System and Method for Instrument Calibration

Abstract

Methods for generating a calibration factor and for calibrating are provided whereby interstitial spacing between support locations of a platform is used to embed a detectable material, emissions from which are used to generate the calibration factor. In some embodiments, the external space, outer perimeter, and other areas of a platform are used to embed detection materials for the normalization, calibration, correction, compensation, or other method of adjustment for detected emission data. The emission data can be taken from an assay, amplification, reaction, analysis, or other process, for example, from a PCR run or other reaction. By calibrating or adjusting the sample support with the detected emission data, a more efficient detection of the assay, amplification, reaction, analysis, or other process, can be achieved.

Description

RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application No. 60/898,064, filed Jan. 29, 2007, entitled “Systems and Methods for Optical and Spectral Calibration of Real-Time PCR Instrumentation,” which is incorporated herein by reference.

FIELD

The present application relates to biological testing devices, instruments that process such devices, and methods that use such devices and instruments.

Introduction

In various known polymerase chain reaction (PCR) implementations, better accuracy in the detection of an amplified signal, and hence original sample quantity, is frequently sought by calibrating each dye, fluorescent material, or other reference material used, in an effort to reduce error in the emission signal detected in each amplification well. This method requires a technician to load one or more sets of liquid pure fluorescent dyes into individual wells of a sample plate, and detect absolute signal levels for those pure dye references. When a calibration protocol for a first dye is completed and a PCR or other run is performed, the plate is disposed of, and then a new plate is used to calibrate for a second dye. A separate plate is used for each dye. The plates are expensive, and calibrating for many dyes requires using many plates. The calibration process can also take up a great deal of time, for example, about 10 minutes to calibrate each plate. The set of dyes loaded in each of the series of calibrations that are conducted on a given plate can differ in volume, concentration, temperature, and other variables of the dye material, and therefore lead to different or varying values in the calibration data. Performing the calibration itself, likewise depends on a trained technician available to conduct the calibration run, and consumes time and dye on each test occasion. Drawbacks of this current calibration method, therefore, include extensive calibration time, lack of uniformity between instruments, lack of uniformity between well plates and dyes, the potential for errors, and the inability to perform PCR, real-time PCR (RT-PCR), assays, processes, or other reactions while calibrating the emission signal. A need exists for calibration and related techniques that address these and other issues.

SUMMARY

According to various embodiments, the present teachings relate to a system and method used in the identification and calibration of signal levels in PCR, RT-PCR, and other assays, amplifications, reactions, processes, and analyses. According to various embodiments, the system and method involve calibrating the signal levels detected from one or more dyes, fluorescent markers, or other reference materials contained in or embedded in interstitial spaces or other spaces of a well plate, sample support, or other platform, outside of sample wells themselves. Herein, such dyes, fluorescent markers, and reference materials are also referred to as detectable materials. According to various embodiments, by embedding one or more detectable materials in the interstitial spaces of a well plate, sample support, or other platform, reaction regions of the platform can be loaded with samples to perform assays, amplifications, reactions, processes, or other analyses, while at the same time using the detectable material contained in the interstitial spaces to calibrate fluorescence or other reference signal levels emitted from the detectable materials. The embedded detectable material can comprise a liquid material, a solid material, or a combination of liquid and solid materials. The embedded detectable material can be permanently inserted, affixed, or otherwise embedded into or onto the platform, for example, in a cavity, recess, or pocket formed in the plastic material of a plastic microtiter plate. The embedded detectable material can be used to generate consistent reference signal levels for calibration purposes, without a need to select and load liquid, dye or other material into or onto the platform before each PCR or other run. These and other features of the present teachings are set forth herein.

FIGURES

FIG. 1 illustrates an exemplary PCR machine and sample well plate, according to various embodiments of the present teachings.

FIG. 2 illustrates a portion of a sample well plate, according to various embodiments.

FIG. 3 illustrates a portion of sample well plate wherein a dye has been embedded in the interstitial spaces of the sample well plate, according to various embodiments.

FIG. 4 illustrates a portion of a sample well plate wherein multiple dyes have been loaded according to a predetermined pattern into the interstitial spaces of the plate around the perimeter of the sample well plate, according to various embodiments.

FIG. 5 illustrates a portion of a sample well plate wherein multiple dyes have been dispersed randomly into the interstitial spaces and the perimeter of a sample well plate, according to various embodiments.

