Apparatus for selective excitation of microparticles

Nucleic acid microparticles are sequenced by performing a sequencing reaction on the microparticles using one or more selectively exciting the microparticles in an excitation pattern, optically imaging the microparticles at a resolution insufficient to resolve individual microparticles, and processing the optical images of the microparticles using information on the excitation pattern to determine the presence or absence of the optical signature, which indicates the sequence information of the nucleic acid. An apparatus for optical excitation of the microparticles comprises an optical fiber delivering a first laser beam, and an interference pattern generation module coupled to the optical fiber. The interference pattern generation module splits the first laser beam into second and third laser beams and generates the excitation pattern for selectively exciting the microparticles by interference between the second and third laser beams.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to U.S. patent application Ser. No. 11/846,049, entitled “Nucleic Acid Sequencing by Selective Excitation of Microparticles,” filed on Aug. 28, 2007.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of nucleic acid sequencing and, more specifically, to a method and system for DNA (deoxyribonucleic acid) sequencing by selective excitation of microparticles.

2. Description of the Related Art

FIG. 1illustrates a conventional method of DNA sequencing with microparticles. The method ofFIG. 1derives DNA sequence data112from a microparticle array102through cycles of sequencing reactions104, non-selective excitation106of the microparticles, and optical signature detection108. Each microparticle in the microparticle array102typically contains DNA molecules with both unknown sequences to be determined and known sequences that are used in the sequencing reactions. Thousands to millions (to potentially billions or more) of these microparticles are distributed and immobilized on the surface of a glass substrate, as conceptually shown inFIG. 2, which illustrates an example of a microparticle array102. The microparticle array102includes DNA sequencing microparticles204distributed and immobilized on a substrate202. The microparticles204can take many forms, such as 1-micron diameter beads covered with DNA molecules amplified by a water-in-oil emulsion PCR (polymerase chain reaction) technique, or clusters of DNA molecules amplified by a bridge amplification technique, or individual unamplified DNA molecules. The microparticles204can be distributed either randomly (e.g., irregularly spaced) or in an orderly pattern (e.g., regularly spaced pattern such as a square grid pattern or a hexagonal grid pattern) on the substrate202. The substrate202is typically made of glass and located inside a flow cell, which allows the microparticles204to be exposed to a series of reagents to perform sequencing reactions. At the end of each cycle of sequencing reactions, each microparticle takes on an optical signature, often as the result of the incorporation of one of the four fluorophores such as Cy3, Cy5, Texas Red, and a fluorescence resonance energy transfer (FRET) pair, that reveals the corresponding bases adenine (abbreviated “a”), cytosine (abbreviated “c”), guanine (abbreviated “g”) and thymine (abbreviated “t”) of the DNA.

FIGS. 3A-3Cillustrate different types of individual sequencing microparticles that can be used for DNA sequencing.FIG. 3Aillustrates an individual microparticle204formed by a 1-micrometer diameter bead302covered with clonal DNA molecules304that have been previously amplified by a water-in-oil emulsion PCR technique. The bead302is attached directly to the substrate202in fluid306.FIG. 3Billustrates an individual microparticle204as a cluster of clonal DNA molecules304attached to the substrate202and placed in fluid306. The DNA molecules have been previously amplified by a bridge amplification technique.FIG. 3Cillustrates an individual microparticle as a single DNA molecule304attached to the substrate202and placed in fluid306. The single DNA molecule304is sequenced without amplification.

Referring back toFIG. 1together with FIGS.2and3A-3C, DNA sequencing with microparticles includes performing a sequencing reaction104on the microparticle array102to cause each microparticle204to take on an optical signature that reveals the DNA sequence information. The microparticle array102is exposed to sequencing reagents, which enables each cycle of sequencing reactions to be performed in a massively parallel manner. For example, one cycle of sequencing reaction can be comprised of hybridizing anchor primers and ligating a pool of fluorescently-labeled query primers. At the end of each cycle of sequencing reactions104, each microparticle takes on an optical signature that reveals the DNA sequence information associated with that microparticle. For example, the optical signature can be the result of the incorporation of one of four fluorophores corresponding to bases “a,” “c,” “g,” and “t” of the DNA304.

The next step is to optically excite106the microparticles204and to detect108the optical signatures of the microparticles. As will be explained with reference toFIGS. 4A-4C, the conventional optical excitation is non-selective. This cycle of reaction104, non-selective excitation106, and optical signature detection108is repeated multiples times to sequence the DNA304in each microparticle204. DNA sequence data112is output from this process.

Conventional DNA sequencing methods with microparticles suffer from low throughput (measured in bases per second) because the rate at which the optical signatures of the microparticles are detected is limited. This is largely due to the use of conventional non-selective excitation patterns, followed by optical imaging using optical microscopy, as used in conventional DNA sequencing methods.FIGS. 4A-4Cillustrate conventional non-selective excitation patterns used to excite the microparticles for subsequent imaging using optical microscopy. Specifically,FIG. 4Aillustrates a wide-field excitation pattern402used with the microparticles204on the substrate202, where all the microparticles in the field of view (FOV) are illuminated.FIG. 4Billustrates line-scanning excitation, where the microparticles204are illuminated by a line of light402that scans the substrate202.FIG. 4Cillustrates spot-scanning excitation, where the microparticles204are illuminated by a spot of light406that scans the substrate202.

