EP2179289A1 - Apparatus and method for digital magnetic beads analysis - Google Patents

Abstract

A Light Transmitted Assay Beads or digital magnetic microbead having a digitally coded structure that is partially transmissive and opaque to light. When hundreds or thousands of LITAB are settled down to the bottom of a microwell in a microplate, the barcode can be decoded by image processed accurately and reliable. Microplate is a standard bioassay format; each plate can have 96, 384, or 1536 patient samples. Therefore, a large number of targets in a sample can be analyzed in one single microwell. The image decoding algorithms comprise of four main processes (1) enhancement of image (2) segmentation of beads (3) extraction of barcode slits, and (4) decoding of barcodes. The bead image is taken from the bottom of an optically clear microplate, and barcode pattern can be decoded by image software. Therefore, the whole bead bioassay experiment can be performed in the microplate without taking the beads out.

Description

Apparatus and Method for Digital Magnetic Beads Analysis

BACKGROUND OF THE INVENTION

[0001] This application is a continuation-in-part application of U.S. Patent Application No. 1 1/580,514, filed October 13, 2006; a continuation-in-part application of U.S. Patent Application No. 1 1/502,606, filed August 9, 2006, which claims the benefit of the priority of Provisional Patent Application No. 60/706,896, filed August 9, 2005; a continuation-in-part of U.S. Patent Application No. 12/069,720, filed February 1 1, 2008; and a continuation-in-part of Provisional Patent Application No. 60/964,108, which was filed August 8, 2007. All other publications and U.S. patent applications disclosed herein below are also incorporated by reference, as if fully set forth herein.

1. FIELD OF THE INVENTION

[0002] The invention relates to carry out multiplexed bioassay with hundreds or thousands of digital magnetic barcode microbeads for proteins, nucleic acids, and molecular diagnostics; and more particularly an optical image decoding algorithm and method is developed to rapidly and simultaneously analyze the barcodes of every single bead in a small microwell, such as microplate. The digital magnetic bead is non-spherical, non-traditional latex microspheres; therefore, the high density optical pattern can be imaged and identified accurately.

2. DESCRIPTION OF RELATED ART

[0003] As current research in genomics and proteomics require multiplexed data, there is a need for technologies that can rapidly screen a large number of targets, such as nucleic acids and proteins, in a very small volume of samples. Microarray, DNA chips, and protein chips, with the ability to screen thousands or millions of targets on a planar platform, require a large volume of sample to cover the large surface. The typical surface area is 1 cm x 1 cm or on a microslide. Distributing a small volume of liquid samples over a relatively large chip surface often encounters the disadvantages of slow diffusion of molecules and non-uniform mixing or distribution over the chip surface. These are the reason, microarray assay require very long reaction time. Furthermore, microarray chip, once it is printed and fabricated, it is impossible to add on one more test into a multiplexed assay.

[0004] Micro bead technology potentially overcomes many of the problems of microarray technology and provides flexibility of library content and amount of beads or bead type in an analysis. Due to its small volume (in the range of picoliter per bead), thousands of beads can be incubated with a very small amount of sample. A number of encoding strategies have been demonstrated include particles with spectrally distinguishable fluorophore, fluorescent semiconductor quantum dots, and metallic rods with either bar coded color (absorption) stripes or black and white strips. Both fluorescence and barcode color strip beads are identified by optical detection in reflective or emissive configuration. The difficulties of reflection configuration are (1) the optical reflection yield is low, especially when the beads are in micrometer scale, (2) the light collection efficiency is poor, and (3) for fluorescence-based encoded beads, the fluorescence bands are very broad and overlapped, thus limit the potential code number. Another drawback of fluorescence-based bead is that most bead-based assay rely on fluorescence readout, thus creating more fluorescence spectral or intensity interference. In the case of multi-metal (Au, Pt, Ni, Ag, etc) color micro rods, the encoding scheme suffers from the difficulty of manufacturing and the number of colors, based on different metal materials, is limited.

[0005] U.S. Pat. No. 6,773,886 issued on August 10, 2004, entire contents of which are incorporated herein by reference, discloses a form of bar coding comprising 30-300 nm diameters by 400-4000 nm multilayer multi metal rods. These rods are constructed by electrodeposition into an alumina mold; thereafter the alumina is removed leaving these small multilayer objects behind. The system can have up to 12 zones encoded, in up to 7 different metals, where the metals have different reflectivity and thus appear lighter or darker in an optical microscope depending on the metal type whereas assay readout is by fluorescence from the target, and the identity of the probe is from the light dark pattern of the barcodes. [0006] U.S. Pat. No. 6,630,307 issued on October 7, 2003, entire contents of which are incorporated herein by reference, discloses semiconductor nano-crystals acting as a barcode, wherein each semiconductor nano-crystal produces a distinct emissions spectrum. These characteristic emissions can be observed as colors, if in the visible region of the spectrum, or may be decoded to provide information about the particular wavelength at which the discrete transition is observed.

[0007] U.S. Pat. No. 6,734,420 issued on May 11 , 2004, entire contents of which are incorporated herein by reference, discloses an identification system comprising a plurality of identifiable elements associated with labels, the labels including markers for generating wavelength/intensity spectra in response to excitation energy, and an analyzer for identifying the elements from the wavelength/intensity spectra of the associated labels.

[0008] U.S. Pat. No. 6,350,620 issued on Feb. 26, 2002, discloses a method of producing a micro carrier by placing a bead between a nickel plate on which the barcode has been electroformed and a second plate, and compressing the barcode onto the surface of the bead to form a microcake-like particle with a barcode.