FIG. 6 illustrates a portion of a sample well plate wherein multiple dyes have been loaded into the interstitial spaces and the perimeter of a sample well plate, some loaded in combination with other dyes, and wherein the dyes are loaded in various shapes, sizes, and locations, according to various embodiments.

DESCRIPTION

According to various embodiments of the present teachings, a method is provided for generating a calibration factor for a signal emitted from a support location of a platform, for example, from a well of a multiwell plate. The method can comprise providing a platform that comprises a top surface, a plurality of support locations, and interstitial space between adjacent ones of the plurality of support locations. At least one detectable material can be embedded in the interstitial space, for example, a fluorescent dye. Emission data detected from the at least one detectable material can be received, for example, by a detector, a calibration factor for a signal emitted from one or more of the plurality of support locations can be generated based on the received emission data. In some embodiments, the method can further comprise calibrating a signal emitted from one or more of the plurality of support locations based on the calibration factor. In some embodiments, the at least one detectable material can comprise a dry material, a wet material, a fluorescent dye, or a combination thereof. The at least one detectable material can comprise a plurality of different detectable materials, and the plurality of different detectable materials can be embedded in the interstitial space such that each detectable material is separated from and spaced apart from the other detectable materials of the plurality. The plurality of different detectable materials can be embedded in the interstitial space such that two or more of the plurality of different detectable materials overlap each other at, at least a portion of the interstitial space. In some embodiments, two or more of the plurality of different detectable materials can be dispersed in equal amounts in the interstitial space. At least one detectable material can be distributed across the interstitial space in a repeating pattern. In some embodiments, at least one detectable material can be dispersed randomly across the interstitial space. In some embodiments, one or more of the plurality of support regions can support or contain the at least one detectable material. In some embodiments, the receiving can comprise receiving fluorescent emissions.

According to yet another embodiment of the present teachings, a method of generating a calibration factor for a signal emitted from a support location of a platform comprises providing a platform comprising a top surface, a plurality of support locations, and a perimeter surrounding the plurality of support locations and that is exclusive of the plurality of support locations, wherein at least one detectable material is embedded in the perimeter. The method can further comprise receiving emission data detected from the at least one detectable material, and generating a calibration factor for a signal emitted from one or more of the plurality of support locations based on the received emission data. The method can further comprise calibrating a signal emitted from one or more of the plurality of support locations based on the calibration factor. The at least one detectable material can comprise a fluorescent dye and the receiving can comprise receiving fluorescent emissions. A plurality of different detectable materials can be used and, in some embodiments, the plurality of different detectable materials can be embedded in the perimeter such that each detectable material is separated from and spaced apart from the other detectable materials of the plurality. Two or more of a plurality of different detectable materials can be provided overlapping each other at, at least a portion of the perimeter. The at least one detectable material can be distributed across the perimeter in a repeating pattern. In some embodiments, one or more of the plurality of support regions can comprise the at least one detectable material supported thereby.

In other embodiments of the present teachings, a platform is provided that comprises a top surface, a plurality of sample support locations formed in or on the top surface, interstitial space between adjacent ones of the plurality of sample support locations, and at least one detectable material embedded in the interstitial space and that emits a detectable signal adapted to generate a calibration factor. The at least one detectable material can comprise an excitable detectable material that emits a detectable signal upon excitation thereof, for example, upon excitation with a laser excitation source. In some embodiments, the platform can comprise a multiwell plate and the interstitial space can comprise areas between adjacent wells of the multiwell plate, exclusive of the wells of the plate. The at least one detectable material can comprise a fluorescent dye. In some embodiments, the at least one detectable material can comprise a plurality of different fluorescent dyes and the plurality of different fluorescent dyes can be embedded in the interstitial space such that either (1) each fluorescent dye of the plurality is separated from and spaced apart from the other, different fluorescent dyes of the plurality, or (2) the different fluorescent dyes overlap one another.

In yet other embodiments of the present teachings, a platform is provided that comprises a top surface, a plurality of sample support locations formed in or on the top surface, a perimeter surrounding the top surface, and at least one detectable material embedded in the perimeter and that emits a detectable signal adapted to generate a calibration factor. The at least one detectable material can comprise an excitable detectable material that can become excited upon excitation from an excitation source, for example, a fluorescent dye that emits an increased level of fluorescence upon excitation from a laser excitation source.