Conventional non-selective excitation patterns can be generated by a variety of means. A wide-field excitation pattern402is typically generated by focusing an arc lamp source through the microscope optical train in a Kohler epi-illumination configuration, or by shining a laser source at a steep angle in an off-axis or total internal reflection (TIR) illumination configuration. A line-scanning excitation pattern404is typically generated by focusing a spatially-coherent laser through the microscope optical train and incorporating a scanning element. A spot-scanning excitation pattern406is typically generated by focusing a spatially-coherent laser source through the microscope optical train in a confocal configuration.

In such conventional DNA sequencing methods, detection of the microparticle optical signatures is typically performed by optical-microscope imaging of the microparticles illuminated with non-selective excitation patterns as shown inFIGS. 4A-4C. The speed of this approach is limited fundamentally for several reasons. First, the field of view (FOV) of an optical microscope is coupled fundamentally to resolution, i.e., the higher the resolution, the smaller the FOV. Similarly, the depth of field (DOF) of an optical microscope is coupled fundamentally to resolution, i.e., the higher the resolution, the smaller the DOF. Because a high-resolution optical microscope is required to resolve the microparticles using conventional sequencing methods, the FOV and DOF are relatively small. Consequently, imaging a microparticle substrate requires the acquisition of hundreds to thousands of smaller images that collectively cover the slide like tiles. Between each image, either the substrate or the optical microscope must be translated and focused precisely with respect to the microscope objective, during which time the optical microscope cannot be acquiring sequence data. Second, a high-resolution image of a microparticle slide is a very inefficient representation of the sequence information contained in the microparticles. For example, in a typical high-resolution image of a microparticle slide, the number of pixels in the image greatly exceeds the number of microparticles in the image. However, assuming that each microparticle can take on one of only four optical signatures, each microparticle carries just 2 bits of sequence information. Consequently, several thousands times more data is acquired than is necessary to generate the sequence information, according to conventional sequencing methods.

Thus, there is a need for a more efficient, faster, and more convenient method of nucleic acid sequencing.

SUMMARY OF THE INVENTION

Embodiments of the present invention include a method of sequencing nucleic acids such as DNA or RNA (ribonecleic acid) with fast speed by selectively exciting the nucleic acid microparticles and using image processing algorithms to extract the optical signatures of the microparticles. The term “nucleic acid” herein includes both DNA and RNA. An advantage of this approach is that it allows a relatively low-resolution optical microscope to image the selectively-excited microparticles, which enables detection of microparticle optical signatures to be performed with an extremely large field of view (FOV) and depth of field (DOF). Another advantage of this approach is that relatively low-resolution images of microparticles with selective excitation are a more efficient data representation of the sequence information than high-resolution images of microparticles with non-selective excitation, which enables the amount of acquired data required for sequencing to be greatly reduced.

In one embodiment, a method for sequencing nucleic acid microparticles comprises performing a sequencing reaction on the nucleic acid microparticles using one or more sequencing reagents, selectively exciting the nucleic acid microparticles in an excitation pattern, optically imaging the excited nucleic acid microparticles at a resolution insufficient to resolve individual microparticles, and processing the optical images of the excited nucleic acid microparticles using information on the excitation pattern to determine the presence or absence of at least an optical signature. The presence or absence of the optical signature indicates sequence information of the nucleic acid. Although the images of the excited nucleic acid microparticles are obtained at a resolution insufficient to resolve individual microparticles, the selective excitation of the nucleic acid microparticles is performed at a resolution sufficient to resolve the individual microparticles. The sequencing reaction, selective excitation, and image processing steps can be repeated using same or different reagents to complete the sequencing.

In one embodiment, an apparatus for optical excitation of the nucleic acid microparticles comprises a laser for generating a first laser beam, an optical fiber coupled to receive the first laser beam, an interference pattern generation module coupled to the first optical fiber and for receiving the first laser beam delivered via the optical fiber, where the interference pattern generation module splits the first laser beam into a second laser beam and a third laser beam and generates the excitation pattern for selectively exciting the target by interference between the second laser beam and the third laser beam.

The interference pattern generation module can include a beam splitter for splitting the first laser beam into the second laser beam and the third laser beam, and a mirror reflecting the third laser beam where the mirror is movable within a range to vary an optical path-length of the third laser beam. Alternatively, the interference pattern generation module can include a beam splitter for splitting the first laser beam into the second laser beam and the third laser beam, and a window coupled to the third laser beam and rotating to modulate the optical phase of the third laser beam.

