STATEMENT REGARDING COPYRIGHTED MATERIAL Portions of the disclosure of this patent document contain material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office file or records, but otherwise reserves all copyright rights whatsoever.

BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates in general to assay devices, and in particular to methods and an apparatus for an assay device implemented in conjunction with an optical bio- disc. More specifically, but without restriction to the particular embodiments hereinafter described in accordance with the best mode of practice, this invention relates to methods and an apparatus for multi-use mapping of an optical bio-disc 2. Discussion of the Background Art CDs and DVDs enable large amounts of data to be stored and quickly retrieved. Audio, visual, and computer program data are frequently stored on CDs or DVDs in a digital format. The end appearance of stored visual data is not readily apparent upon visual inspection. Thus, to extract visual information from a disc, a CD or DVD reader collects a stream of data that contains an encoding of the visual image.

The functional aspects of a CD or DVD can be better understood with a review of CDs.

A CD is a fairly simple piece of plastic, about four one-hundredths (4/100) of an inch (1.2 mm) thick. Most of a CD consists of an injection-molded piece of clear

polycarbonate plastic. During manufacturing, this plastic is impressed with microscopic bumps arranged as a single, continuous, extremely long spiral track of data. Once the clear piece of polycarbonate is formed, a thin, reflective aluminum layer is sputtered onto the disc, covering the bumps. Then a thin acrylic layer is sprayed over the aluminum to protect it. The label is then printed onto the acrylic.

A CD has a single spiral track of data, circling from the inside of the disc to the outside. The fact that the spiral track starts at the center means that the CD can be smaller than 4.8 inches (12 cm) if desired. The data tracks are approximately 0.5 microns wide, with 1.6 microns separating one track from the next, and the elongated bumps that make up the track are each 0.5 microns wide, a minimum of 0.83 microns long and 125 nanometers high. Frequently, data is described as encoded in pits on a CD instead of bumps. Usually, the marks appear as pits on the aluminum side, but on the side the laser reads from, they are bumps. However, the scheme can be reversed.

The CD player has the job of finding and reading the data stored on the CD.

Considering how small the bumps are, the CD player is an exceptionally precise piece of equipment. The drive consists of three fundamental components. A drive motor spins the disc and is precisely controlled to rotate, typically, between 200 and 500 rpm depending on which track is being read. A laser and a lens system focus in on and read the bumps. A tracking mechanism moves the laser assembly so that the laser's beam can follow the spiral track. The tracking system has to be able to move the laser at micron resolutions.

Inside the CD player, there is frequently a degree of computer technology involved in forming the data into understandable data blocks and sending them either to the DAC (in the case of an audio CD) or to the computer (in the case of a CD-ROM drive). The fundamental job of the CD player is to focus the laser on the track of bumps. The laser beam passes through the polycarbonate layer, reflects off the aluminum layer and hits an opto-electronic device that detects changes in light. The bumps reflect light differently than the lands (the rest of the aluminum layer), and the opto-electronic sensor detects that change in reflectivity. The electronics in the drive interpret the changes in reflectivity in order to read the bits that make up the bytes.

One difficult task is keeping the laser beam centered on the data track. This centering is the job of the tracking system. The tracking system, as it plays the CD,

has to continually move the laser outward. As the laser moves outward from the center of the disc, the bumps move past the laser faster. This happens because the linear, or tangential, speed of the bumps is equal to the radius times the speed at which the disc is revolving (rpm). Therefore, as the laser moves outward, the spindle motor must slow the speed of the CD. That way, the bumps travel past the laser at a constant speed, and the data comes off the disc at a constant rate.

SUMMARY OF THE INVENTION It is an object of the present invention to overcome limitations in the known art.

The embodiments of the present invention provide methods and an apparatus for an assay device for multi-use mapping of an optical bio-disc. The following embodiments discusses the techniques for mapping available ports of the optical bio- disc. According to one embodiment, a user has to identify a port that has been used when the optical bio-disc is initialized. The above embodiment is necessary when the optical bio-disc is designed for multiple uses during different sessions over extended periods of time. According to another embodiment, the assay device is designed to be ca able to logically map the available ports and provide logic to the software that maps the optical bio-disc.

According to another embodiment, the optical bio-disc drive first interrogates the bio-disc and looks for signs if a port has been violated. According to another embodiment, the disc drive looks for the presence of an analyte on the bio-disc.

According to another embodiment, if an analyte is found on the bio-disc, then the assay device creates a data file that relates available sample areas on the bio-disc to a customer interface. The sample areas are preferably encoded in the bio-disc so the software can identify a known area. The sample area is also preferably labeled so that a user can interrogate a response from the software. According to another embodiment, the logic from the encoded sample area matches the visible labels.

According to another embodiment, used areas on a bio-disc are also provided with a visual response that prevents the user from trying to reuse a used area.

This invention or different aspects thereof may be readily implemented in, adapted to, or employed in combination with the discs, assays, and systems disclosed in the following commonly assigned and co-pending patent applications : U. S. Patent Application Serial No. 09/378,878 entitled"Methods and Apparatus for Analyzing

Operational and Non-operational Data Acquired from Optical Discs"filed August 23, 1999; U. S. Provisional Patent Application Serial No. 60/150,288 entitled"Methods and Apparatus for Optical Disc Data Acquisition Using Physical Synchronization Markers" filed August 23,1999 ; U. S. Patent Application Serial No. 09/421, 870 entitled "Trackable Optical Discs with Concurrently Readable Analyte Material"filed October 26,1999 ; U. S. Patent Application Serial No. 09/643,106 entitled"Methods and Apparatus for Optical Disc Data Acquisition Using Physical Synchronization Markers" filed August 21,2000 ; U. S. Patent Application Serial No. 09/999,274 entitled"Optical Biodiscs with Reflective Layers"filed November 15,2001 ; U. S. Patent Application Serial No. 09/988, 728 entitled"Methods and Apparatus for Detecting and Quantifying Lymphocytes with Optical Biodiscs"filed November 16,2001 ; U. S. Patent Application Serial No. 09/988,850 entitled"Methods and Apparatus for Blood Typing with Optical Bio-discs"filed November 19,2001 ; U. S. Patent Application Serial No. 09/989, 684 entitled"Apparatus and Methods for Separating Agglutinants and Disperse Particles" filed November 20,2001 ; U. S. Patent Application Serial No. 09/997,741 entitled"Dual Bead Assays Including Optical Biodiscs and Methods Relating Thereto"filed November 27,2001 ; U. S. Patent Application Serial No. 09/997,895 entitled"Apparatus and Methods for Separating Components of Particulate Suspension"filed November 30,2001 ; U. S. Patent Application Serial No. 10/005, 313 entitled"Optical Discs for Measuring Analytes"filed December 7,2001 ; U. S. Patent Application-Serial No.

10/006,371 entitled"Methods for Detecting Analytes Using Optical Discs and Optical Disc Readers"filed December 10,2001 ; U. S. Patent Application Serial No.