Various embodiments of the present teachings relate to systems and methods for calibration of PCR, RT-PCR, or other instruments used to detect various reactions, amplifications, assays, processes, and analyses. In some embodiments, the calibration systems and methods according to the present teachings, can be implemented in or applied to biological scanning systems, assays, reactions, analyses, or other processes in which a read head containing a photodetector, photosensor, or other optical detector, for example, a photodiode, can read the fluorescent output or other emissions from a sample well, container, or other support region. The detector can then travel to a next well or location to read the spectral dye or other output at that location, and step or repeat across a plate or other container or platform to take spectra from the entire group of sample wells, containers, or other support regions on the platform. The sample platform can be or include, for example, any type of plate, sample support, or other platform or structure capable of holding, supporting, separating, containing, storing, or enclosing a material, for example, a microtiter well plate having a standard 96-well format or another number of wells. The platform or support can comprise other supports or structures, for example, a plate or surface adapted to receive or support samples on solid microsupports in a planar array, a platform comprising one or more flow cells, a hybridization array, or the like.

According to various embodiments, calibration or adjustment systems can be implemented in, or applied to, PCR or RT-PCR imaging systems, or other apparatuses in which a photodetector, for example, a CCD, CID, or other detector can image all sample wells contained in that well plate, sample support, or other platform, at one time or substantially at one time. For example, the detector can take a spectral image of all the wells of a standard microtitre plate. Various types of instruments, systems, apparatuses, or other devices can be used, for example, a PCR machine 102 that is capable of holding a well plate 104, as illustrated in FIG. 1.

According to various embodiments, each sample well, container, or other support region, in a well plate, sample support, or other platform, can contain samples, for example, samples of DNA fragments or other materials. These samples can comprise one or more spectrally distinct dye, fluorescent marker, or other reference material that can be attached or hybridized to the sample for detection and/or analysis of the sample. A calibration or adjustment can be performed to normalize, correct, adjust, compensate, or otherwise increase the consistency or accuracy of readings detected from the sample wells, containers, or other support regions, on the plate or other platform. The calibration can be performed to identify and characterize the behavior of detected responses of the detectable material in one or more wells or other sample regions, for example, to establish a maximum and minimum detected range of values for readings from a given well or wells, based on pure-dye emission data collected from that well or those wells. The calibration or adjustment can correct or compensate for variations due to or affected by factors including, but not limited to, differences in signal strength, dye or sample concentrations, contaminations, spectral or amplitude distortions, deviations in optical path, plate geometry, fluorescent noise floor, sample population or size, or other variations or anomalies that can arise from dye to dye, from well to well, from plate to plate, or from instrument to instrument, among other corrections.

According to various embodiments of the present teachings, the calibration or adjustment can comprise using emission data detected from a sample support, well plate, or other platform, and calibrating or adjusting the detection apparatus for variations in response to individual dyes, fluorescent markers, reference materials, or other detectable materials. According to various embodiments of the present teachings, the calibration or adjustment can comprise generating and applying a correction or calibration factor to maintain detected signal values within a validated range, for example, for one or more wells. This can be done, for example, by measuring the output from an assay, amplification, or other reaction, analysis, or process that involves such detectable materials. In some embodiments, an embedded dye reference can be encapsulated in interstitial regions between sample wells or between other sample support regions within the support.

According to various embodiments, each detectable material embedded in, encapsulated in, or affixed to an interstitial space in a sample plate, container, or other support can comprise one or more dyes, fluorescent markers, or other reference material. By embedding a predetermined amount of detectable material, the quantity of detectable material does not change during an assay, amplification, reaction, analysis, or other process or series of processes. This feature can be advantageous in the calibration process, because it reduces the amount of human error that can be created when the dyes or other detectable materials are manually embedded. It will be appreciated that each embedded cavity of space can be adapted or prepared to contain a plurality of dyes, fluorescent markers, other reference materials, or a combination thereof.

According to various embodiments, assays can be detected by an optical detection system comprising a set of filters, lenses, channels, or a combination thereof. The filters, lenses, or channels can correspond to the emission wavelength of the detectable material. The detectable material can comprise one or more dyes such as VIC, FAM, TAMRA, NED, JOE, ROX, or a combination thereof. According to various embodiments, the embedded dye or other material can comprise a liquid material, a solid material, or a combination thereof. The detected emission signal or signals emitted by or detected from the embedded detectable material can be used to determine a spectral calibration matrix for a respective detectable material in a respective location, for example, a spectral calibration matrix used to calibrate emission signals from a particular dye in a particular well. Techniques for generating a spectral calibration matrix are described, for example, in U.S. Pat. No. 6,991,712 B2 to Sharaf et al., and in U.S. Patent Application Publication No. 2006/0138344 A1, entitled “Spectral Calibration Method and System for Multiple Instruments” to Gunstream et al., each of which is incorporated herein by reference in its entirety.