The nucleic acid sequencing method of the present invention is fast and efficient for at least two reasons. First, since the FOV and DOF for detection of microparticle optical signatures are increased, the mechanical motion required for scanning and focusing is greatly reduced. Second, since the amount of acquired data required for sequencing is reduced (by use of low-resolution images insufficient to resolve individual microparticles enabled by use of the information on the excitation pattern in processing the optical images), the time required for sequencing is greatly reduced.

DETAILED DESCRIPTION OF EMBODIMENTS

FIG. 5illustrates a method of nucleic acid (e.g., DNA or RNA) sequencing by selective excitation of microparticles using structured illumination. Although the disclosure herein describes the nucleic acid sequencing method in the context of DNA sequencing, the use of the term “sequencing” is not intended to limit the scope of the present invention to DNA sequencing, and “sequencing” herein does include RNA sequencing or other types of nucleic acid sequencing. “Sequencing” or “sequence” herein is also intended to cover all sequence variations, such as single nucleotide polymorphisms (SNPs), gene copy number variations, single base duplications, inversions, insertions and deletions and all the applications of such sequencing, such as genotyping, gene expression analysis, and medical applications.

The microparticle array502with the DNA first undergoes a cycle of sequencing reactions504. The sequencing reactions504include exposing the microparticles to a series of reagents and incubating the microparticles at a series of temperatures. As the end-product of the sequencing reaction cycle, each microparticle takes on an optical signature that reveals the sequence information associated with that microparticle. The optical signature can be the result of the incorporation of one or more optically detectable labels, such as fluorescent dyes, colloidal gold, and quantum dots. For example, each sequencing reaction504can be designed such that the optical signature is the result of the incorporation of one of four fluorophores corresponding to the bases “a,” “c,” “g,” and “t” of the DNA304. Note that the microparticles can be one of the types shown inFIGS. 3A-3Cor some other type suitable for DNA sequencing. Also note that a number of additional conventional steps may have to be performed to prepare the microparticle array with the DNA from the DNA sample, which are not the subject of the present invention and are not described herein.

In contrast to conventional DNA sequencing methods, the microparticles204are then selectively excited506with a selective excitation pattern, as is explained in more detail with reference toFIGS. 6A-6C. The selectively-excited microparticle array is then imaged508using an optical microscope. Then, it is determined510whether the current selective excitation pattern is the last pattern to apply. If it is not the last pattern, the excitation pattern is then changed512, and the excite-and-image cycle in steps506,508,510is repeated.

FIGS. 6A-6Cillustrate how the microparticles are selectively excited by a sequence of excitation patterns as shown in the excite-and-image cycle in steps506,508,510. Referring toFIG. 6A, a selective excitation pattern602is generated on the microparticles204at time N. Referring toFIG. 6B, another selective excitation pattern604is generated on the microparticles204at time N+1. Referring toFIG. 6C, still another selective excitation pattern606is generated on the microparticles204at time N+2.

Note that these selective excitation patterns602,604,606are “selective” in that they excite the microparticles204in a non-trivial sequence of patterns. In contrast, the conventional excitation patterns shown inFIGS. 4A-4Cindiscriminately excite a region of space in a trivial pattern with the goal of producing an image (i.e., a photographic replica) of the region as a function of space and/or time. For example, the wide-field scanning excitation ofFIG. 4Aproduces an image of the region as a function of space in two dimensions. The line-scanning excitation ofFIG. 4Bproduces an image of the region as a function of space in one dimension and time in one dimension. The spot-scanning excitation ofFIG. 4Cproduces an image of the region as a function of time in two dimensions. In other words, conventional excitation is used to generate a single high-resolution image that is a photographic replica of the microparticle array502. In contrast, selective excitation according to the present invention is used to generate a sequence of low-resolution images of the microparticle array502in which the low-resolution images are not photographic replicas of the microparticle array502; instead, selective excitation encodes the sequence information in the set of low-resolution images (with the resolution not being high enough to resolve the individual microparticles204), and the images are processed using knowledge of the selective excitation patterns to decode the sequence information. Also, the use of conventional excitation entails little more than simply translating a trivial excitation pattern, such as a rectangle402, a line404, or a circle406. In contrast, the use of selective excitation according to the present invention entails more complex pattern changes, such as changes in feature size and/or orientation, and the use of more complex patterns.

As will be explained with reference toFIGS. 7A-7B, in one embodiment the selective excitation patterns602,604,606are generated by a synthetic aperture optics apparatus for structured illumination (also referred to as patterned excitation or standing wave excitation). The selective excitation patterns602,604,606are generated optimized to the microparticle array502. For example, as will be explained with reference toFIG. 10, the selective excitation patterns602,604,606determine the distribution of samples in the frequency domain. The extent of the distribution of samples in the frequency domain is matched to the feature size608of the microparticle array. The selective excitation of the nucleic acid microparticles204is performed at a resolution sufficient to resolve the individual microparticles. For example, a sine wave illumination with a period that is twice the spacing between the microparticle centers204may be used to generate the selective excitation patterns602,604,606, although a variety of other illumination periods can be used in other examples.