[0009] U.S. Pub. No. US2005/0003556 Al, entire contents of which are incorporated herein by reference, discloses an identification system using optical graphics, for example, bar codes or dot matrix bar codes and color signals based on color information signal for producing the affinity reaction probe beads. The color pattern is decoded in optical reflection mode.

[0010] U.S. Pub. No. US2005/0244955, entire contents of which are incorporated herein by reference, discloses a micro-pallet which includes a small flat surface designed for single adherent cells to plate, a cell plating region designed to protect the cells, and shaping designed to enable or improve flow-through operation. The micro-pallet is preferably patterned in a readily identifiable manner and sized to accommodate a single cell to which it is comparable in size.

[0011] Magnetic beads are used widely in high throughput automated operation. The magnetic beads, are paramagnetic, that is, they have magnetic property when placed within a magnetic field, but retain no residual magnetism when removed from the magnetic field. This allows magnetic collection of microbeads and resuspension of the beads when the magnetic field is removed. Collection and resuspension of the digital magnetic beads can be repeated easily and rapidly any number of times. The common robotic automation is simply putting a 96-well, 384- well or 1536-well microplate on a magnetic stand facilitated with magnetic pins to activate the magnetic field. This enables washing of unbound molecules from the beads, changing buffer solution, or removing any contaminant in the solution. For example, in the case of DNA or RNA assay, the unbound or non-specific nucleotides can be removed after hybridization. While in the case of protein assay, the unbound or non-specific antibodies or antigens can be removed after the antibody-antigen reaction. Extensive washing often required during molecular biology applications to be conducted swiftly, efficiently, and with minimal difficulty. While magnetic beads are widely used in the bioassays, no magnetic beads with high density barcode are available.

[0012] What is needed is a digitally encoded magnetic micro bead that provides high optical contrast and high signal-to-noise for reliable decoding, and also provides magnetic property for high-throughput automated washing in the microplate format. The decoding method should be simple and robust. The data process should be rapid and accurate.

SUMMARY OF THE INVENTION

[0013] The present invention improves on the prior art systems and the parent application, to provide a high throughput process of determining the code represented by the simultaneous imaging of a plurality of digital coded beads. According to the present invention, the microbead analytical system comprises a support (e.g., a plate of wells or a microslide) supporting at least one bead, wherein the bead is provided with an indicia representing a digital code; an imaging device obtaining an image of the support and the bead including the indicia; and a decoding system configured to analyze the image obtained by the imaging device to recognize and isolate the bead from one another and from the support in the background, and determining the digital code represented by the indicia provided on the bead. The image processing approach of the present invention is different from prior art scanning of bar code on a pallet, in which the pallet is not imaged along with the background support, hence the pallet is not recognized and isolated from any background or rest of any other image. The present invention is particularly useful to efficiently analyze a plurality of microbeads, each having an indicia representing a digital code, wherein the image obtained by the imaging device include the plurality of beads, including the indicia on each bead, and wherein the decoding system is configured to analyze the image to recognize and isolate the plurality of beads, and determining the digital code represented by the indicia provided on each bead. By recognizing and isolating each bead from each other and the background in the overall image of the plurality of beads, the digital code represented on each bead can be determined.

[0014] In one aspect, the present invention is directed to an image processing method for decoding digitally coded beads used in bioassays, which conduct imaging of the beads in their steady or static state, instead of a moving state in which the beads are moving relative to the imaging optics (e.g., the beads being carried in a flow pass the imaging optics, or the beads are being scanned by a moving light beam). A plurality of beads can be distributed on a planar surface (e.g., a glass microslide), and imaged simultaneously in two dimension with an imaging device (e.g., a wide viewing image camera), thereby allowing a plurality of beads to be decoded to improve detection throughput. The digital encoding may be observable by imaging based on emission, reflection and/or transmission of light with respect to the beads.

[0015] In another aspect, the bead imaging aspect of the present invention can be applied to beads in a moving state in which the beads are moving relative to the imaging optics (e.g., the beads being carried in a flow pass the imaging optics, or the beads are being scanned by a moving light beam), as long as the bead imaging system is able to clearly snap a static image of the beads while the beads are in motion.

[0016] In another aspect of the present invention, the digital coding of the beads comprises a bar code pattern. The bar code pattern with a series of narrow and wide bands provides an unambiguous signal and differentiation for O's and l 's. The position of the slits on the pallet will determine which of the bits is the least significant (LSB) and most significant bit (MSB). The LSB will be placed closer to the edge of the pallet to distinguish it from the MSB at the other, longer end. In one embodiment, the coded bead comprises a body having a bar code image of a series of alternating light and dark sections (e.g., light transmissive and opaque sections) with relative widths (e.g., a series of narrow section representing a "0" code and wide sections representing a "1" code, or vice versa). The digital bar code pattern is imaged with the imaging device.

[0017] The image decoding process comprises four main sub-processes (1) Enhancement of image (2) Segmentation of beads (3) Extraction of barcode slits, and (4) Decoding of barcodes. In one embodiment, decoding of the images of the beads involves a sequence of steps that are undertaken to resolve the images of the beads, and extract digitally encoded information. Such steps may include: (1) gray scale conversion to preserve bead image details; (2) background subtraction to obtain uniform background for bead images; (3) bead image edge detection; (4) if bead images are touching, additional filtering process to separate the touching bead images; (5)^' labeling of individual bead images (e.g., using color codes or numbers); (6) alignment transformation of the bead images and representing the digital encoding (e.g., a bar code pattern) in each bead image by intensity (e.g., an intensity plot); and (7) analyzing the intensity to determine the digital code (e.g., identify the narrow and wide bands in a bar code pattern).