According to some embodiments, the detected emission signal from the interstitially embedded detectable material can be used to calibrate a range of maximum and minimum values for a location on a sample plate, flow cell, or other platform, or for an entire sample plate, flow cell, or other platform. Herein, the term “emission” is used to exemplify a signal detected and/or calibrated according to various embodiments of the present teachings. It is to be understood that by “emission” the present teachings are referring to not only electromagnetic radiation but rather are also referring to any physical or chemical signal or other data that can be read, detected, imaged, or surmised from one or more area of interest, for example, a support region such as a well of a multi-well plate. “Emission” herein is intended to encompass electromagnetic radiation, optical signals, chemiluminescent signals, fluorescent signals, radiation transmission values, and radiation absorption values.

According to various embodiments, calibrating or adjusting the amplitude response or spectral response to one or more dyes, fluorescent markers, or other reference materials, by conducting an end-point PCR, RT-PCR, or other reaction, amplification, assay, analysis, or other process, can provide a more accurate representation of the dye signal, output, or other measurement. Detection of reference dye embedded in interstitial spaces between wells, embedded in other areas outside of wells, or embedded in other sample retaining areas, can be done at an endpoint, on a real-time basis, on a cycle-per-cycle basis, or at any other time, point, or points during the reaction, amplification, assay, analyses, or other process. By calibrating or adjusting the amplitude or spectral response based on embedded dye or other detectable material at each cycle, a more precise calibration, compensation, adjustment, normalization, or other correction can be performed.

According to various embodiments of the present teachings, the calibration or adjustment can comprise utilizing a universal reference standard or group of standards or other normalization methods that can calibrate the dye, fluorescent marker, or other reference material, signals or relationships. The universal reference standard can comprise, for example, a set of layers of solid material, each of which provides a spectral response associated with a particular dye.

A universal reference standard can be used to calibrate multiple dyes, fluorescent markers, or other reference material responses produced during a reaction, assay, amplification, analysis, or other process, and multiple different detectable materials can be calibrated at the same time. For example, the reference standard can be formulated to generate a response similar to, or exactly the same as, the absorption and emission behavior of one or more fluorescent dyes. According to various embodiments, the reference standard can comprise a material or set of materials whose amplitude and spectral response is not necessarily identical to the operating dyes that would be used in a system, but that can have expected and/or known relationships to those of the operating dyes. In such embodiments, a transform can be applied to the detected amplitude and spectral response curves to produce characteristics that match the target dye or dyes. With this universal reference standard or group of standards, a well plate, sample support, or other platform, or location in or on a well plate, sample support, or other platform, can be calibrated for one or more detectable materials and multiple different detectable materials can be calibrated at the same time.

According to some embodiments, the spectral response of the universal reference standard or standards need not match all of the individual dyes. If the spectral behavior of the universal reference standard does not match all subject dyes, the behavior or response of one or more dyes can be predicted or generated using a transformation, alteration, or other method, based upon the universal reference standard. The universal reference standard can comprise solid material, liquid material, gaseous material, one fluorescent dye or material, multiple fluorescent dyes or materials, or a combination of such materials.

According to embodiments of the present teachings, the calibration or adjustment can comprise suspending, inserting, encapsulating, layering, enclosing, or using some other method of embedding each dye, fluorescent marker, or other reference material into or onto the plate or other support. The material of the plate or other support can comprise, for example, a solid material, for example, a plastic, a polymer, a resin, graphite, a combination thereof, or other material or medium. The suspension, insertion, application, or other method of encapsulation or embedding of the one or more detectable materials in the support can enable the detectable material to exhibit persistently similar spectral responses to wet dye, media, or other material over the life of the support. The use of a dry chemical or solid material can, for example, help to reduce or eliminate the drawbacks of wet chemistry, such as evaporation, condensation, spillage, temperature-related variances, and other drawbacks or effects.

According to some embodiments of the present teachings, the dyes, fluorescent markers, or other reference materials used to calibrate the detected signal response can be placed, loaded, inserted, enclosed, encased, built into, or otherwise embedded in spaces or locations other than the sample wells, containers, or other support regions themselves. For example, the interstitial space between or adjacent one or more wells of a multi-well plate can be embedded with one or more reference dyes. The dye or other material can be embedded in a cavity or space formed in the material of the plate or support, for example, a recess or cavity formed by injection molding, milling, stamping, or other techniques.