10/006,620 entitled"Multiple Data Layer Optical Discs for Detecting Analytes"filed December 10,2001 ; U. S. Patent Application Serial No. 10/006, 619 entitled"Optical Disc Assemblies for Performing Assays"filed December 10,2001 ; U. S. Patent Application Serial No. 10/020, 140 entitled"Detection System For Disk-Based Laboratory and Improved Optical Bio-Disc Including Same"filed December 14,2001 ; U. S. Patent Application Serial No. 10/035,836 entitled"Surface Assembly for Immobilizing DNA Capture Probes and Bead-Based Assay Including Optical Bio-Discs and Methods Relating Thereto"filed December 21,2001 ; U. S. Patent Application Serial No. 10/038,297 entitled"Dual Bead Assays Including Covalent Linkages for Improved Specificity and Related Optical Analysis Discs"filed January 4,2002 ; U. S.

Patent Application Serial No. 10/043,688 entitled"Optical Disc Analysis System

Including Related Methods for Biological and Medical Imaging"filed January 10,2002 ; U. S. Provisional Application Serial No. 60/348, 767 entitled"Optical Disc Analysis System Including Related Signal Processing Methods and Software"filed January 14, 2002 U. S. Patent Application Serial No. 10/086,941 entitled"Methods for DNA Conjugation Onto Solid Phase Including Related Optical Biodiscs and Disc Drive Systems"filed February 26,2002 ; U. S. Patent Application Serial No. 10/087, 549 entitled"Methods for Decreasing Non-Specific Binding of Beads in Dual Bead Assays Including Related Optical Biodiscs and Disc Drive Systems"filed February 28,2002 ; U. S. Patent Application Serial No. 10/099,256 entitled"Dual Bead Assays Using Cleavable Spacers and/or Ligation to Improve Specificity and Sensitivity'Including Related Methods and Apparatus"filed March 14,2002 ; U. S. Patent Application Serial No. 10/099,266 entitled"Use of Restriction Enzymes and Other Chemical Methods to Decrease Non-Specific Binding in Dual Bead Assays and Related Bio-Discs, Methods, and System Apparatus for Detecting Medical Targets"also filed March 14,2002 ; U. S.

Patent Application Serial No. 10/121,281 entitled"Multi-Parameter Assays Including Analysis Discs and Methods Relating Thereto"filed April 11,2002 ; U. S. Patent Application Serial No. 10/150, 575 entitled"Variable Sampling Control for Rendering Pixelization of Analysis Results in a Bio-Disc Assembly and Apparatus Relating Thereto"filed May 16,2002 ; U. S. Patent Application Serial No. 10/150,702 entitled "Surface Assembly For Immobilizing DNA Capture Probes in Genetic Assays Using Enzymatic Reactions to Generate Signals in Optical Bio-Discs and Methods Relating Thereto"filed May 16,2002 ; U. S. Patent Application Serial No. 10/194, 418 entitled "Optical Disc System and Related Detecting and Decoding Methods for Analysis of Microscopic Structures"filed July 12,2002 ; U. S. Patent Application Serial No.

10/194,396 entitled"Multi-Purpose Optical Analysis Disc for Conducting Assays and Various Reporting Agents for Use Therewith"also filed July 12,2002 ; U. S. Patent Application Serial No. 10/199,973 entitled"Transmissive Optical Disc Assemblies for Performing Physical Measurements and Methods Relating Thereto"filed July 19, 2002; U. S. Patent Application Serial No. 10/201,591 entitled"Optical Analysis Disc and Related Drive Assembly for Performing Interactive Centrifugation"filed July 22, 2002; U. S. Patent Application Serial No. 10/205,011 entitled"Method and Apparatus for Bonded Fluidic Circuit for Optical Bio-Disc"filed July 24,2002 ; U. S. Patent Application Serial No. 10/205,005 entitled"Magnetic Assisted Detection of Magnetic

Beads Using Optical Disc Drives"also filed July 24,2002 ; U. S. Patent Application Serial No. 10/230,959 entitled"Methods for Qualitative and Quantitative Analysis of Cells and Related Optical Bio-Disc Systems"filed August 29,2002 ; U. S. Patent Application Serial No. 10/233,322 entitled"Capture Layer Assemblies for Cellular Assays Including Related Optical Analysis Discs and Methods"filed August 30,2002 ; U. S. Patent Application Serial No. 10/236,857 entitled"Nuclear Morphology Based Identification and Quantification of White Blood Cell Types Using Optical Bio-Disc Systems"filed September 6,2002 ; U. S. Patent Application Serial No. 10/241,512 entitled"Methods for Differential Cell Counts Including Related Apparatus and Software for Performing Same"filed September 11,2002 ; U. S. Patent Application Serial. No. 10/279,677 entitled"Segmented Area Detector for Biodrive and Methods Relating Thereto"filed October 24,2002 ; U. S. Patent Application Serial No.

10/293,214 entitled"Optical Bio-Discs and Fluidic Circuits for Analysis of Cells and Methods Relating Thereto"filed on November 13,2002 ; U. S. Patent Application Serial No. 10/298,263 entitled"Methods and Apparatus for Blood Typing with Optical Bio- Discs"filed on November 15,2002 ; U. S. Patent Application Serial No. 10/307,263 entitled"Magneto-Optical Bio-Discs and Systems Including Related Methods"filed November 27,2002 ; U. S. Patent Application Serial No. 10/341,326 entitled"Method and Apparatus for Visualizing Data"filed January 13,2003 ; U. S. Patent Application Serial No. 10/345,122 entitled"Methods and Apparatus for Extracting Data From an Optical Analysis Disc"filed on January 14,2003 ; U. S. Patent Application Serial No.

10/347,155 entitled"Optical Discs Including Equi-Radial and/or Spiral Analysis Zones and Related Disc Drive Systems and Methods"filed on January 15,2003 ; U. S. Patent Application Serial No. 10/347,119 entitled"Bio-Safe Dispenser and Optical Analysis Disc Assembly"filed January 17,2003 ; U. S. Patent Application Serial No. 10/348,049 entitled"Multi-Purpose Optical Analysis Disc for Conducting Assays and Related Methods for Attaching Capture Agents"filed on January 21,2003 ; U. S. Patent Application Serial No. 10/348,196 entitled"Processes for Manufacturing Optical Analysis Discs with Molded Microfluidic Structures and Discs Made According Thereto"filed on January 21,2003 ; U. S. Patent Application Serial No. 10/351,604 entitled"Methods for Triggering Through Disc Grooves and Related Optical Analysis Discs and System"filed on January 23,2003 ; U. S. Patent Application Serial No.

10/351,280 entitled"Bio-Safety Features for Optical Analysis Discs and Disc System

Including Same"filed on January 23,2003 ; U. S. Patent Application Serial No.