[0018J The accuracy in the decoding of images of static beads can be better than decoding of images of moving beads. The accuracy of decoding is very important for clinical diagnostics, because false identification can lead to mis-diagnosis and mis-therapy.

[0019] In the illustrated embodiments, the present invention is directed to a Light Transmitted Assay Beads (LITAB) or micropallet that is digitally coded as represented by an image that provides for high contrast and high signal-to-noise optical detection to facilitate identification of the bead. The image is implemented by a physical structure having a pattern that is partially substantially transmissive (e.g., transparent, translucent, and/or pervious to light), and partially substantially opaque (e.g., reflective and/or absorptive to light) to light. The pattern of transmitted light is determined (e.g., by scanning or imaging), the code represented by the image on the coded bead can be decoded. In one embodiment, the coded bead comprises a body having a series of alternating light transmissive and opaque sections, with relative positions, widths and spacing resembling a ID or 2D bar code image (e.g., a series of narrow slits (e.g., 5 microns in width) representing a "0" code and wide slits (e.g., 10 microns in width) representing a "1" code, or vice versa). To decode the image, the alternating transmissive and opaque sections of the body are scanned and imaged (e.g., with a CCD sensor) to determine the code represented by the image determined from the transmitted light.

[0020] In a further aspect of the present invention, a number of digital magnetic beads can be distributed on a planar surface and detected simultaneously with an image camera. The planar surface can be on a microslide or at the bottom of a microplate. The barcode patterns, representing digital signal such as "0" and "1", of each bead are determined by image processing.

[0021] In a further aspect of the present invention, the digital barcode beads have paramagnetic property, that is, they have magnetic property when placed within a magnetic field, but retain no residual magnetism when removed from the magnetic field. Magnetic beads allow washing in a microplate by collection of beads with an external magnet, and resuspension of beads when the magnetic field is removed. Multiple digital magnetic beads allow multiplexed assays to be performed in a single well. Microplate is a standard high throughput format in clinical diagnostics; each plate has 96, 384, or 1 ,536 patient samples.

[0022] In a further aspect of the present invention, a digital magnetic microbead analytical system comprises: (a) a slide or microplate with a plurality of wells; (b) at least one digital magnetic bead on the surface of the slide or settled at the bottom of the wells of the microplate, (c) an optical detector, located above or under the slide or the microplate, imaging the at least one magnetic microbead; and (d) a digital processing system implemented with an image software to process the image pattern of at least one magnetic microbead. In one embodiment, the number of wells is between about 96, 384, or 1536 wells.

[0023] In a further aspect of the present invention, both bar-code image and fluorescence image are taken under a microscope and camera simultaneously. Therefore, the whole bead experiment can be performed in the microplate without taking the beads out. The barcode is used to identify which molecular probe is immobilized on the bead, while the fluorescence is used to detect the positive or negative reaction. A large number of targets can be analyzed simultaneously.

[0024] In a further aspect of the present invention, the digital magnetic microbeads comprise a first layer; a second layer; and an intermediate layer between the first layer and the second layer, the intermediate layer having an encoded pattern defined thereon, wherein the intermediate layer is partially substantially transmissive and partially substantially opaque to light, representing a code corresponding to each of the microbeads.

[0025] In a further aspect of the present invention, the intermediate layer comprises a series of alternating substantially light transmissive sections and substantially light opaque sections defining the encoded pattern. The relative positions, widths and/or spacing between the transmissive sections and/or opaque sections represent a binary code. The substantially light opaque sections comprise a light blocking material. The body of each microbead has a longest orthogonal axis of 1 mm or less.

[0026] In a further aspect of the present invention, the first layer and the second layer of the digital magnetic beads are functionalized with a material selected from the group consisting of proteins, nucleic acids, small molecules, chemicals, and combinations thereof.

[0027] In one embodiment, the body of the coded bead may be configured to have at least two orthogonal cross sections that are different in relative geometry and/or size. Further, the geometry of the cross sections may be symmetrical or non-symmetrical, and/or regular or irregular shape. In one embodiment, the longest orthogonal axis of the coded bead is less than 1 mm.

[0028] In a further aspect of the present invention, the light transmissive sections are defined by slits through the intermediate layer, and the light opaque sections are defined by a light reflective material and/or a light absorptive material. The slits comprise slits of a first width and slits of a second width, and wherein the first width represents a "0" and the second width represents a "1" in a binary code. The first width is about 1 to 10 microns and the second width is about 1 to 50 microns, and wherein the first width is narrower than the second width. The binary codes can be decoded by image software.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] For a fuller understanding of the scope and nature of the invention, as well as the preferred mode of use, reference should be made to the following detailed description read in conjunction with the accompanying drawings. In the following drawings, like reference numerals designate like or similar parts throughout the drawings.

[0030] FIG. 1 illustrates the process for preparing Light Transmitted Assay Beads (LITAB) for bioassay, in accordance with one embodiment of the present invention: (a) Multiple LITAB in a microwell of a microplate, (b) LITAB for bioassay, and (c) a schematic representing a photo image of LITABs.

[0031] FIG. 2(a) is a top view of a LITAB in accordance with one embodiment of the present invention; FIG. 2(b) is a sectional view taken along line A-A in FIG. 2(a); FIG. 2(c) shows the transmitted digital signal of a barcoded bead representing 001 1111001 on an image camera.

[0032] FIG. 3 illustrates an optical signal pattern representing light transmitted through the pattern of slits in a LITAB with the least significant bit (LSB) or the most significant bit (MSB).