Also, the sequence and number of the excitation patterns are designed to ensure that relatively low-resolution images of the excited microparticle array502still produce an efficient yet complete and accurate representation of the optical signatures of the microparticles204, and correspondingly the sequence information in the microparticle array502. For example, as will be explained with reference toFIG. 10, the selective excitation patterns602,604,606determine the distribution of samples in the frequency domain. The number of samples in the frequency domain is matched to the feature density of the optical signatures of the microparticle array502.

Referring back toFIG. 5, after the last excite-and-image cycle is complete at the last excitation pattern510, it is determined514whether the current FOV is the last FOV for the microparticle array502. If the current FOV is not the last FOV, the microparticle array502is translated (move stage)516to the next FOV and the excite-and-image cycles in steps506,508,510,512are repeated for that next FOV. For example, the microparticle array502can be moved516to expose another FOV of the microparticle array502. Alternatively, the structured illumination apparatus that generated the selective excitation patterns can be moved to expose another FOV of the microparticle array502.

After the entire microparticle array502has been imaged (i.e., after the last FOV in step514), it is determined518whether the microparticle array should undergo another cycle of sequencing reactions. If the current reaction504is not the last reaction, a new cycle of sequencing reactions504is performed with same or different sequencing reagents, and then steps506through518are repeated. After the final sequencing-reaction cycle518, image processing520is performed on the optical signature data of the microparticle array502to extract the sequence data522contained in the DNA molecules in the microparticle array502.

FIG. 7Aillustrates a synthetic aperture optics structured illumination apparatus for selectively exciting the microparticles, according to one embodiment of the present invention. At a high level, the structured illumination apparatus generates multiple mutually-coherent laser beams, the interference of which produces interference patterns. Such interference patterns are projected onto the microparticle array502and selectively excite the microparticles204. Using the interference of multiple laser beams to generate the interference patterns is advantageous for many reasons. For example, this enables high-resolution excitation patterns with extremely large FOV and DOF. Although the structured illumination apparatus ofFIG. 7A(andFIG. 7B) is described herein with the example of generating excitation patterns for the microparticle array502, it should be noted that the structured illumination apparatus ofFIG. 7A(andFIG. 7B) can be used for any other type of application to generate excitation patterns for any other type of target.

Referring toFIG. 7A, the structured illumination apparatus700includes a laser702, a beam splitter704, shutters705,707, fiber couplers708,709, a pair of optical fibers710,711, and a pair of interference pattern generation modules712,713. The beam703of the laser702is split by the beam splitter704into two beams740,742. A pair of high-speed shutters705,707is used to switch each beam740,742“on” or “off” respectively, or to modulate the amplitude of each beam740,742, respectively. Such switched laser beams are coupled into a pair of polarization-maintaining optical fibers711,710via fiber couplers709,708. Each fiber711,710is connected to a corresponding interference pattern generation module713,712, respectively. The interference pattern generation module713includes a collimating lens714′, a beam splitter716′, and a translating mirror718′, and likewise the interference pattern generation module712includes a collimating lens714, a beam splitter716, and a translating mirror718.

The beam744from the optical fiber710is collimated by the collimating lens714and split into two beams724,726by the beam splitter716. The mirror718is translated by an actuator720to vary the optical path-length of the beam726. Thus, an interference pattern722is generated on the substrate202in the region of overlap between the two laser beams724,726, with the pattern changed by varying the optical path-length of one of the beams726(i.e., by modulating the optical phase of the beam726by use of the translating mirror718).

Similarly, the beam746from the optical fiber711is collimated by the collimating lens714′ and split into two beams728,730by the beam splitter716′. The mirror718′ is translated by an actuator720′ to vary the optical path-length of the beam728. Thus, the interference pattern722is generated on the substrate202in the region of overlap between the two laser beams728,730, with the pattern changed by varying the optical path-length of one of the beams728(i.e., by modulating the optical phase of the beam728by use of the translating mirror718). In fact, the interference pattern722is generated on the substrate202in the region of overlap between the four laser beams726,724,728,730. In one embodiment, for generating sinusoidal interference patterns, only two phases (0 and 90 degrees) or three phases (0, 120, and 240 degrees) are used and are sufficient for selective excitation of microparticles.

While this implementation illustrated inFIG. 7Ais used for its simplicity, various other approaches can be used within the scope of the present invention. For example, the amplitude, polarization, direction, and wavelength, in addition to or instead of the optical amplitude and phase, of one or more of the beams724,726,728,730can be modulated to change the excitation pattern722. Also, the structured illumination can be simply translated with respect to the microparticle array to change the excitation pattern. Similarly, the microparticle array can be translated with respect to the structured illumination to change the excitation pattern. Also, various types of optical modulators can be used in addition to or instead of the translating mirrors718,718′, such as acousto-optic modulators, electro-optic modulators, and micro-electro-mechanical systems (MEMS) modulators. In addition, although the structured illumination apparatus ofFIG. 7A(andFIG. 7B) is described herein as using a laser702as the illumination source for coherent electro-magnetic radiation, other types of coherent electro-magnetic radiation sources such as an SLD (super-luminescent diode) may be used in place of the laser702.