10/351,244 entitled"Manufacturing Processes for Making Optical Analysis Discs Including Successive Patterning Operations and Optical Discs Thereby Manufactured" filed on January 24,2003 ; U. S. Patent Application Serial No. 10/353,777 entitled "Processes for Manufacturing Optical Analysis Discs with Molded Microfluidic Structures and Discs Made According Thereto"filed on January 27,2003 ; U. S. Patent Application Serial No. 10/353,839 entitled"Method and Apparatus for Logical Triggering"filed on January 28,2003 ; and U. S. Patent Application Serial No.

10/356,666 entitled"Methods For Synthesis of Bio-Active Nanoparticles and Nanocapsules For Use in Optical Bio-Disc Assays and Disc Assembly Including Same"filed January 30,2003. All of these applications are herein incorporated by reference in their entireties. They thus provide background and related disclosure as support hereof as if fully repeated herein.

The above described methods and apparatus according to the present invention as disclosed herein can have one or more advantages which include, but, are not limited to, simple and quick on-disc processing without the necessity of an experienced technician to run the test, small sample volumes, use of inexpensive materials, and use of known optical disc formats and drive manufacturing. These and other features and advantages will be better understood by reference to the following detailed description when taken in conjunction with the accompanying drawing figures and technical examples.

BRIEF DESCRIPTION OF THE DRAWINGS Further objects of the present invention together with additional features contributing thereto and advantages accruing therefrom will be apparent from the following description of the preferred embodiments of the invention which are shown in the accompanying drawing figures with like reference numerals indicating like components throughout, wherein: Fig. 1 is a pictorial representation of a bio-disc system; Fig. 2 is an exploded perspective view of a reflective bio-disc; Fig. 3 is a top plan view of the disc shown in Fig. 2; Fig. 4 is a perspective view of the disc illustrated in Fig. 2 with cut-away sections showing the different layers of the disc;

Fig. 5 is an exploded perspective view of a transmissive bio-disc; Fig. 6 is a perspective view representing the disc shown in Fig. 5 with a cut- away section illustrating the functional aspects of a semi-reflective layer of the disc; Fig. 7 is a graphical representation showing the relationship between thickness and transmission of a thin gold film ; Fig. 8 is a top plan view of the disc shown in Fig. 5; Fig. 9 is a perspective view of the disc illustrated in Fig. 5 with cut-away sections showing the different layers of the disc including the type of semi-reflective layer shown in Fig. 6; Fig. 10 is a perspective and block diagram representation illustrating the system of Fig. 1 in more detail ; Fig. 11 is a partial cross sectional view taken perpendicular to a radius of the reflective optical bio-disc illustrated in Figs. 2,3, and 4 showing a flow channel formed therein; Fig. 12 is a partial cross sectional view taken perpendicular to a radius of the transmissive optical bio-disc illustrated in Figs. 5,8, and 9 showing a flow channel formed therein and a top detector; Fig. 13 is a partial longitudinal cross sectional view of the reflective optical bio- disc shown in Figs. 2,3, and 4 illustrating a wobble groove formed therein; Fig. 14 is a partial longitudinal cross sectional view of the transmissive optical bio-disc illustrated in Figs. 5,8, and 9 showing a wobble groove formed therein and a top detector; Fig. 15 is a view similar to Fig. 11 showing the entire thickness of the reflective disc and the initial refractive property thereof; Fig. 16 is a view similar to Fig. 12 showing the entire thickness of the transmissive disc and the initial refractive property thereof; Fig. 17 is a pictorial graphical representation of the transformation of a sampled analog signal to a corresponding digital signal that is stored as a one-dimensional array; Fig. 18 is a perspective view of an optical disc with an enlarged detailed view of an indicated section showing a captured white blood cell positioned relative to the tracks of the bio-disc yielding a signal-containing beam after interacting with an incident beam;

Fig. 19A is a graphical representation of a white blood cell positioned relative to the tracks of an optical bio-disc; Fig. 19B is a series of signature traces derived from the white blood cell of Fig.

19A ; Fig. 20 is a graphical representation illustrating the relationship between Figs.

20A, 20B, 20C, and 20D; Figs. 20A, 20B, 20C, and 20D, when taken together, form a pictorial graphical representation of transformation of the signature traces from Fig. 19B into digital signals that are stored as one-dimensional arrays and combined into a two- dimensional array for data input; Fig. 21 is a logic flow chart depicting the principal steps for data evaluation according to processing methods and computational algorithms related to the present invention; Fig. 22 is a flowchart illustrating disc initialization according to an embodiment of the present invention; Fig. 23 is a flowchart illustrating a disc program logic according to an embodiment of the present invention; and Fig. 24 is an illustration of a bio-disc with an analyte on its mapping area.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to disc drive systems, optical bio-discs, image processing techniques, counting methods and related software, x, y, and z. Each of these aspects of the present invention is discussed below in further detail.

Drive System and Related Discs Fig. 1 is a perspective view of an optical bio-disc 110 according to the present invention as implemented to conduct the cell counts and differential cell counts disclosed herein. The present optical bio-disc 110 is shown in conjunction with an optical disc drive 112 and a display monitor 114. Further details relating to this type of disc drive and disc analysis system are disclosed in commonly assigned and co- pending U. S. Patent Application Serial No. 10/008, 156 entitled"Disc Drive System and Methods for Use with Bio-discs"filed November 9,2001 and U. S. Patent Application Serial No. 10/043, 688 entitled"Optical Disc Analysis System Including

Related Methods For Biological and Medical Imaging"filed January 10,2002, both of which are herein incorporated by reference.

Fig. 2 is an exploded perspective view of the principal structural elements of one embodiment of the optical bio-disc 110. Fig. 2 is an example of a reflective zone optical bio-disc 110 (hereinafter"reflective disc") that may be used in the present invention. The principal structural elements include a cap portion 116, an adhesive member or channel layer 118, and a substrate 120. The cap portion 116 includes one or more inlet ports 122 and one or more vent ports 124. The cap portion 116 may be formed from polycarbonate and is preferably coated with a reflective surface 146 (Fig.

4) on the bottom thereof as viewed from the perspective of Fig. 2. In the preferred embodiment, trigger marks or markings 126 are included on the surface of the reflective layer 142 (Fig. 4). Trigger markings 126 may include a clear window in all three layers of the bio-disc, an opaque area ; or a reflective or semi-reflective area encoded with information that sends data to a processor 166, as shown Fig. 10, that in turn interacts with the operative functions of the interrogation or incident beam 152, Figs. 6 and 10.

The second element shown in Fig. 2 is an adhesive member or channel layer 118 having fluidic circuits 128 or U-channels formed therein. The fluidic circuits 128 are formed by stamping or cutting the membrane to remove plastic film and form the shapes as indicated. Each of the fluidic circuits 128 includes a flow channel 130 and a return channel 132. Some of the fluidic circuits 128 illustrated in Fig. 2 include a mixing chamber 134. Two different types of mixing chambers 134 are illustrated. The first is a symmetric mixing chamber 136 that is symmetrically formed relative to the flow channel 130. The second is an off-set mixing chamber 138. The off-set mixing chamber 138 is formed to one side of the flow channel 130 as indicated.