[0033] FIG 4 illustrates the steps of forming a bead in accordance with one embodiment of the present invention.

[0034] FIG 5, illustrates a metal layer as a layer sandwiched between two polymeric layers that may provide the same surface chemistry for molecule immobilization.

[0035] FIG. 6 illustrates the microscopic image of the barcode microbeads. [0036] FIG. 7 illustrates the use of edge detection technique to outline the objects in the image.

[0037] FIG. 8 illustrates segregated beads are separated using Watershed lines.

[0038] FIG. 9 illustrates the method of aligning each microbead to the major axis, and the gray scale image is converted into a matrix, which contained the pixel values.

[0039] FIG. 10 shows that the bead image is converted to gray scale intensity as a function of pixel values.

[0040] FIG. 11 illustrates the digital code being determined based on the widths of the barcode present in each microbead.

[0041] FIG. 12 illustrates the LITAB analytical system in accordance with one embodiment of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0042] The present description is of the best presently contemplated mode of carrying out the invention. This description is made for the purpose of illustrating the general principles of the invention and should not be taken in a limiting sense. The scope of the invention is best determined by reference to the appended claims.

[0043] The detailed descriptions of the process of the present invention are presented largely in terms of methods or processes, symbolic representations of operations, functionalities and features of the invention. These method descriptions and representations are the means used by those skilled in the art to most effectively convey the substance of their work to others skilled in the art. A software implemented method or process is here, and generally, conceived to be a self-consistent sequence of steps leading to a desired result. These steps require physical manipulations of physical quantities. Often, but not necessarily, these quantities take the form of electrical or magnetic signals capable of being stored, transferred, combined, compared, and otherwise manipulated.

[0044] Useful devices for performing the software implemented operations of the present invention include, but are not limited to, general or specific purpose digital processing and/or computing devices, which devices may be standalone devices or part of a larger system. The devices may be selectively activated or reconfigured by a program, routine and/or a sequence of instructions and/or logic stored in the devices. In short, use of the methods described and suggested herein is not limited to a particular processing configuration.

[0045] For purposes of illustrating the principles of the present invention and not by limitation, the present invention is described herein below by reference to a micro bead that is in the shape of a pallet, and by reference to bioanalysis. However, it is understood that the present invention is equally applicable to micro beads of other overall geometries, and which are applied for other applications requiring identification based on the identity of the beads, without departing from the scope and spirit of the present invention. To facilitate discussion below, the micro bead of the present invention is referred to as a LITAB, which stands for a Light Transmitted Assay Beads.

[0046] LITAB is digitally coded as represented by an image that provides for high contrast and high signal-to-noise optical detection to facilitate identification of the bead. The image is implemented by a physical structure having a pattern that is partially substantially transmissive (e.g., transparent, translucent, and/or pervious to light), and partially substantially opaque (e.g., reflective and/or absorptive to light) to light. The pattern of transmitted light is determined (e.g., by scanning or imaging), and the code represented by the image on the coded bead can be decoded. Various barcode patterns, such as circular, square, or other geometrical shapes, can be designed as long as it represented a "1" or "0" and can be recognized by the decoder. However, LITAB is not spherical shape; it is different from the conventional latex-based spherical beads.

[0047] In one embodiment, the coded bead comprises a body having a series of alternating light transmissive and opaque sections, with relative positions, widths and/or spacing resembling a ID or 2D bar code image (e.g., a series of narrow slits (e.g., about 1 to 5 microns in width) representing a "0" code and wide slits (e.g., about 1 to 10 microns in width) representing a "1" code, or vice versa, to form a binary code). FIG. 2 illustrates a coded bead, LITAB 1 1 in accordance with one embodiment of the present invention. The LITAB 1 1 has a body 25 in the shape of a flat pallet or disc. The body of the coded bead may be configured to have at least two orthogonal cross sections that are different in relative geometry and/or size. Further, the geometry of the cross sections may be symmetrical or non-symmetrical, and/or regular or irregular shape. In this particular embodiment, all three orthogonal axes are of different lengths, and the geometries of all three orthogonal cross sections are symmetrical and of regular shape. FIG. 2(a) shows that the planar geometry resembles a symmetrical stretched or elongated oval shaped pallet. FIG. 2(b) shows the cross section showing the longitudinal (or longest) axis. A series of wide and narrow slits 23 and 24 are provided through the body 25, which may be made of or coated with a substantially light opaque material (e.g., reflective or absorptive). The wide and narrow slits 23 and 24 represent a logical "1" and "0", respectively, or vice versa, and collectively represent a binary code (each slit representing a bit). In this embodiment, the code is analogous to a bar code. The narrow slits 24 may have a width of 5 microns, and the wide slits 23 may have a width of 10 microns. For a LITAB having an overall dimension of 100 x 30 x 10 μm to 300 μm x 100 μm x 40 μm, at least about 10 slits may be provided on the LITAB pallets to encode 6 bits to 12 bits or more, allowing 64 to 4,096 or more unique codes. In one embodiment, the longest orthogonal axis of the coded bead is less than 1 mm.

[0048] While the illustrated embodiment shows a pattern of slits of spaced apart narrow and wide width, it is also possible to use a pattern of slits having a constant width which are spaced apart at narrow and wide spacings between adjacent slits to represent 1 's and O's, without departing from the scope and spirit of the present invention. FIG. 2(c) shows the transmission peaks of a single bead detected by a CCD (charge coupled device) and displayed on the computer screen. When the bead is illuminated with a light beam, based on the either the "total intensity" of the transmission peak or the "bandwidth" of the transmission peak from the slit, the digital barcode either 0 or 1 can be determined by an imaging camera and a digital signal processor. As shown in the figure, the barcode patterns can be easily identified based on the peak widths. Specifically as illustrated in the embodiment shown in Fig. 2(c), the beads show 10-bit barcodes representing 0011 1 1 1001.