In some embodiments, a detectable dye or other material can be embedded in equally or regularly spaced positions between or adjacent a plurality of wells of a multiwell plate. In some embodiments, the dye or other detectable material can be embedded in or around a periphery of the plate or other support. In some embodiments, the dye or other material can be embedded in different combinations of areas or locations of the plate or other support. In various methods of the present teachings, the embedded dye or other material can be irradiated with an excitation beam before, during, or after PCR or another reaction, amplification, or process, and the emission data detected from those embedded material locations can be used to calibrate or adjust readings taken from the subject well plate, sample support, or other platform.

According to various embodiments, a sample platform adapted to contain an embedded detectable material is illustrated in FIG. 2. As shown, sample wells 206 can be formed in sample platform 200. The sample platform 200 can comprise a top surface 202 that contains a plurality of openings 204, for sample wells 206, and one or more interstitial spaces 208 adjacent openings 204. Sample platform 200 can also contain an underside 212 and a perimeter 214 that exists on all sides (not shown) of the sample support, in the area between top surface 202 and underside 212. Calibration or adjustment operations, according to the present teachings, can comprise utilizing interstitial space 208, underside 212, space 216 inside the wells, perimeter 214, or any combination thereof, to store, encapsulate, insert, affix, or otherwise embed calibration dyes, fluorescent markers, reference materials, or other detectable materials. The dye, fluorescent marker, or other reference material can comprise a dry dye, a solid dye, a wet dye, a gaseous material, or any combination thereof.

According to various embodiments, a detector can take an emission data reading of emissions from sample platform 200, and from the embedded dyes, fluorescent markers, or other reference materials. Such detectable materials, when located in one or more interstitial spaces 208, can generate fluorescence, or other emission signals having an amplitude, intensity, spectral response, time-varying value, or other behavior that can be detected. These detected emissions can be used to perform a calibration to a standard range, spectral response, or other characteristic or value. According to various embodiments, a calibration factor, numerical reference, spectral response curve, or other correction can be generated, approximated, determined and/or estimated, based on the emission data detected from the detectable material in the one or more interstitial spaces 208 of sample platform 200. This calibration factor can then be used to calibrate the emission data.

In some embodiments, the embedded dyes, fluorescent material, or other reference materials used to perform calibration or adjustment can be spaced throughout the well plate, sample support, or other platform in a predetermined pattern. An example of an illustrative configuration is shown in FIG. 3, where a portion of a sample well plate 300 is shown, and a first dye D1 is embedded in an interstitial space 304, adjacent openings 306, in a top surface 308 of sample well plate 300. The predetermined pattern is not necessarily limited to one detectable material, but can comprise a number of detectable materials, for example, distributed throughout interstitial space 304 or other available space external to, under, or inside the sample wells, containers, other support regions, or distributed throughout any combination thereof. An example of a system using multiple detectable materials is the multiple-dye configuration illustrated in FIG. 4. A portion of a sample well plate 400 is shown, and a first dye D1 is embedded in a perimeter 402 of a portion of sample well plate 400. First dye D1 is distributed in perimeter 402 in a predetermined pattern. A second dye D2 and a third dye D3 can be embedded one after the other in a repeating pattern in an interstitial space 408, adjacent the openings 404, in a top surface 406 of sample well plate 400. A fourth dye D4 and more of second dye D2 are embedded in interstitial space 408 such that D4 is distributed to form a row on sample well plate 400, D2 is distributed to form an adjacent row on sample well plate 400, and the pattern repeats itself across the entire sample well plate 400. While all these illustrative patterns are located or configured together on the same sample well plate in FIG. 4, the patterns can be of any configuration, and other patterns can be located throughout the entire plate, sample support, or other platform. According to various embodiments, these examples are just illustrative possibilities and are not meant to limit the predetermined or known format, pattern, numbering, shape, size, or platform, or the type of dye, fluorescent marker, reference material, or other detectable material used.

According to various embodiments, the dyes, fluorescent markers, and other reference materials can be embedded in or on the well plate, support, or other platform in a regular pattern, irregular pattern, or random pattern. Each different dye can be presented in equal or different numbers of interstitial spaces throughout the well plate, platform, or other container. According to various embodiments, more than one dye, fluorescent marker, or other reference material can be incorporated into the same interstitial space cavity, or other area of a well plate, sample support, or other platform.