The third element illustrated in Fig. 2 is a substrate 120 including target or capture zones 140. The substrate 120 is preferably made of polycarbonate and has a reflective layer 142 deposited on the top thereof, Fig. 4. The target zones 140 are formed by removing the reflective layer 142 in the indicated shape or alternatively in any desired shape. Alternatively, the target zone 140 may be formed by a masking technique that includes masking the target zone 140 area before applying the reflective layer 142. The reflective layer 142 may be formed from a metal such as aluminum or gold.

Fig. 3 is a top plan view of the optical bio-disc 110 illustrated in Fig. 2 with the reflective layer 142 on the cap portion 116 shown as transparent to reveal the fluidic circuits 128, the target zones 140, and trigger markings 126 situated within the disc.

Fig. 4 is an enlarged perspective view of the reflective zone type optical bio- disc 110 according to one embodiment of the present invention. This view includes a portion of the various layers thereof, cut away to illustrate a partial sectional view of each principal layer, substrate, coating, or membrane. Fig. 4 shows the substrate 120 that is coated with the reflective layer 142. An active layer 144 is applied over the reflective layer 142. In the preferred embodiment, the active layer 144 may be formed from polystyrene. Alternatively, polycarbonate, gold, activated glass, modified glass, or modified polystyrene, for example, polystyrene-co-maleic anhydride, may be used. In addition, hydrogels can be used. Alternatively as illustrated in this embodiment, the plastic adhesive member 118 is applied over the active layer 144. The exposed section of the plastic adhesive member 118 illustrates the cut out or stamped U- shaped form that creates the fluidic circuits 128. The final principal structural layer in this reflective zone embodiment of the present bio-disc is the cap portion 116. The cap portion 116 includes the reflective surface 146 on the bottom thereof. The reflective surface 146 may be made from a metal such as aluminum or gold.

Referring now to Fig. 5, there is shown an exploded perspective view of the principal structural elements of a transmissive type of optical bio-disc 110 according to the present invention. The principal structural elements of the transmissive type of optical bio-disc 110 similarly include the cap portion 116, the adhesive or channel member 118, and the substrate 120 layer. The cap portion 116 includes one or more inlet ports 122 and one or more vent ports 124. The cap portion 116 may be formed from a polycarbonate layer. Optional trigger markings 126 may be included on the surface of a thin semi-reflective layer 143, as best illustrated in Figs. 6 and 9. Trigger markings 126 may include a clear window in all three layers of the bio-disc, an opaque area, or a reflective or semi-reflective area encoded with information that sends data to the processor 166, Fig. 10, which in turn interacts with the operative functions of the interrogation beam 152, Figs. 6 and 10.

The second element shown in Fig. 5 is the adhesive member or channel layer 118 having fluidic circuits 128 or U-channels formed therein. The fluidic circuits 128 are formed by stamping or cutting the membrane to remove plastic film and form the

shapes as indicated. Each of the fluidic circuits 128 includes the flow channel 130 and the return channel 132. Some of the fluidic circuits 128 illustrated in Fig. 5 include the mixing chamber 134. Two different types of mixing chambers 134 are illustrated. The first is the symmetric mixing chamber 136 that is symmetrically formed relative to the flow channel 130. The second is the off-set mixing chamber 138. The off-set mixing chamber 138 is formed to one side of the flow channel 130 as indicated.

The third element illustrated in Fig. 5 is the substrate 120 which may include the target or capture zones 140. The substrate 120 is preferably made of polycarbonate and has the thin semi-reflective layer 143 deposited on the top thereof, Fig. 6. The semi-reflective layer 143 associated with the substrate 120 of the disc 110 illustrated in Figs. 5 and 6 is significantly thinner than the reflective layer 142 on the substrate 120 of the reflective disc 110 illustrated in Figs. 2,3 and 4. The thinner semi-reflective layer 143 allows for some transmission of the interrogation beam 152 through the structural layers of the transmissive disc as shown in Figs. 6 and 12. The thin semi-reflective layer 143 may be formed from a metal such as aluminum or gold.

Fig. 6 is an enlarged perspective view of the substrate 120 and semi-reflective layer 143 of the transmissive embodiment of the optical bio-disc 110 illustrated in Fig.

5. The thin semi-reflective layer 143 may be made from a metal such as aluminum or gold. In the preferred embodiment, the thin semi-reflective layer 143 of the transmissive disc illustrated in Figs. 5 and 6 is approximately 100-300 A thick and does not exceed 400 A. This thinner semi-reflective layer 143 allows a portion of the incident or interrogation beam 152 to penetrate and pass through the semi-reflective layer 143 to be detected by a top detector 158, Figs. 10 and 12, while some of the light is reflected or returned back along the incident path. As indicated below, Table 1 presents the reflective and transmissive characteristics of a gold film relative to the thickness of the film. The gold film layer is fully reflective at a thickness greater than 800 A. While the threshold density for transmission of light through the gold film is approximately 400 A.

In addition to Table 1, Fig. 7 provides a graphical representation of the inverse relationship of the reflective and transmissive nature of the thin semi-reflective layer 143 based upon the thickness of the gold. Reflective and transmissive values used in the graph illustrated in Fig. 7 are absolute values.

TABLE 1 Au Film Reflection and Transmission (Absolute Values) Thickness Thickness (nm) Reflectance Transmittance (Angstroms) 0 0 0. 0505 0.9495 50 5 0. 1683 0.7709 100 10 0.3981 0.5169 150 15 0.5873 0.3264 200 20 0.7142 0.2057 250 25 0.7959 0.1314 300 30 0.8488 0.0851 350 35 0. 8836 0. 0557 400 40 0.9067 0.0368 450 45 0.9222 0.0244 500 50 0. 9328 0.0163 550 55 0. 9399 0.0109 600 60 0. 9448 0.0073 650 65 0.9482 0.0049 700 70 0.9505 0.0033 750 75 0.9520 0.0022 800 80 0.9531 0.0015 With reference next to Fig. 8, there is shown a top plan view of the transmissive type optical bio-disc 110 illsutrated in Figs. 5 and 6 with the transparent cap portion 116 revealing the fluidic channels, the trigger markings 126, and the target zones 140 as situated within the disc.