[0049] To decode the image, the alternating transmissive and opaque sections of the body are imaged with light (e.g., using a CCD sensor) to determine the code represented by the image determined from the transmitted light. For illustration purposes, FIG. 3 shows a series of signal pulses representing the detection of light transmitted through the slits 23 and 24 in the LITAB 1 1 in FIG. 2(a). The signals correspond to the contrast of transmitted versus blocked light across the longitudinal axis of the LITAB 1 1. The width of each signal pulses represents a "1" or a "0" in the code of the LITAB 1 1. In the particular illustrated example, the wider widths represent 1 's and the narrow widths represent O's. The relative positions of the slits on the LITAB 11 determine which of the bits is the least significant bit (LSB) or the most significant bit (MSB). In one embodiment, the least significant bit was placed closer to one edge or end of the LITAB 1 1 to distinguish it from the most significant bit at the opposing edge or end. The concept of decoding the signal pulses is analogous to decoding for a traditional bar code.

[0050] It is noted that in an alternate embodiment, the substantial transmissive section need not be a slit through the entire thickness of the body of the LITAB. The slit may be completely or partially filled with a substantially transparent or translucent material, which nonetheless provides substantially light transmissivity, compared to the opaque section. For example, the LITAB may have a transparent body, covered with a light blocking material (e.g., a reflective material, or a light reflective or absorptive dye) that has openings defining slits exposing the transparent body. Light imaged on this LITAB would transmit light through the body at sections not covered by the blocking material (i.e., the slits), and block light in the covered section. In another embodiment, the coded LITAB can be provided with a reflective thin film or coating, (e.g., plating or coating the surface of the LITAB with a metal thin film, or providing an intermediate, sandwiched layer of paramagnetic metal thin film, or coating with a light absorptive dye) to improve contrast between transmitted versus blocked/reflected light and optical efficiency for image recognition and decoding. [0051] The LITAB may be fabricated using conventional methods used in thin film formation in a clean room microfabrication facility. The structure of the LITAB may be obtained using processes that may include conventional photo-lithography, printing, silk-screening, curing, developing, etching (e.g., chemical etching, ion etching, and/or other removing processes), plating, dicing, and other process steps well known in the art for such types of structure and the material involved. Referring to FIG. 4(a) to (d), in one embodiment of the process for fabricating the LITAB, a layer 52 of Ti (e.g., 100 nm) is deposited by e-beam evaporation on a substrate 50, e.g., a clean glass slide (e.g., about 1 mm thick). Ti functions as a conducting seed layer as well as a surrogate releasing layer. The body 25 of the LITAB may be formed using a layer of polymeric material. For example, a photoresist photopolymer (e.g., SU-8 and the like, as known in the art), may be utilized in creating the LITABs. A layer 21 of polymeric material is spin-coated on the Ti layer 52, and the slits 23 and 24 are formed in such layer using standard photolithographic procedures. For example, the slits 23 and 24 may be defined by UV-light irradiation using a photomask (not shown) defining the desired pattern of wide and narrow slits, and the planar shape of the LITAB body 25. An array of LITABs may be formed on a single substrate, each having a different slit pattern representing a different code. The photomask may also define the periphery of the array of LITAB bodies, such that the LITAB bodies are separated from one another at the end of the same photolithographic process that defines the slits. Because SU-8 is transparent, an e-beam evaporator is utilized to deposit a metal layer, such as gold (Au, 0.1 μm) top layer 22 (see also FIG. 2(b)) on the SU-8 layer 21 supported on the substrate 50. The individual LITAB bodies 25 (shown in FIG. 2(b)) are finally freed from the underlying substrate 50 by dissolving the surrogate Ti layer 52 with an etching solution containing hydrofluoric acid (HF). In this way, the gold pattern on the LITAB blocks light by reflecting light (directed to both from the side exposed and the side adjacent to the SU-8 layer 21), and slits not covered by gold layer transmit light. Because the gold layer 22 blocks the light, while the open slits transmit the light, LITAB "bar codes" provide high optical signal, and high optical contrast when the transmitted light is detected.

[0052] FIG. 5 shows an alternate embodiment of a LITAB 80, which may include a metal layer 81 as an intermediate layer sandwiched between two polymer layers 82. A barcode pattern is fabricated on the metal layer 81. For example, slits 84 of different widths and/or spacings are formed in the metal layer 81. In the illustrated embodiment, the polymer (photopolymer: SU-8) layers 82 are closed layers (i.e., no slits). The process for forming the LITAB 80 may include first forming a first photopolymer layer 82, then forming the metal layer 81 followed by etching the slits 84 therein. A second photopolymer layer 82 is formed on the metal layer 81 (e.g., by spin coating and curing), which fills the slits 84. Alternatively, the slits 84 may be first filled with another transparent material, before forming the second photopolymer layer 82. With this embodiment, surface condition could be made the same for both exposed planar surfaces of the LITAB, to provide similar surface coating and immobilization conditions. The other embodiment is to coat the LITAB with polymer or functional molecules, such as biotin, carboxylated, or streptavidin; therefore, the whole bead has the same condition for molecular immobilization.