According to various embodiments, the dyes, fluorescent markers, or other reference materials used to perform calibrations or adjustments can be randomly distributed throughout the interstitial space or other available area of the well plate, sample support, or other platform. In some embodiments, a random distribution of embedded detectable materials can be combined with one or more predetermined pattern of embedded detectable materials. An example of this is illustrated in FIG. 5, where a portion of a sample well plate 500 is shown, and multiple dyes are embedded in various shapes, sizes, and/or locations throughout the plate. A first dye D1 can be embedded using various shapes and patterns and in various locations throughout the well plate 500. The locations include an interstitial space 502, adjacent openings 504, in a top surface 506 of well plate 500. A second dye D2 can be embedded in interstitial space 502 and on a perimeter 508 of well plate 500. It will be appreciated that these examples are illustrative only and are not meant to limit the format, pattern, number, shape, or platform, or the type of dye, fluorescent marker, reference material, or other detectable material used. Although a well plate is depicted, it is to be understood that other platforms could be used, for example, flow cells or hybridization arrays.

According to some embodiments, the embedded detectable material located between the sample wells, containers, other support regions, or located in other areas, can comprise a liquid phase material, a solid phase material, a dry phase material, or any combination thereof. The dyes, fluorescent markers, or other reference materials can be embedded independently of each other or they can be combined with each other.

Further examples are illustrated in FIG. 6, where a portion of a sample well plate 600 is shown, and a first dye D1, a second dye D2, and a third dye D3, are embedded in an interstitial space 602 adjacent openings 604 in a top surface 606 and on a perimeter 608 of sample well plate 600. Dye D1 is shown embedded by itself at location 610, embedded with dye D2 at location 612, and embedded with a dye D3 at location 614. At location 616, dye D1, dye D2, and dye D3 are all embedded. Dye D2 is also located throughout sample well plate 600 and is embedded by itself at location 618, and combined with dye D3 at location 620, in the various shapes and sizes shown. Dye D3 is located throughout sample well plate 600 and is embedded by itself at location 622, in the various shapes and sizes shown. It will be appreciated that these examples are illustrative only and are not meant to limit the format, pattern, number, shape, or platform, or the type of dye, fluorescent marker, or other reference material used. In some embodiments, the plate can be reused a plurality of times, and because the quantity of reaction material in the well plate, sample support, or other platform is permanently embedded therein or thereon, the platform can reliably emit the same or very similar spectral response each time it is used.

According to some embodiments, by using the interstitial space to embed reference dyes for calibration, the individual sample wells, containers, or other support regions of a platform can be loaded with an analyte, dye, or other reaction material, for example, samples of DNA fragments. In various embodiments, the reaction material is labeled with one or more spectrally distinct dye, fluorescent marker, or other reference material. The instrument, apparatus, device, or other system, can perform a reaction, assay, amplification, analysis, or other process, while at the same time calibrating, adjusting, compensating, or correcting for the well plate, sample support, or other platform by taking readings of the emission signals of the embedded detectable material.

According to various embodiments of the present teachings, the calibration or adjustment can comprise a calibration based on measurements of emission signals of multiple detectable materials that are loaded in sample wells, containers, or other support regions of a platform. These support regions can be distributed and between sample wells, containers, or other support regions on the same platform which are loaded with the same type of detectable material, within a different type of detectable material, or with a combination thereof. The loaded sample wells, containers, or other support regions used for calibration or adjustment can be spread across the entire platform. For example, a multi-well plate can comprise a plurality of wells, some of the plurality of wells can comprise empty wells, some of the wells can comprise a first dye D1, and some can comprise a different, second dye D2, or a different number of dyes.

According to some embodiments, the well plate, sample support, or other platform, can be left completely empty. By leaving the well plate empty, the machine, system, apparatus, device, or other instrument, can be calibrated or adjusted for unwanted signals coming from the platform itself. Such unwanted signals can include, but are not limited to, spurious signal contributions such as the residual fluorescence contributed by the plastic or other material of the platform, fluorescence from a running buffer or other non-reactant liquid material, or from the thickness, temperature, design, or other property of the platform. Using a combination of separate detectable materials and empty support regions, a calibration or adjustment of a machine, device, apparatus, system, or other instrument, can be performed based on the respective emission signals detected from the detectable materials, at the same time that a calibration is performed to remove unwanted background signals from the platform.

According to various embodiments, the system can use a method of interpolation, spatial interpolation, or another calculation method which takes known data values to determine, estimate, or approximate unknown data values. The method can determine, estimate, or approximate the well calibration or adjustment value for those sample wells, containers, or other support regions in a platform that are left empty, or that contain a detectable material.