Also, althoughFIG. 7Aillustrates use of four beams724,726,728,730to generate the interference pattern722, larger number of laser beams can be used by splitting the source laser beam into more than two beams. For example, 64 beams may be used to generate the interference pattern722. In addition, the beam combinations do not need to be restricted to pair-wise combinations. For example, three beams724,726,728, or three beams724,726,730, or three beams724,728,730, or three beams726,729,730, or all four beams724,726,728,730can be used to generate the interference pattern722. Typically, a minimal set of beam combinations is chosen as necessary to maximize speed. In one embodiment, the number of beam combinations is matched to the amount of unknown information in the microparticle array502. For example, once the locations of the microparticles in the microparticle array502are known, the optical signatures of the microparticles, and subsequently the sequence information, can be determined using a relatively small number of excitation patterns. Also, the beams can be collimated, converging, or diverging. Although different from the specific implementations ofFIGS. 7A and 7Band for different applications, additional general background information on generating interference patterns using multiple beam pairs can be found in (i) U.S. Pat. No. 6,016,196, issued on Jan. 18, 2000 to Mermelstein, entitled “Multiple Beam Pair Optical Imaging,” (ii) U.S. Pat. No. 6,140,660, issued on Oct. 31, 2000 to Mermelstein, entitled “Optical Synthetic Aperture Array,” and (iii) U.S. Pat. No. 6,548,820, issued on Apr. 15, 2003 to Mermelstein, entitled “Optical Synthetic Aperture Array,” all of which are incorporated by reference herein.

FIG. 7Billustrates a different type of interference pattern generation module that can be used with the apparatus ofFIG. 7A, according to another embodiment of the present invention. The interference pattern generation module750ofFIG. 7Bcan be used in place of the interference pattern generation module712or713ofFIG. 7A. AlthoughFIG. 7Billustrates the situation where the interference pattern generation module712ofFIG. 7Ais replaced by the interference pattern generation module of750ofFIG. 7B, the interference pattern generation module713can be similarly replaced by the interference pattern generation module of750ofFIG. 7B.

Referring toFIG. 7B, the output beam770of the fiber710is collimated by the collimator754and split by the beam splitter756into two beams772,774. An optical window760is inserted into the optical path of one beam774and rotated, using a galvanometer, to modulate the optical path-length of the beam774, thereby modulating the optical phase of the corresponding beam774and generate a modulated beam776. Having only one phase modulator (rotating optical window760) for every two beams makes the design of the interference pattern generation module750compact and efficient. Two stationary mirrors758,762reflect the beams772,776, respectively, to generate an interference pattern780while maintaining approximately matching optical path-lengths.

The structured illumination apparatus ofFIGS. 7A and 7Bhave a number of technical advantages compared to conventional structured illumination apparatuses. These advantages include that:

(i) The use of the interference of multiple lasers beams to generate high-resolution excitation patterns enables an extremely large FOV and DOF that is not achievable using a conventional lens projection system.

(ii) The optical fibers710,711used to deliver the laser beams eliminate the transmission of vibration from the laser702source, which provides better pointing stability and better manufacturability.

(iii) The modularized design using interference pattern generation modules provide more flexible beam geometry design, which enables large numbers of beams to be used by reducing complexity and cost of manufacturing.

(iv) The optical fiber-based design enables a compact and lightweight apparatus that provides better mechanical and thermal stability. The compact assembly also minimizes free-space beam propagation, reducing disturbances caused by atmospheric turbulence.

(v) The interference pattern generation module ofFIG. 7Bprovides nominally matched optical path-lengths, which eliminates the need for a single longitudinal mode laser source.

(vi) The optical fiber-based design performs the beam-splitting into two stages (one stage at beam splitter704and another stage at the beam splitters716,716′ in the interference module generation modules712,713), such that temperature variations and mechanical disturbances of the fibers do not affect the interference pattern significantly.
(vii) Because only two phases (0 and 90 degrees) or three phases (0, 120, and 240 degrees) are used for generating sinusoidal interference patterns, as opposed to 8 or more phases, the time required for data acquisition is greatly reduced.
(viii) The use of shutters and rotating optical windows for amplitude and phase modulation enables a compact and lightweight apparatus with low cost, simple control electronics, and high optical efficiency. For example, the optical efficiency of the interference pattern generation module750shown inFIG. 7Bcan be greater than 95%.