[0053] A paramagnetic material is imbedded in the intermediate layer in the LITAB, and thus sandwiched between the first layer and second layer of polymer films. Paramagnetic materials include magnesium, molybdenum, lithium, aluminum, nickel, and tantalum. It is noted that the paramagnetic material on the LITAB would also function as a light blocking material, so a reflective layer is not necessary. The present invention would allow decoding based on transmitted light, even in the presence of the paramagnetic material. However, for prior art, there are magnetic beads and barcode beads, no magnetic material has been incorporated into the barcode microbeads. This is because the magnetic material being inherently dark brown, would not be compatible with the reflective bar code, which requires alternating dark and white lines.

[0054] FIG. 1 illustrates an embodiment for preparing LITAB for bioassays. As shown in FIG. l(a), the LITABs 1 1 allow multiplexed homogeneous bioassays on micro-volume samples. A mixture of LITABs 1 1 corresponding to different codes 14 are introduced into a small volume of biological sample 12 in a tube or microwell plate 13. The LITABs can be optically decoded easily and rapidly thereafter. In one embodiment, FIG. l(b) shows one LITAB 11 functionalizing with nucleic acid probe 15 for target hybridization 16 and fluorescence detection 17. Several materials are available for bead immobilization. In one embodiment, the LITAB may be coated with a covalent DNA-binding agent used in microarray. The probe beads were subsequently hybridized in solution to a complementary oligo target which carried a covalently bound fluorophore at its 5' end. FIG. l(c) is LITABs imaged on a CCD with a microscope. Different objectives provide different field of views.

[0055] The randomly oriented microbeads can be decoded on a support, such as a slide or in the bottom of a microplate by imaging processing method. When beads are finally settled down and distributed on the bottom of a planar surface in a microplate, multiple beads can be decoded simultaneously with a wide viewing or scanning image camera. Microplate is a standard format for high throughput clinical assays. Each well is used for one sample; each plate holds 96, 384, or 1,536 patient samples for 96-well, 384-well, and 1,536-well, respectively. Therefore, an experiment can be performed in the microplate without taking the beads out, the image of the microbeads can be taken in the steady state with a better accuracy and sensitivity for decoding. The accuracy of decoding is very important for clinical diagnostics, because any false identification can lead to mis-diagnosis and mis-therapy. Due to the small bead size, hundreds or even thousands of beads can be displayed in the bottom of a microwell with minimal overlap. To minimize bead overlap, depend on the area of the microwell, the total number of beads is limited to a certain number. Both bar-code image and fluorescence image can be constructed on a conventional microscope or an inverted fluorescence microscope.

[0056] One embodiment illustrated in Fig. 12, the digital magnetic LITAB analytical system 100 has a light source 102 for bead pattern illumination and an optical CCD 104 for capturing images of beads 11 at the bottom of the support (e.g., a microwell 106 in the illustrated embodiment). The bottom of the microwell 106 is transparent or translucent, allowing sufficient light to pass through to the beads. A scanning or translation mechanism 1 10 (schematically represented in Fig. 12) moves the microwell 106 relative to the optical detector 104 (e.g., a CCD) and light source to image the desired wells. The optical detector 104 can be used for both barcode image and fluorescence detection. A IM pixels CCD should have sufficient pixels to resolve the barcode pattern 14 on beads 11. Lens and optical filters 108 are used to collect and select the excitation and fluorescence wavelength. Control of the system 100 and/or image processing is implemented by a processing device 112, which is configured with the functionality and features to carry out the process steps in accordance with the disclosure of the present invention herein. The image of the beads may be stored and/or printed/displayed, before being processed by the processing device 1 12, or the signal or data representing the image of the beads may be fed to the processing device 1 12 to be proceeded in real time or near real time.

[0057] In another embodiment (not shown), two light sources are used, one light source 102 below the microwell for barcode illumination in transmission mode and one light source 103 above the microwell for fluorescence excitation in incident or reflection mode. Barcode illumination light source can be a white light, while fluorescence excitation light source need a wavelength matches with the absorption of the fluorophore. By measuring the fluorescence intensity, we can identify which beads have positive biochemical reaction. Measurement of fluorescence intensity may be made by the same or a different optical detector from that which images the beads. By decoding the digital barcode image, we can identify which biological probe is immobilized on the surface of that microbead. The choice of light source depends on the fluorophore. For example, a Mercury light source facilitated with an optical filter cube offers UV to visible light excitation. A red diode laser (665 nm), and compact Argon Laser (488 nm) or green laser (530 nm) are common laser light sources for variety of fluorophores (e.g. phycoerythrin (PE), Cy3, and Cy 5, etc.). Optical filter sets are designed to select particular excitation and fluorescence wavelength for various fluorophores.

[0058] As soon as a barcode image is obtained from the CCD, the image data is rapidly processed by the image software. They are many image-decoding algorithms. Depend on the image patterns, different algorithms may vary in terms of decoding speed or accuracy. One of the image decoding algorithms comprise of four main processes (1) Enhancement of image (2) Segmentation of beads (3) Extraction of barcode slits, and (4) Decoding of barcodes. Some of these processing are carried out using the mathematical software, such as toolkits available from "The Mathworks, Inc." (e.g., MATLAB^® Version 7.4.0.287 (R2007a); January 29, 2007), The functions of these processes are explained in the following sections.

[0059] (1) Enhancement of image: The performance of the decoding of beads depends heavily upon the quality of the image. The accuracy of the decoding process can be improved by imaging enhancement. This image enhancement using image intensity normalization to provide uniform intensity background. Non-uniform background is often due to the non-homogeneous illumination. To achieve high image contrast of the beads, the homogeneous background should be produced first by background subtraction and normalization.