Fig. 9 is an enlarged perspective view of the optical bio-disc 110 according to the transmissive disc embodiment of the present invention. The disc 110 is illustrated with a portion of the various layers thereof cut away to show a partial sectional view of each principal layer, substrate, coating, or membrane. Fig. 9 illustrates a transmissive disc format with the clear cap portion 116, the thin semi-reflective layer 143 on the substrate 120, and trigger markings 126. In this embodiment, trigger markings 126 include opaque material placed on the top portion of the cap. Alternatively, the trigger marking 126 may be formed by clear, non-reflective windows etched on the thin reflective layer 143 of the disc, or any mark that absorbs or does not reflect the signal coming from the trigger detector 160, Fig. 10. Fig. 9 also shows, the target zones 140 formed by marking the designated area in the indicated shape or alternatively in any desired shape. Markings to indicate target zone 140 may be made on the thin semi- reflective layer 143 on the substrate 120 or on the bottom portion of the substrate 120

(under the disc). Alternatively, the target zones 140 may be formed by a masking technique that includes masking the entire thin semi-reflective layer 143 except the target zones 140. In this embodiment, target zones 140 may be created by silk screening ink onto the thin semi-reflective layer 143. In the transmissive disc format illustrated in Figs. 5,8, and 9, the target zones 140 may alternatively be defined by address information encoded on the disc. In this embodiment, target zones 140 do not include a physically discernable edge boundary.

With continuing reference to Fig. 9, an active layer 144 is illustrated as applied over the thin semi-reflective layer 143. In the preferred embodiment, the active layer 144 is a 10 to 200 pm thick layer of 2% polystyrene. Alternatively, polycarbonate, gold, activated glass, modified glass, or modified polystyrene, for example, polystyrene-co- maleic anhyride, may be used. In addition, hydrogels can be used. As illustrated in this embodiment, the plastic adhesive member 118 is applied over the active layer 144. The exposed section of the plastic adhesive member 118 illustrates the cut out or stamped U-shaped form that creates the fluidic circuits 128.

The final principal structural layer in this transmissive embodiment of the present bio-disc 110 is the clear, non-reflective cap portion 116 that includes inlet ports 122 and vent ports 124.

Referring now to Fig. 10, there is a representation in perspective and block diagram illustrating optical components 148, a light source 150 that produces the incident or interrogation beam 152, a return beam 154, and a transmitted beam 156.

In the case of the reflective bio-disc illustrated in Fig. 4, the return beam 154 is reflected from the reflective surface 146 of the cap portion 116 of the optical bio-disc 110. In this reflective embodiment of the present optical bio-disc 110, the return beam 154 is detected and analyzed for the presence of signal elements by a bottom detector 157. In the transmissive bio-disc format, on the other hand, the transmitted beam 156 is detected, by a top detector 158, and is also analyzed for the presence of signal elements. In the transmissive embodiment, a photo detector may be used as a top detector 158.

Fig. 10 also shows a hardware trigger mechanism that includes the trigger markings 126 on the disc and a trigger detector 160. The hardware triggering mechanism is used in both reflective bio-discs (Fig. 4) and transmissive bio-discs (Fig.

9). The triggering mechanism allows the processor 166 to collect data only when the

interrogation beam 152 is on a respective target zone 140. Furthermore, in the transmissive bio-disc system, a software trigger may also be used. The software trigger uses the bottom detector to signal the processor 166 to collect data as soon as the interrogation beam 152 hits the edge of a respective target zone 140. Fig. 10 further illustrates a drive motor 162 and a controller 164 for controlling the rotation of the optical bio-disc 110. Fig. 10 also shows the processor 166 and analyzer 168 implemented in the alternative for processing the return beam 154 and transmitted beam 156 associated the transmissive optical bio-disc.

As shown in Fig. 11, there is presented a partial cross sectional view of the reflective disc embodiment of the optical bio-disc 110 according to the present invention. Fig. 11 illustrates the substrate 120 and the reflective layer 142. As indicated above, the reflective layer 142 may be made from a material such as aluminum, gold or other suitable reflective material. In this embodiment, the top surface of the substrate 120 is smooth. Fig. 11 also shows the active layer 144 applied over the reflective layer 142. As also shown in Fig. 11, the target zone 140 is formed by removing an area or portion of the reflective layer 142 at a desired location or, alternatively, by masking the desired area prior to applying the reflective layer 142.

As further illustrated in Fig. 11, the plastic adhesive member 118 is applied over the active layer 144. Fig. 11 also shows the cap portion 116 and the reflective surface 146 associated therewith. Thus when the cap portion 116 is applied to the plastic adhesive member 118 including the desired cutout shapes, flow channel 130 is thereby formed. As indicated by the arrowheads shown in Fig. 11, the path of the incident beam 152 is initially directed toward the substrate 120 from below the disc 110. The incident beam then focuses at a point proximate the reflective layer 142.

Since this focusing takes place in the target zone 140 where a portion of the reflective layer 142 is absent, the incident continues along a path through the active layer 144 and into the flow channel 130. The incident beam 152 then continues upwardly traversing through the flow channel to eventually fall incident onto the reflective surface 146. At this point, the incident beam 152 is returned or reflected back along the incident path and thereby forms the return beam 154.

Fig. 12 is a partial cross sectional view of the transmissive embodiment of the bio-disc 110 according to the present invention. Fig. 12 illustrates a transmissive disc format with the clear cap portion 116 and the thin semi-reflective layer 143 on the

substrate 120. Fig. 12 also shows the active layer 144 applied over the thin semi- reflective layer 143. In the preferred embodiment, the transmissive disc has the thin semi-reflective layer 143 made from a metal such as aluminum or gold approximately 100-300 Angstroms thick and does not exceed 400 Angstroms. This thin semi- reflective layer 143 allows a portion of the incident or interrogation beam 152, from the light source 150, Fig. 10, to penetrate and pass upwardly through the disc to be detected by a top detector 158, while some of the light is reflected back along the same path as the incident beam but in the opposite direction. In this arrangement, the return or reflected beam 154 is reflected from the semi-reflective layer 143. Thus in this manner, the return beam 154 does not enter into the flow channel 130. The reflected light or return beam 154 may be used for tracking the incident beam 152 on pre-recorded information tracks formed in or on the semi-reflective layer 143 as described in more detail in conjunction with Figs. 13 and 14. In the disc embodiment illustrated in Fig. 12, a physically defined target zone 140 may or may not be present.

Target zone 140 may be created by direct markings made on the thin semi-reflective layer 143 on the substrate 120. These marking may be formed using silk screening or any equivalent method. In the alternative embodiment where no physical indicia are employed to define a target zone (such as, for example, when encoded software addressing is utilized) the flow channel 130 in effect may be employed as a confined target area in which inspection of an investigational feature is conducted.

Fig. 13 is a cross sectional view taken across the tracks of the reflective disc embodiment of the bio-disc 110 according to the present invention. This view is taken longitudinally along a radius and flow channel of the disc. Fig. 13 includes the substrate 120 and the reflective layer 142. In this embodiment, the substrate 120 includes a series of grooves 170. The grooves 170 are in the form of a spiral extending from near the center of the disc toward the outer edge. The grooves 170 are implemented so that the interrogation beam 152 may track along the spiral grooves 170 on the disc. This type of groove 170 is known as a"wobble groove". A bottom portion having undulating or wavy sidewalls forms the groove 170, while a raised or elevated portion separates adjacent grooves 170 in the spiral. The reflective layer 142 applied over the grooves 170 in this embodiment is, as illustrated, conformal in nature.