FIGS. 7C,7D, and7E illustrate how the beams from the structured illumination apparatus ofFIG. 7AorFIG. 7Bcan be coupled into fluid to illuminate the microparticles, according to embodiments of the present invention. Specifically,FIG. 7Cillustrates the laser beams724entering the fluid306through a window792to illuminate and selectively excite the microparticles204on the substrate202of an upright microparticle array502. The microparticles204can be imaged through the window792.FIG. 7Dillustrates the laser beams724entering the fluid306through the back side of the substrate202(i.e., the side of the substrate202opposite to the side where the microparticles204are placed) to illuminate an inverted microparticle array502. The microparticles204can be imaged through the substrate202.FIG. 7Eillustrates the laser beams724entering the fluid306through a coupling prism794to illuminate and selectively excite the microparticles204on the substrate202of an inverted microparticle array502in a TIR (total internal reflection) illumination configuration off of the substrate202. The microparticles204can be imaged through the substrate202. For clarity, only one beam is illustrated inFIGS. 7C-7E.

FIG. 8illustrates the image processing520ofFIG. 5for sequencing in more detail, according to one embodiment of the present invention. The raw images802of the selectively-excited microparticles output from the optical imaging508(FIG. 5) are input into image synthesis algorithms804. Note that the raw images802are low-resolution images—the selective excitation506(FIG. 5) and the knowledge of the selective excitation patterns eliminate the need for high-resolution images. The low resolution of the raw images is insufficient to resolve the individual microparticles. However, the image synthesis algorithms804process the raw images802, together with information806about the excitation patterns used to excite the microparticles and information808about the microparticle array502(such as the sizes and locations of the microparticles), to generate a synthetic high-resolution image810of the microparticle array502. The synthetic image810is input into microparticle signature identification (MSI) algorithms812that process the synthetic image810together with the information808about the microparticle array502to determine the optical signature814of each microparticle. The microparticle optical signature data814is input into base-calling algorithms816that produce a sequence522of DNA bases. While the implementation ofFIG. 8is advantageous due to its transparency and simplicity, various other approaches can be used for the image processing520.

The image synthesis algorithms804inFIG. 8take advantage of the property that the interference pattern generation modules712,713(FIG. 7) generate excitation patterns that are well-approximated as sinusoids or sums of sinusoids.FIG. 9illustrates how the detected optical pattern can be modeled as a product of the microparticle fluorophore distribution function and the excitation pattern. As shown one-dimensionally inFIG. 9, the optical patterns802(FIG. 8) detected by the optical imaging508(FIG. 5) can then be expressed as the product of a microparticle fluorophore distribution function and a sinusoidal function (or sums of sinusoidal functions), which lends itself to a Fourier sum representation. For example, the detected optical pattern902can be a product of the microparticle fluorophore distribution function (f(x))908and the excitation pattern (g1(x))910. The detected optical pattern904can be a product of the same microparticle fluorophore distribution function (f(x))908and a different excitation pattern (g2(x))912(which in this example is 180 degrees out of phase with the excitation pattern (g1(x))910). The detected optical pattern906can be a product of the same microparticle fluorophore distribution function (f(x))908and another different excitation pattern (g3(x))914(which in this example has a different period compared to the excitation patterns910,912).

Once the raw image data802is expressed as a Fourier sum, the problem of generating a synthetic high-resolution image810can be cast as a general Fourier-inversion problem, which can be solved using a great variety of well-known methods. Note that a more general implementation of the image synthesis algorithms does not assume that the excitation patterns are sinusoids or sums of sinusoids. In this case, the raw image802data can be expressed as a more general matrix multiplication. The problem of generating a synthetic high-resolution image810can then be cast as a general matrix-inversion problem, which can be solved using a great variety of well-known methods.

FIG. 10illustrates the relationship between the sampling pattern in the frequency domain and the geometry of laser beams used to generate the excitation patterns. As shown inFIG. 10, the distribution of such samples in the frequency domain is determined by the geometry of the laser beams in the structured illumination apparatus. For example, if the laser beams have k-vectors in the geometry1002, the frequency samples would have a rectilinear distribution1004(also referred to as a 2DFT distribution, a Cartesian distribution, or a uniform distribution). For another example, if the laser beams have k-vectors in the geometry1006, the frequency samples would have a non-rectilinear distribution1008.

The MSI algorithms812and base-calling algorithms816inFIG. 8take as input the high-resolution synthetic images810of the microparticle array and produce a sequence522of DNA bases for each microparticle.FIG. 11conceptually illustrates this process of identifying the DNA bases for each microparticle, according to one embodiment of the present invention. Referring toFIG. 11, each of the 12 images1102through1122shows the same FOV, and there are two sequencing microparticles1172,1174in each FOV in this example.

The synthetic image set1150illustrates the microparticles1172,1174after a first sequencing reaction cycle. During that first reaction cycle, the microparticles1172,1174take on one of four optical signatures corresponding to a first unknown DNA base for each microparticle1172,1174. The synthetic image set1150is comprised of four high-resolution synthetic images810, each of which is optimized to detect one of the four optical signatures. The first image1102of the set1150is optimized to detect the optical signature corresponding to the DNA base “a.” The second image1104of the set1150is optimized to detect the optical signature corresponding to the DNA base “t.” The third image1106of the set1150is optimized to detect the optical signature corresponding to the DNA base “g.” The fourth image1106of the set1150is optimized to detect the optical signature corresponding to the DNA base “c.”