According to some embodiments, interpolation based on emission signal readings from interstitial spacings can be used during a reaction, assay, amplification, analysis, or other process, for example, during a PCR run, or during an RT-PCR run. A calibration or adjustment can be performed using dyes, fluorescent markers, or other reference materials inside sample wells, containers, or other support regions of a platform, and/or a calibration or adjustment can be performed using dyes, fluorescent markers, or other reference materials interstitially spaced and/or embedded throughout other areas of the same platform. The incorporation of interpolative techniques and calibrative techniques using interstitial spacing, can conserve time and expense because calibration can be performed for a liquid, dye, fluorescent marker, reference material, or other detectable material, or other detectable material in wells or supports of a platform, at the same time that a calibration can be performed based on the detectable material interstitially spaced throughout the platform.

According to various embodiments of the present teachings, the calibration or adjustment can comprise a uniformity correction of a real-time PCR instrument. The uniformity correction or calibration can compensate for optical, spectral, or other sources of non-uniformity across multiple instruments. The uniformity correction or calibrations can correct or compensate for non-uniform variations between instruments, optical paths, plates, dyes, samples, detectors, filters, or other factors. The calibration or adjustment can comprise utilizing a uniformity platform to calibrate for spectral non-uniformity or intensity deviations. The uniformity platform can comprise, for example, one dye, or multiple dyes, for example, mixed together at equimolar concentrations. The calibration or adjustment can further comprise taking images of multiple locations of the uniformity platform and correcting for location-to-location variations, for example, on a filter, channel, or other basis. According to some embodiments, the method can also, or instead, correct for well-to-well differences on a dye basis. The calibration or adjustment can also comprise utilizing a reference standard platform, to correct for non-uniformity. In some embodiments, the calibration or adjustment can utilize a reference standard having a response, or a calculated response, that is similar to an expected response from a desired dye or from a set of dyes.

According to various embodiments, the calibrations or adjustments based on embedded detectable materials can comprise normalizing a pure dye calibration to the highest filter signal and ignoring relative intensities. According to various embodiments, the calibration can comprise correcting for non-uniformity using relative intensities. According to various embodiments, the calibration or adjustment can comprise utilizing a calibration dye or other fluorescent generator mix with a sample at every location of a platform to correct for optical non-uniformity or other sources of non-uniformity. The optical uniformity correction can comprise detecting only a subset of locations of a platform, and utilizing interpolation across undetected locations.

According to various embodiments, the calibration or adjustment can comprise detecting locations of a well plate, sample support, or other platform, comprising uniformity and spectral calibration locations such as sample wells, containers, or other support regions that are distributed throughout the platform. Multiple interpolations can be done to perform spectral and/or intensity calibrations, and uniformity calibrations, both on the same platform.

According to various embodiments, various exemplary teachings are illustrated in the document entitled “Applied Biosystems Step One Real-Time PCR System Getting Started Guide,” (Applied Biosystems, Foster City, Calif.), which is incorporated herein in its entirety by reference.

Various embodiments of the present teachings can be implemented, in whole or in part, in digital electronic circuitry, or in computer hardware, firmware, software, or combinations thereof. Apparatuses of the present teachings can be implemented in a computer program, software, code, or an algorithm embodied in machine-readable media. Such media can comprise, for example, electronic memory, CD-ROM, DVD discs, hard drives, or other storage devices or media, used for execution by a programmable processor. Various method steps can be performed by a programmable processor to generate output data by executing a program of instructions, functions, or processes on input data.

The present teachings can be implemented in one or more computer programs that are executable on a programmable system that can include at least one programmable processor coupled to receive and transmit data and instructions, to and from a data storage system or memory. The system can include at least one input device, such as, a keyboard or mouse, and at least one output device, such as, a display or printer. Each computer program, software, code, or algorithm, can be implemented in a high-level procedural or object-oriented programming language, or in assembly, machine, or other low-level language, if desired. The code or language can be a compiled, interpreted, or otherwise processed for execution.

According to various embodiments, processes, methods, techniques, and algorithms can be executed on processors that can include, for example, both general and special purpose microprocessors, such as those manufactured by Intel Corp. (Santa Clara, Calif.) or AMD Inc. (Sunnyvale, Calif.). The processors can also include digital signal processors, programmable controllers, or other processors or devices. According to various embodiments, a processor can receive instructions and data from a read-only memory and/or a random access memory. A computer implementing one or more aspects of the present teachings can, in some embodiments, comprise one or more mass storage devices for storing data files, such as magnetic disks, internal hard disks, removable disks, magneto-optical disks, CD-ROM disks, DVD disks, Blu-Ray disks, or other storage disks or media.

