUM> may also include an optics housing <NUM>. In certain embodiments, optical system <NUM> includes a radiation shield <NUM> comprising a sensor aperture <NUM> disposed along emission optical path <NUM> and at least one blocking structure <NUM> disposed to cooperate with sensor aperture <NUM> such that the only radiation from excitation beams <NUM>, and reflected off an illuminated surface or area <NUM>, to pass through sensor aperture <NUM> is radiation that has also reflected off at least one other surface of, or within, the optics housing <NUM>. In other words, radiation shield <NUM> is configured such that radiation from excitation beams <NUM> reflected illuminated area <NUM> are blocked from directly passing through aperture <NUM> and, therefore, from passing into sensor lens <NUM> and onto optical detector <NUM>. In certain embodiments, illuminated area <NUM> comprises the area defined by all the apertures <NUM> of heated cover <NUM> corresponding to the plurality of reaction regions <NUM>.

In the illustrated embodiment of <FIG>, blocking structure <NUM> comprises a shelf <NUM>. Dashed lines or rays 484a and 484b may be used to illustrate the effectiveness of blocking structure <NUM> in preventing light directly reflected from illuminated area <NUM> from passing through sensor aperture <NUM> and onto senor lens <NUM> and/or optical sensor <NUM>. Ray 484a originates from an edge of illuminated area <NUM> an just passes shelf <NUM>, but does not pass through sensor aperture <NUM>. Ray 484b is another ray originating from the same edge of illuminated area <NUM> that is blocked by shelf <NUM>. As can be seen, this ray would have entered through sensor aperture <NUM> were it not for the presences of shelf <NUM>.

With continued reference to <FIG>, in certain embodiments, optical system <NUM> may further comprise an energy or power detection unit comprising a power or energy sensors <NUM> optically coupled to one end of a light pipe <NUM>. An opposite end <NUM> of light pipe <NUM> is configured to be illuminated by excitation beams <NUM>. Light pipe end <NUM> may be illuminated either directly by radiation contained in excitation beams <NUM> or indirectly, for example, by radiation scattered by a diffuse surface. In certain embodiments, sensor <NUM> is located outside of the excitation optical path <NUM> from excitation source <NUM>. Additionally or alternatively, sensor <NUM> is located outside optics housing <NUM> and/or is located at a remote location outside instrument housing <NUM>. In the illustrated embodiment shown in <FIG>, light pipe end <NUM> is disposed near or adjacent mirror <NUM> and may be oriented so that the face of the light pipe is perpendicular, or nearly perpendicular, to the surface of mirror <NUM> that reflects excitation beams <NUM>. The inventors have discovered that the low amount of energy or power intercepted by light pipe <NUM> when oriented in this way is sufficient for the purpose of monitoring the energy or power of excitation beams <NUM>. Advantageously, by locating sensor <NUM> outside the optical path of excitation beams a more compact optical system <NUM> may be provided.

In certain embodiments, light pipe <NUM> comprises a single fiber or a fiber bundle. Additionally or alternatively, light <NUM> may comprise a rod made of a transparent or transmissive material such as glass, Plexiglas, polymer based material such as acrylic, or the like.

Referring to <FIG> instrument <NUM> comprises a position source <NUM> configured to emit radiation <NUM> and a corresponding position sensor <NUM> configured to receive radiation <NUM> from position source <NUM>. Position source <NUM> and position sensor <NUM> may be configured to produce a position signal indicative of a position of an optical element <NUM> disposed along an optical paths. Instrument <NUM> comprises radiation shields <NUM> configured to block at least some radiation <NUM> from position source <NUM>.

The above presents a description of the best mode contemplated of carrying out the present invention to enable any person skilled in the art to which it pertains to make and use this invention. This invention is, however, susceptible to modifications and alternate constructions from that discussed above which are fully equivalent. Consequently, it is not the intention to limit this invention to the particular embodiments disclosed. On the contrary, the intention is to cover modifications and alternate constructions coming within the scope of the invention according to the appended claims.

Claim 1:
An instrument (<NUM>) for biological analysis, comprising:
an excitation source (<NUM>);
an optical sensor (<NUM>) configured to receive emissions from biological samples in response to the excitation source;
an excitation optical system (<NUM>) disposed along an excitation optical path;
an emission optical system (<NUM>) disposed along an emission optical path;
a plurality of emission filters (<NUM>) which are moveable into and out of the emission optical path;
a rotatable filter wheel (<NUM>) to which the plurality of emission filters are mounted and configured to move each of the filters into and out of the emission beam path by rotation of the rotatable filter wheel;
a position source (<NUM>) configured to emit radiation (<NUM>) and a corresponding position sensor (<NUM>) configured to receive radiation from the position source,
the position source and the position sensor together arranged at a radial position with respect to the filter wheel beyond the rotatable filter wheel and configured to produce a position signal indicative of a position of an emission filter of the rotatable filter wheel;
a housing enclosing a peripheral portion of the filter wheel, at which portion the emission filters are mounted, the position source and the position sensor being provided to the housing;
and radiation shields (<NUM>) extending from the housing of the rotatable filter wheel towards the rotatable filter wheel at spaced positions between the position source and the emission beam path so as to block at least some radiation from the position source from reaching the emission beam path.