The second synthetic image set1160illustrates the microparticles1172,1174after a second sequencing reaction cycle. During that second reaction cycle, the microparticles1172,1174take on one of four optical signatures corresponding to a second unknown DNA base for each microparticle1172,1174. The synthetic image set1160consists of four high-resolution synthetic images810, each of which is optimized to detect one of the four optical signatures. The first image1110of the set1160is optimized to detect the optical signature corresponding to the DNA base “a.” The second image1112of the set1160is optimized to detect the optical signature corresponding to the DNA base “t.” The third image1113of the set1160is optimized to detect the optical signature corresponding to the DNA base “g.” The fourth image1114of the set1160is optimized to detect the optical signature corresponding to the DNA base “c.”

The third synthetic image set1170illustrates the microparticles1172,1174after a third sequencing reaction cycle. During that third reaction cycle, the microparticles1172,1174take on one of four optical signatures corresponding to a third unknown DNA base for each microparticle1172,1174. The synthetic image set1170consists of four high-resolution synthetic images810, each of which is optimized to detect one of the four optical signatures. The first image1116of the set1170is optimized to detect the optical signature corresponding to the DNA base “a.” The second image1118of the set1170is optimized to detect the optical signature corresponding to the DNA base “t.” The third image1120of the set1170is optimized to detect the optical signature corresponding to the DNA base “g.” The fourth image1122of the set1170is optimized to detect the optical signature corresponding to the DNA base “c.”

In the synthetic image set1150corresponding to the first unknown DNA bases, the first microparticle1172is brightest in image1102corresponding to “a”, and the second microparticle1174is brightest in the image1106corresponding to “g” but no microparticle is bright in the images1104,1108corresponding to “t” and “c,” respectively. In the synthetic image set1160corresponding to the second unknown DNA bases, both microparticles1172,1174are brightest in the image1110corresponding to “a” but no microparticle is bright in the images1112,1113,1114corresponding to “t,” “g” and “c,” respectively. In the synthetic image set1170corresponding to the third unknown DNA bases, the first microparticle1172is brightest in the image1118corresponding to “t” and the second microparticle1174is brightest in the image1122corresponding to “c” but no microparticle is bright in the images1116,1120corresponding to “a” and “g,” respectively. Thus, in this simple conceptual example, the DNA sequence associated with the first microparticle1172is “aat” and the DNA sequence associated with the second microparticle1174is “gac.” Although the example inFIG. 11is illustrated above as using the brightness of the imaged microparticles to determine the sequence information, the sequence information can also be derived by detecting different spectral characteristics of the imaged microparticles.

In embodiments other than this simple conceptual example, the sequencing reaction cycles occur more or less frequently, and the number of optical signatures is more or less than four. For example, in one embodiment, sequencing reactions can occur between high-resolution synthetic images1102,1104rather than between synthetic image sets1150,1160. In another embodiment, each microparticle1172,1174takes on just one optical signature, and the absence of an optical signature conveys sequence information. In still another embodiment, each microparticle1172,1174simultaneously takes on multiple optical signatures. In still another embodiment, the simple one-to-one correspondence between optical signatures and DNA bases is replaced by a more sophisticated scheme for encoding for DNA sequence information with optical signatures (e.g., two-base encoding). Note that there are a number of other conventional sequencing chemistries. The sequencing method of the present invention can be used and is compatible with the majority of sequencing chemistries.

Referring back toFIG. 8, the MSI algorithms812identify the microparticles1172,1174in each high-resolution synthetic image1102through1122, and extract the optical signature data for each microparticle. In practice, this can be accomplished by thresholding the image to identify the microparticles visible in each synthetic image, and then fitting the image of each microparticle with a two-dimensional Gaussian function to estimate location and brightness.

Referring back toFIG. 8, the base-calling algorithms816take as input the microparticle optical signature data814and generate a DNA sequence for each microparticle. The first step is to track the location of each microparticle through the sets of microparticle optical signature data814. In practice, the position of the microparticle array is not identical in each imaging cycle. Consequently, the sets of microparticle optical signature data814typically require spatial registration. In one embodiment, spatial registration is aided by the use of registration microparticles mixed in with the sequencing microparticles. Typically, the registration microparticles are 1-micron diameter beads that are several times brighter than the sequencing microparticles. The brightness of the registration microparticles makes them easy to distinguish from the sequencing microparticles, and the ratio of registration microparticles to sequencing microparticles is low (approximately 1-to-1000) such that the sequencing throughput is not significantly affected. After the registration step, the second step is to determine the optical signature of each microparticle for each set of microparticle optical signature data814. As stated above, each microparticle can take on one of four optical signatures in each set of microparticle optical signature data814. The four optical signatures correspond to the four possible base calls (i.e., “a”, “t”, “g”, and “c”). A quality metric is typically assigned to each base call. Note that a variety of conventional algorithms can be used to interpret the raw base calls, which are not the subject of the present invention and are not described herein.