Fig. 13 also shows the active layer 144 applied over the reflective layer 142. As shown in Fig. 13, the target zone 140 is formed by removing an area or portion of the

reflective layer 142 at a desired location or, alternatively, by masking the desired area prior to applying the reflective layer 142. As further illustrated in Fig. 13, the plastic adhesive member 118 is applied over the active layer 144. Fig. 13 also shows the cap portion 116 and the reflective surface 146 associated therewith. Thus, when the cap portion 116 is applied to the plastic adhesive member 118 including the desired cutout shapes, the flow channel 130 is thereby formed.

Fig. 14 is a cross sectional view taken across the tracks of the transmissive disc embodiment of the bio-disc 110 according to the present invention as described in Fig. 12, for example. This view is taken longitudinally along a radius and flow channel of the disc. Fig. 14 illustrates the substrate 120 and the thin semi-reflective layer 143. This thin semi-reflective layer 143 allows the incident or interrogation beam 152, from the light source 150, to penetrate and pass through the disc to be detected by the top detector 158, while some of the light is reflected back in the form of the return beam 154. The thickness of the thin semi-reflective layer 143 is determined by the minimum amount of reflected light required by the disc reader to maintain its tracking ability. The substrate 120 in this embodiment, like that discussed in Fig. 13, includes the series of grooves 170. The grooves 170 in this embodiment are also preferably in the form of a spiral extending from near the center of the disc toward the outer edge. The grooves 170 are implemented so that the interrogation beam 152 may track along the spiral. Fig. 14 also shows the active layer 144 applied over the thin semi-reflective layer 143. As further illustrated in Fig. 14, the plastic adhesive member or channel layer 118 is applied over the active layer 144. Fig. 14 also shows the cap portion 116 without a reflective surface 146. Thus, when the cap is applied to the plastic adhesive member 118 including the desired cutout shapes, the flow channel 130 is thereby formed and a part of the incident beam 152 is allowed to pass there through substantially unreflected.

Fig. 15 is a view similar to Fig. 11 showing the entire thickness of the reflective disc and the initial refractive property thereof. Fig. 16 is a view similar to Fig. 12 showing the entire thickness of the transmissive disc and the initial refractive property thereof. Grooves 170 are not seen in Figs. 15 and 16 since the sections are cut along the grooves 170. Figs. 15 and 16 show the presence of the narrow flow channel 130 that is situated perpendicular to the grooves 170 in these embodiments. Figs. 13,14, 15, and 16 show the entire thickness of the respective reflective and transmissive

discs. In these figures, the incident beam 152 is illustrated initially interacting with the substrate 120 which has refractive properties that change the path of the incident beam as illustrated to provide focusing of the beam 152 on the reflective layer 142 or the thin semi-reflective layer 143.

Counting Methods and Related Software By way of illustrative background, a number of methods and related algorithms for white blood cell counting using optical disc data are herein discussed in further detail. These methods and related algorithms are not limited to counting white blood cells, but may be readily applied to conducting counts of any type of particulate matter including, but not limited to, red blood cells, white blood cells, beads, and any other objects, both biological and non-biological, that produce similar optical signatures that can be detected by an optical reader.

For the purposes of illustration, the following description of the methods and algorithms related to the present invention as described with reference to Figs. 17-21, are directed to cell counting. With some modifications, these methods and algorithms can be applied to counting other types of objects similar in size to cells. The data evaluation aspects of the cell counting methods and algorithms are described generally herein to provide related background for the methods and apparatus of the present invention. Methods and algorithms for capturing and processing investigational data from the optical bio-disc has general broad applicability and has been disclosed in further detail in commonly assigned U. S. Provisional Application No.

60/291,233 entitled"Variable Sampling Control For Rendering Pixelation of Analysis Results In Optical Bio-Disc Assembly And Apparatus Relating Thereto"filed May 16, 2001 which is herein incorporated by reference and the above incorporated U. S.

Provisional Application No. 60/404,921 entitled"Methods For Differential Cell Counts Including Related Apparatus And Software For Performing Same". In the following discussion, the basic scheme of the methods and algorithms with a brief explanation is presented. As illustrated in Fig. 10, information concerning attributes of the biological test sample is retrieved from the optical bio-disc 110 in the form of a beam of electromagnetic radiation that has been modified or modulated by interaction with the test sample. In the case of the reflective optical bio-disc discussed in conjunction with Figs. 2,3, 4,11, 13, and 15, the return beam 154 carries the information about the

biological sample. As discussed above, such information about the biological sample is contained in the return beam essentially only when the incident beam is within the flow channel 130 or target zones 140 and thus in contact with the sample. In the reflective embodiment of the bio-disc 110, the return beam 154 may also carry information encoded in or on the reflective layer 142 or otherwise encoded in the wobble grooves 170 illustrated in Figs. 13 and 14. As would be apparent to one of skill in the art, pre-recorded information is contained in the return beam 154 of the reflective disc with target zones, only when the corresponding incident beam is in contact with the reflective layer 142. Such information is not contained in the return beam 154 when the incident beam 152 is in an area where the information bearing reflective layer 142 has been removed or is otherwise absent. In the case of the transmissive optical bio-disc discussed in conjunction with Figs. 5,6, 8,9, 12,14, and 16, the transmitted beam 156 carries the information about the biological sample.

With continuing reference to Fig. 10, the information about the biological test sample, whether it is obtained from the return beam 154 of the reflective disc or the transmitted beam 156 of the transmissive disc, is directed to a processor 166 for signal processing. This processing involves transformation of the analog signal detected by the bottom detector 157 (reflective disc) or the top detector 158 (transmissive disc) to a discrete digital form.

Referring next to Fig. 17, the signal transformation involves sampling the analog signal 210 at fixed time intervals 212, and encoding the corresponding instantaneous analog amplitude 214 of the signal as a discrete binary integer 216.

Sampling is started at some start time 218 and stopped at some end time 220. The two common values associated with any analog-to-digital conversion process are sampling frequency and bit depth. The sampling frequency, also called the sampling rate, is the number of samples taken per unit time. A higher sampling frequency yields a smaller time interval 212 between consecutive samples, which results in a higher fidelity of the digital signal 222 compared to the original analog signal 210. Bit depth is the number of bits used in each sample point to encode the sampled amplitude 214 of the analog signal 210. The greater the bit depth, the better the binary integer 216 will approximate the original analog amplitude 214. In the present embodiment, the sampling rate is 8 MHz with a bit depth of 12 bits per sample, allowing an integer sample range of 0 to 4095 (0 to (2n-1), where n is the bit depth.