[0060] (2) Segmentation of beads: The goal of image segmentation is to outline the beads in the image for further analysis. Basic segmentation routines track boundaries such as lines, curves in images that can locate the beads in the image. In accordance with one embodiment, a watershed algorithm in MATLAB is applied to isolate the bead orientation. The watershead algorithm in MATLAB^® requires the images to be black and white images and so the image is converted to black and white image during the portion of the image processing. Because the higher density of black pixels (due to opaque area) correspond to edges of the beads, the watershed transform finds ridgelines in an image and treat the surfaces enclosed by dense pixels as beads. Alternately, a new watershed algorithm in MATLAB^® can be modified to use grayscale images, so that the segmentation and final decoding of the beads are done at higher accuracy. Because LITABs have constant area and therefore each bead is separated and filtered from the image based on their areas.

[0061] (3). Extraction of barcode slits: After segmentation of the beads, each bead is processed separately in order to extract the barcodes. The areas of beads, extracted from the main image, are considered as subimages and are processed one by one. The subimages show the orientation of the beads in random direction with major and minor axes. The angles made by the major axis of the beads with the x- axis of the image are calculated. Extraction of slits from the beads is performed after rotating the major axis of the beads to x-axis. After the rotation of the beads, the borders of the beads are eliminated and the intensity values are averaged along the length (y- axis) of the bars. Though these subimages are 2-D in nature, it carries only transmission intensity information. The intensity plot along the x-axis of the beads shows peaks with two widths (narrow and wide) corresponding to 1 and 0 bits.

[0062] (4). Decoding of bits: In order to decode the barcode, the widths of the transmission intensity peaks are analyzed. A half maximum line is used to calculate the widths of the peaks. In order to extract the binary bit information, five pixels are sufficient to describe the narrow slit ('0') of the beads. The widths are translated to binary bits using a tolerance of 10%. Depending on the rising and falling edges of the intensity peaks in reference to the peaks representing the end edges of the bead, we can identify as most significant bit (MSB) or least significant bit (LSB) of the barcodes. Based on the identification of the two widths, the binary digit can be decoded.

[0063] In accordance with the illustrated embodiment, image processing software consists of the following detailed step-by-step procedure for image decoding:

1. Gray scale conversion and preserve bead image details: The images are read and converted into gray scale image as shown in the FIG 6. Some of the images processing algorithms used in this programming are from MATLAB^® and since they require the images to be black and white images. So during the course of image processing, the processed images are further converted to black and white images when required.

2. Background subtraction to obtain uniform background: The non-uniform optical illumination of the background is eliminated. To get uniform background each image is processed using structuring element and image arithmetic. The image is subtracted from its background and the intensity of the image is adjusted in order to do further processing.

3. Bead image edge detection: The edges detected are shown in FIG 7. This process recognizes and isolates the beads from the background or the rest of the image. A 'sobeF filtering technique is used to suppress all the edge boundaries of the bead and to highlight the slits within each bead.

4. Labeling of individual bead images: Once the beads are separated as individual regions in the image, the regions are labeled (FIG. 8) using a RGB color code or by numbering each bead. Different parameters such as area, angle of orientation, pixel list are calculated for each bead using the region pops algorithm.

5. Alignment transformation of the bead images and representing the digital encoding by intensity plot: To align each microbead to the major axis (FIG. 9), the original gray scale image is taken and converted into a matrix, which contained the pixel values. Pixel index list of the microbeads are obtained from the region properties. The gray scale image is replaced with the pixel index list of each microbead and the rest of the pixels are made zero. Using the angle of orientation of each microbead calculated earlier, each bead is rotated along the major axis.

6. Analyzing the intensity to determine the digital code: The microbead images are displayed by plotting the intensity versus pixel number as shown in FIG.10. Each intensity plot is analyzed to get the barcode in the microbead. A line is drawn parallel to the x-axis and the x values are taken for each intersection of the line with the plot. The line is drawn at the average value of peaks. That means sum of all the points in the peaks divided by the number of points. Then the differences between two consecutive x values are obtained. The alternative values are the widths of the barcodes in each microbead. The direction of the barcode is found by calculating the spacing between the edges of the barcode and the first slit. Longer distance corresponds to the forward direction of reading the barcodes. If a bead appears reversely in the image the barcode bits are reversed. The decoded results are displayed as shown in FIG. 1 1. 7. Segmentation of touching beads: When two beads are touching along its edges, the barcodes are not overlapping and so decoding the beads is possible. But the two beads would be segmented as a single bead. In order to disintegrate such touching beads 'watershed transformation' is utilized using Distance Transform and Watershed lines. Distance Transformation is mapping representation of digital image which supplies each pixel of the image with the distance to the nearest boundary pixel. An Image data may be interpreted as a topographic surface where the gray-level peaks in the gradient of the image represent watersheds. In the segmentation, the slits of these beads are dilated and the distance transform is used to find the watershed lines. The watershed line creation requires the images in black and white and so each image is converted into black and white based on threshold intensity. Rectangular structural elements of the barcode slits are assumed in order to carryout the image dilation. Once the watershed lines are created the edge detected image are superimposed with the watershed lines to get the resultant separated beads. These separated beads though irregular at the edges, the barcodes are intact for decoding.

[0064] Though rectangular shaped beads are addressed in this patent, this also can be extended to curved rectangular shaped beads. That is to say the bead is composed of a rectangle and two semi circles on either ends. One of the semi circles is larger than the other to show MSB and LSB. In order to decode the barcodes, image processing is done to erode the bead except the semicircles. A filter based on area can extract only the MSB semicircle from the beads. This solid semi circle image is reduced to edges image using 'Sobel filter'. The whole image consists of a curve forming semicircle and a straight line. Using Hough transform in MATLAB^® the straight lines are identified. Based on the straight-lines, boxes of lengths of the barcode portion of the beads are constructed towards the direction perpendicular to the straight lines but away from the semicircular regions. Extraction of the barcode is accomplished by plotting the intensity inside the constructed boxes.