According to various embodiments, memory or storage devices suitable for storing, encoding, or embodying computer program instructions or software and data can comprise, for example, all forms of volatile and non-volatile memory. This type of memory can comprise, for example, semiconductor memory, random access memory, electronically programmable memory (EPROM), electronically erasable programmable memory (EEPROM), flash memory, optical memory, and magnetic memory such as memory stored on magnetic disks, internal hard disks, removable disks, and magneto-optical disks. Any of the foregoing can be supplemented by, or incorporated in, ASICs. In some embodiments, processors, workstations, personal computers, storage arrays, servers, and other computer, information, or communication resources used to implement features of the present teachings, can be networked or network-accessible.

It will be appreciated that while various embodiments described above involve the calibration of one or more aspects of instrument reading, dye selection, support preparation, or other calibrations, in various embodiments more than one type of calibration can be performed, simultaneously or in sequence.

Other embodiments will be apparent to those skilled in the art from consideration of the present specification and practice of the present teachings disclosed herein. It is intended that the present specification and examples be considered as exemplary only.

Claims

1. A method of generating a calibration factor for a signal emitted from a support location of a platform, comprising:

providing a platform comprising a top surface, a plurality of support locations, and interstitial space between adjacent ones of the plurality of support locations;

embedding at least one detectable material in the interstitial space;

receiving emission data detected from the at least one detectable material; and

generating a calibration factor for a signal emitted from one or more of the plurality of support locations based on the received emission data.

2. The method of claim 1, further comprising calibrating a signal emitted from one or more of the plurality of support locations based on the calibration factor.

3. The method of claim 1, wherein the at least one detectable material comprises a dry material.

4. The method of claim 1, wherein the at least one detectable material comprises a fluorescent dye and the receiving comprises receiving fluorescent emissions.

5. The method of claim 1, wherein the at least one detectable material comprises a plurality of different detectable materials.

6. The method of claim 1, wherein the at least one detectable material is distributed across the interstitial space in a repeating pattern.

7. The method of claim 1, wherein one or more of the plurality of support regions comprises the at least one detectable material supported thereby.

8. A method of generating a calibration factor for a signal emitted from a support location of a platform, comprising:

providing a platform comprising a top surface, a plurality of support locations, and a perimeter surrounding the plurality of support locations and that is exclusive of the plurality of support locations;

embedding at least one detectable material in the perimeter;

receiving emission data detected from the at least one detectable material; and

generating a calibration factor for a signal emitted from one or more of the plurality of support locations based on the received emission data.

9. The method of claim 8, further comprising calibrating a signal emitted from one or more of the plurality of support locations based on the calibration factor.

10. The method of claim 8, wherein the at least one detectable material comprises a fluorescent dye and the receiving comprises receiving fluorescent emissions.

11. The method of claim 8, wherein the at least one detectable material comprises a plurality of different detectable materials.

12. The method of claim 11, wherein the plurality of different detectable materials are embedded in the perimeter such that each detectable material is separated from and spaced apart from the other detectable materials of the plurality.

13. The method of claim 8, wherein the at least one detectable material is distributed across the perimeter in a repeating pattern.

14. The method of claim 8, wherein one or more of the plurality of support regions comprises the at least one detectable material supported thereby.

15. A platform comprising:

a top surface;

a plurality of sample support locations formed in or on the top surface;

interstitial space between adjacent ones of the plurality of sample support locations; and

at least one excitable detectable material embedded in the interstitial space and that emits a detectable signal adapted to generate a calibration factor.

16. The platform of claim 15, wherein the platform comprises a multiwell plate and the interstitial space comprises areas between adjacent wells of the multiwell plate.

17. The platform of claim 15, wherein the at least one detectable material comprises a fluorescent dye.

18. The platform of claim 15, wherein the at least one detectable material comprises a plurality of different fluorescent dyes.

19. The platform of claim 18, wherein the plurality of different fluorescent dyes are embedded in the interstitial space such that each fluorescent dye of the plurality is separated from and spaced apart from the others different fluorescent dyes of the plurality.

20. A platform comprising:

a top surface;

a plurality of sample support locations formed in or on the top surface;

a perimeter surrounding the top surface; and

at least one excitable detectable material embedded in the perimeter and that emits a detectable signal adapted to generate a calibration factor.