This combination may change to accommodate the particular accuracy necessary in other embodiments. By way of example and not limitation, it may be desirable to increase sampling frequency in embodiments involving methods for counting beads, which are generally smaller than cells. The sampled data is then sent to processor 166 for analog-to-digital transformation.

During the analog-to-digital transformation, each consecutive sample point 224 along the laser path is stored consecutively on disc or in memory as a one- dimensional array 226. Each consecutive track contributes an independent one- dimensional array, which yields a two-dimensional array 228 (Fig. 20A) that is analogous to an image.

Fig. 18 is a perspective view of an optical bio-disc 110 of the present invention with an enlarged detailed perspective view of the section indicated showing a captured white blood cell 230 positioned relative to the tracks 232 of the optical bio-disc. The white blood cell 230 is used herein for illustrative purposes only. As indicated above, other objects or investigational features such as beads or agglutinated matter may be utilized herewith. As shown, the interaction of incident beam 152 with white blood cell 230 yields a signal-containing beam, either in the form of a return beam 154 of the reflective disc or a transmitted beam 156 of the transmissive disc, which is detected by either of detectors 157 or 158.

Fig. 19A is another graphical representation of the white blood cell 230 positioned relative to the tracks 232 of the optical bio-disc 110 shown in Fig. 18. As shown in Figs. 18 and 19A, the white blood cell 230 covers approximately four tracks A, B, C, and D. Fig. 19B shows a series of signature traces derived from the white blood cell 210 of Figs. 19 and 19A. As indicated in Fig. 19B, the detection system provides four analogue signals A, B, C, and D corresponding to tracks A, B, C, and D.

As further shown in Fig. 19B, each of the analogue signals A, B, C, and D carries specific information about the white blood cell 230. Thus as illustrated, a scan over a white blood cell 230 yields distinct perturbations of the incident beam that can be detected and processed. The analog signature traces (signals) 210 are then directed to processor 166 for transformation to an analogous digital signal 222 as shown in Figs. 20A and 20C as discussed in further detail below.

Fig. 20 is a graphical representation illustrating the relationship between Figs.

20A, 20B, 20C, and 20D. Figs. 20A, 20B, 20C, and 20D are pictorial graphical

representations of transformation of the signature traces from Fig. 19B into digital signals 222 that are stored as one-dimensional arrays 226 and combined into a two- dimensional array 228 for data input 244.

With particular reference now to Fig. 20A, there is shown sampled analog signals 210 from tracks A and B of the optical bio-disc shown in Figs. 18 and 19A.

Processor 166 then encodes the corresponding instantaneous analog amplitude 214 of the analog signal 210 as a discrete binary integer 216 (see Fig. 17). The resulting series of data points is the digital signal 222 that is analogous to the sampled analog signal 210.

Referring next to Fig. 20B, digital signal 222 from tracks A and B (Fig. 20A) is stored as an independent one-dimensional memory array 226. Each consecutive track contributes a corresponding one-dimensional array, which when combined with the previous one-dimensional arrays, yields a two-dimensional array 228 that is analogous to an image. The digital data is then stored in memory or on disc as a two- dimensional array 228 of sample points 224 (Fig. 17) that represent the relative intensity of the return beam 154 or transmitted beam 156 (Fig. 18) at a particular point in the sample area. The two-dimensional array is then stored in memory or on disc in the form of a raw file or image file 240 as represented in Fig. 20B. The data stored in the image file 240 is then retrieved 242 to memory and used as data input 244 to analyzer 168 shown in Fig. 10.

Fig. 20C shows sampled analog signals 210 from tracks C and D of the optical bio-disc shown in Figs. 18 and 19A. Processor 166 then encodes the corresponding instantaneous analog amplitude 214 of the analog signal 210 as a discrete binary integer 216 (Fig. 17). The resulting series of data points is the digital signal 222 that is analogous to the sampled analog signal 210.

Referring now to Fig. 20D, digital signal 222 from tracks C and D is stored as an independent one-dimensional memory array 226. Each consecutive track contributes a corresponding one-dimensional array, which when combined with the previous one-dimensional arrays, yields a two-dimensional array 228 that is analogous to an image. As above, the digital data is then stored in memory or on disc as a two- dimensional array 228 of sample points 224 (Fig. 17) that represent the relative intensity of the return beam 154 or transmitted beam 156 (Fig. 18) at a particular point in the sample area. The two-dimensional array is then stored in memory or on disc in

the form of a raw file or image file 240 as shown in Fig. 20B. As indicated above, the data stored in the image file 240 is then retrieved 242 to memory and used as data input 244 to analyzer 168 Fig. 10.

The computational and processing algorithms of the present invention are stored in analyzer 168 (Fig. 10) and applied to the input data 244 to produce useful output results 262 (Fig. 21) that may be displayed on the display monitor 114 (Fig. 10).

With reference now to Fig. 21 there is shown a logic flow chart of the principal steps for data evaluation according to the processing methods and computational algorithms related to the present invention. A first principal step of the present processing method involves receipt of the input data 244. As described above, data evaluation starts with an array of integers in the range of 0 to 4096.

The next principle step 246 is selecting an area of the disc for counting. Once this area is defined, an objective then becomes making an actual count of all white blood cells contained in the defined area. The implementation of step 246 depends on the configuration of the disc and user's options. By way of example and not limitation, embodiments of the invention using discs with windows such as the target zones 140 shown in Figs. 2 and 5, the software recognizes the windows and crops a section thereof for analysis and counting. In one preferred embodiment, such as that illustrated in Fig. 2, the target zones or windows have the shape of 1x2 mm rectangles with a semicircular section on each end thereof. In this embodiment, the software crops a standard rectangle of 1x2 mm area inside a respective window. In an aspect of this embodiment, the reader may take several consecutive sample values to compare the number of cells in several different windows.

In embodiments of the invention using a transmissive disc without windows, as shown in Figs. 5,6, 8, and 9, step 246 may be performed in one of two different manners. The position of the standard rectangle is chosen either by positioning its center relative to a point with fixed coordinates, or by finding reference mark which may be a spot of dark dye. In the case where a reference mark is employed, a dye with a desired contrast is deposited in a specific position on the disc with respect to two clusters of cells. The optical disc reader is then directed to skip to the center of one of the clusters of cells and the standard rectangle is then centered around the selected cluster.

As for the user options mentioned above in regard to step 246, the user may specify a desired sample area shape for cell counting, such as a rectangular area, by direct interaction with mouse selection or otherwise. In the present embodiment of the software, this involves using the mouse to click and drag a rectangle over the desired portion of the optical bio-disc-derived image that is displayed on a monitor 114.

Regardless of the evaluation area selection method, a respective rectangular area is evaluated for counting in the next step 248.