[0065] In order to reduce the amount of computational time for the image processing, a number of attempts have been made to improve the algorithm. The image resolution is reduced from lum/pixel to lOum/pixel in order to do the initial processing for segmentation of the beads. When the beads are segmented, the barcodes are extracted using the high resolution image. Another attempt is to convert the image decoding algorithm from MATLAB^® to C and pre- compile the C program before execution. This tremendously improves the speed of execution of the image decoding software. Finally the decoding program is run with a co-processor such as NVIDIA and using a CUDA library to couple the program with the co-processor. This system has as many as 128 co-processors to execute the program in a parallel fashion. [0066] The process and system of the present invention has been described above in terms of functional modules. It is understood that unless otherwise stated to the contrary herein, one or more functions may be integrated in a single physical device or a software module in a software product, or a function may be implemented in separate physical devices or software modules, without departing from the scope and spirit of the present invention. It will be further appreciated that the line between hardware and software is not always sharp.

[0067] It is appreciated that detailed discussion of the actual implementation of each step that comprises the process is not necessary for an enabling understanding of the invention. The actual implementation is well within the routine skill of a programmer and computer engineer, given the disclosure herein of the system attributes, functionality and inter-relationship of the various software and hardware components in the system. A person skilled in the art, applying ordinary skill can practice the present invention without undue experimentation. [0068] While the invention has been described with respect to the described embodiments in accordance therewith, it will be apparent to those skilled in the art that various modifications and improvements may be made without departing from the scope and spirit of the invention. Accordingly, it is to be understood that the invention is not to be limited by the specific illustrated embodiments, but only by the scope of the appended claims.

Claims

CLAIMS:

1. A microbead analytical system, comprising: a support supporting at least one bead, wherein the bead is provided with an indicia representing a digital code; an imaging device obtaining an image of the support and the bead including the indicia; and a decoding system configured to analyze the image obtained by the imaging device to recognize and isolate the bead from the rest of the image, and determining the digital code represented by the indicia provided on the bead.

2. The microbead analytical system as in claim 1, wherein the support supporting a plurality of microbeads, each having an indicia representing a digital code, wherein the image obtained by the imaging device include the plurality of beads, including the indicia on each bead, and wherein the decoding system is configured to analyze the image to recognize and isolate the plurality of beads from one another and from the support in the background, and determining the digital code represented by the indicia provided on each bead.

3. The microbead analytical system as in claim 2, wherein the support comprises a plate having a plurality of wells, and wherein the plurality of beads are support in a well.

4. The microbead analytical system as in claim 2, further comprising a light source, illuminating the plurality of beads for imaging.

5. The microbead analytical system as in claim 4, wherein the light source is positioned to transmit light through the plurality of beads to the imaging device.

6. The microbead analytical system as in claim 4, wherein the light source is positioned to incident light onto the plurality of beads.

7. The microbead analytical system as in claim 6, wherein the beads include a material that produces an optical signal, and wherein the imaging device comprises an optical device that detects the optical signal.

8. The microbead analytical system as in claim 2, wherein the beads each comprises: a first layer; a second layer; and an intermediate layer between the first layer and the second layer, the intermediate layer having an encoded pattern defined thereon, wherein the intermediate layer is partially substantially transmissive and partially substantially opaque to light, representing the digital code corresponding to the bead.

9. The microbead analytical system as in claim 8, wherein the intermediate layer comprises a series of alternating substantially light transmissive sections and substantially light opaque sections defining the encoded pattern.

10. The microbead analytical system as in claim 9, wherein relative positions, widths and/or spacing between the transmissive sections and/or opaque sections represent a binary code.

11. The microbead analytical system as in claim 8, wherein the light transmissive sections are defined by slits through the intermediate layer, and the light opaque sections are defined by a light reflective material and/or a light absorptive material.

12. The microbead analytical system as in claim 11 , wherein the slits comprise slits of a first width and slits of a second width, and wherein the first width represents a "0" and the second width represents a "1" in a binary code.

13. The microbead analytical system as in claim 2, wherein the decoding system is configured to recognize and isolate a particular bead from the rest of the image, and determine the digital code represented by the indicia provided on the bead, by: (1) gray scale conversion to preserve bead image details; (2) background subtraction to obtain uniform background for bead images; (3) bead image edge detection; (4) if bead images are touching, additional filtering process to separate the touching bead images; (5) labeling of individual bead images; (6) alignment transformation of the bead images and representing the digital encoding in each bead image by intensity; and (7) analyzing the intensity to determine the digital code.

14. A method of analyzing microbeads, comprising: supporting a plurality of beads, wherein the beads are provided with an indicia representing a digital code; obtaining an image of the support and the bead including the indicia; and analyzing the image to recognize and isolate the bead from the rest of the image, and determining the digital code represented by the indicia provided on the bead.

15. The method of claim 14, wherein the analyzing step comprises the following steps:

(1) gray scale conversion to preserve bead image details;

(2) background subtraction to obtain uniform background for bead images;

(3) bead image edge detection;

(4) if bead images are touching, additional filtering process to separate the touching bead images;

(5) labeling of individual bead images;

(6) alignment transformation of the bead images and representing the digital encoding in each bead image by intensity; and

(7) analyzing the intensity to determine the digital code.