FIG. 12illustrates a process for calibrating the structured illumination apparatus shown inFIG. 5. Calibration is done in order to know the excitation patterns with a certain degree of accuracy so that DNA sequence data can be successfully generated through image processing. For example, this excitation pattern data806(FIG. 8) is an input to the image synthesis algorithms804(FIG. 8). The typical calibration parameters in a synthetic aperture optics structured illumination apparatus as inFIGS. 7A and 7Bare the direction, wavefront, shape, amplitude, polarization, wavelength, and relative optical phase of each beam724,726,728,730. Referring toFIG. 12, in one embodiment, calibration is performed by placing a calibration target1202in place of the microparticle array. A calibration target1202can in theory be any target with known features. For example, the calibration target1202can be a random array of fluorescent 1-micrometer diameter beads with substantially identical brightness. Similar to the process illustrated inFIG. 5, the calibration target1202is selectively excited506with an excitation pattern. The selectively-excited calibration target microparticle array is then imaged508using an optical microscope. The excitation pattern is then changed512, and the excite-and-image cycle is repeated until the last pattern510is reached. After the last excite-and-image cycle is complete510, the images are then processed520. Based on knowledge of the calibration target1202and the old calibration parameters1206for the structured illumination apparatus, the content of the calibration images can be predicted. The discrepancy between the predicted calibration images (based on the old calibration parameters) and the measured calibration images through image processing520is used to generate new calibration parameters1204.

FIG. 13conceptually illustrates the hardware system used for sequencing by selective excitation of microparticles, according to one embodiment of the present invention. The hardware system includes a flow cell1302, a reagent handling module1304, a temperature control module1306, a selective microparticle excitation module1308, an optical imaging module1310, an image processing module1312, and a sequence data storage module1314. The microparticle array502is typically located inside a flow cell1302that allows the microparticles to be exposed to sequencing reagents. The reagent handling module1304applies the reagents to expose the microparticle array502to the sequencing reagents. The temperature control module1306controls the reaction temperature of the flow cell1302at temperatures appropriate for reactions with the sequencing reagents. The selective microparticle excitation module1308selectively excites the microparticles as explained above, and the optical imaging module1310obtains images of the selectively-excited microparticles. The images of the selectively-excited microparticles are analyzed using image processing algorithms in the image processing module1312to extract the optical signatures of the microparticles and to generate the sequence information. The sequence information is stored in the sequence data storage module1314.

FIG. 14illustrates the control system architecture for the sequencing hardware ofFIG. 13. The control system architecture includes a data acquisition computer1402with a user interface1404, a frame grabber1410, a camera1418, stage controllers with a programmable logic controller (PLC)1414, (scanning) stages1420, shutters1422, beam modulators1424, an autosampler1426, pumps1428, temperature controllers1430, a focus controller1432, and an image processing computer cluster1406. The frame grabber1410is connected to the data acquisition computer1402through a peripheral component interconnect (PCI) interface1412. The frame grabber1410and the camera1418together obtain images of the selectively-excited microparticles under control of the data acquisition computer1402. For high speed, a PLC1414is used to control the shutters1422and beam modulators1424to change the excitation patterns, move the stages1420, and trigger the camera1418to ensure tight synchronization. The PLC1414is connected to the data acquisition computer1402through a Firewire interface1416. Other less-critically-timed hardware such as the autosampler1426, pumps1428, temperature controllers1430, and the focus controller1432are controlled through a slower interface such as RS-232. The autosampler1426samples the sequencing reagents. The pumps1428pump the sequencing reagents into the flow cell1302to expose the microparticle array502to the sequencing reagents. The temperature controllers1430control the reaction temperature of the flow cell1302at temperatures appropriate for reactions with the sequencing reagents. The focus controller1432dynamically adjusts the focus of the optical imaging module1310to keep the microparticle array502in focus as the stages1420move. The data acquisition computer1402runs the user interface (UI)1404, controls the variety of hardware, and receives image data from the camera1418. The image data is sent over an Ethernet interface1408to a cluster of computers1406for image processing. The scanning stages1420and the focus controller1432enable large area microparticle arrays spanning multiple fields of view to be imaged.

Upon reading this disclosure, those of skill in the art will appreciate still additional alternative structural and functional designs for a method and system for nucleic acid sequencing through selective excitation of microparticles. For example, an excitation pattern can also be produced using a spatial light modulator (such as a liquid-crystal modulator or a MEMS-mirror-array modulator) and a projection lens. Thus, while particular embodiments and applications of the present invention have been illustrated and described, it is to be understood that the invention is not limited to the precise construction and components disclosed herein and that various modifications, changes and variations which will be apparent to those skilled in the art may be made in the arrangement, operation and details of the method and apparatus of the present invention disclosed herein without departing from the spirit and scope of the invention as defined in the appended claims.