The third principal step in Fig. 21 is step 248, which is directed to background illumination uniformization. This process corrects possible background uniformity fluctuations caused in some hardware configurations. Background illumination uniformization offsets the intensity level of each sample point such that the overall background, or the portion of the image that is not cells, approaches a plane with an arbitrary background value Vbackground. While Vbackground may be decided in many ways, such as taking the average value over the standard rectangular sample area, in the present embodiment, the value is set to 2000. The value V at each point P of the selected rectangular sample area is replaced with the number (Vbackground + (V- average value over the neighborhood of P) ) and truncated, if necessary, to fit the actual possible range of values, which is 0 to 4095 in a preferred embodiment of the present invention. The dimensions of the neighborhood are chosen to be sufficiently larger than the size of a cell and sufficiently smaller than the size of the standard rectangle.

The next step in the flow chart of Fig. 21 is a normalization step 250. In conducting normalization step 250, a linear transform is performed with the data in the standard rectangular sample area so that the average becomes 2000 with a standard deviation of 600. If necessary, the values are truncated to fit the range 0 to 4096. This step 250, as well as the background illumination uniformization step 248, makes the software less sensitive to hardware modifications and tuning. By way of example and not limitation, the signal gain in the detection circuitry, such as top detector 158 (Fig.

18), may change without significantly affecting the resultant cell counts.

As shown in Fig. 21, a filtering step 252 is next performed. For each point P in the standard rectangle, the number of points in the neighborhood of P, with dimensions smaller than indicated in step 248, with values sufficiently distinct from Vbackground is calculated. The points calculated should approximate the size of a cell in

the image. If this number is large enough, the value at P remains as it was; otherwise it is assigned to Vbackground. This filtering operation is performed to remove noise, and in the optimal case only cells remain in the image while the background is uniformly equal Vbackground.

An optional step 254 directed to removing bad components may be performed as indicated in Fig. 21. Defects such as scratches, bubbles, dirt, and other similar irregularities may pass through filtering step 252. These defects may cause cell counting errors either directly or by affecting the overall distribution in the images histogram. Typically, these defects are sufficiently larger in size than cells and can be removed in step 254 as follows. First a binary image with the same dimensions as the selected region is formed. A in the binary image is defined as white, if the value at the corresponding point of the original image is equal to Vbackground, and black otherwise.

Next, connected components of black points are extracted. Then subsequent erosion and expansion are applied to regularize the view of components. And finally, components that are larger than a defined threshold are removed. In one embodiment of this optional step, the component is removed from the original image by assigning the corresponding sample points in the original image with the value Vbackground. The threshold that determines which components constitute countable objects and which are to be removed is a user-defined value. This threshold may also vary depending on the investigational feature being counted i. e. white blood cells, red blood cells, or other biological matter. After optional step 254, steps 248,250, and 252 are preferably repeated.

The next principal processing step shown in Fig. 21 is step 256, which is directed to counting cells by bright centers. The counting step 256 consists of several substeps. The first of these substeps includes performing a convolution. In this convolution substep, an auxiliary array referred to as a convolved picture is formed.

The value of the convolved picture at point P is the result of integration of a picture after filtering in the circular neighborhood of P. More precisely, for one specific embodiment, the function that is integrated, is the function that equals v-2000 when v is greater than 2000 and 0 when v is less than or equal to 2000. The next substep performed in counting step 256 is finding the local maxima of the convolved picture in the neighborhood of a radius about the size of a cell., Next, duplicate local maxima

with the same value in a closed neighborhood of each other are avoided. In the last substep in counting step 256, the remaining local maxima are declared to mark cells.

In some hardware configurations, some cells may appear without bright centers. In these instances, only a dark rim is visible and the following two optional steps 258 and 260 are useful.

Step 258 is directed to removing found cells from the picture. In step 258, the circular region around the center of each found cell is filled with the value 2000 so that the cells with both bright centers and dark rims would not be found twice.

Step 260 is directed to counting additional cells by dark rims. Two transforms are made with the image after step 258. In the first substep of this routine, substep (a), the value v at each point is replaced with (2000-v) and if the result is negative it is replaced with zero. In substep (b), the resulting picture is then convolved with a ring of inner radius R1 and outer radius R2. R1 and R2 are, respectively, the minimal and the maximal expected radius of a cell, the ring being shifted, subsequently, in substep (d) to the left, right, up and down. In substep (c), the results of four shifts are summed. After this transform, the image of a dark rim cell looks like a four petal flower. Finally in substep (d), maxima of the function obtained in substep (c) are found in a manner to that employed in counting step 256. They are declared to mark cells omitted in step 256.

After counting step 256, or after counting step 260 when optionally employed, the last principal step illustrated in Fig. 21 is a results output step 262. The number of cells found in the standard rectangle is displayed on the monitor 114 shown in Figs. 1 and 5, and each cell identified is marked with a cross on the displayed optical bio-disc- derived image.

Additional computer science methodologies and apparatus directed to extracting and visualizing data from bio-discs and/or optical analysis discs are discussed in commonly assigned U. S. Patent Application Serial No. 10/341,326 entitled"Method and Apparatus for Visualizing Data"filed January 13,2003 and U. S.

Patent Application Serial No. 10/345,122 entitled"Methods and Apparatus for Extracting Data From an Optical Analysis Disc"filed on January 14,2003 both of which have been herein incorporated by reference.

Multi-Use Mapping of an Optical Bio-Disc The embodiments of the present invention are methods and an apparatus for an assay device for multi-use mapping of an optical bio-disc. In the following description, numerous specific details are set forth to provide a more thorough description of embodiments of the invention. It will be apparent, however, to one skilled in the art, that the embodiments of the present invention may be practiced without these specific details. In other instances, well known features have not been described in detail so as not to obscure the invention.

Disc Mapping : The present invention provides an independent mapping step when the bio-disc is first initialized. Since the possibility of changing the disc configuration each time the disc is removed from the drive, it is necessary to provide a user with a mapping procedure each time the drive is initialized by the disc.

According to an embodiment of the present invention one way to initialize a disc is to follow a sequence of commands that include a pseudo code on the disc. This code describes all of the possible testing positions on the disc. A manufactured area in each of the sample positions respond to a violation of the sample boundary that defines the potential addition of an analyte to that position. The initial scan of the disc provides a software routine with a map of used and unused areas of the disc.

Pseudo-Code And Mapping : The above embodiment is illustrated in Fig. 22, where at step 100 the pseudo code is included on the disc. At step 110, a check is made to see if there have been any violations of the sample boundary of the disc. If there is a violation, then at step 120 a manufactured area on the disc responds to the violation. If not, then at step 130 the initial scan of the disc generates a software routine with a map of all used and unused areas on the disc.

An example of using pseudo code variables for a mapping of"use"is shown below, where A, B, etc. are the variables along with their Cartesian co-ordinates.

A = (0,1) Top versus bottom detector.

B = (0.. n) Layer number.

C = (OX) Radial position/Range of mapping band (id-od) D = (OX) Number of members in the band.

E = (OX) Interrogation technique Finding A Potential Analvte : According to an embodiment of the present invention, a chemistry is added to the channel that responds to the presence of any

potential analyte. Since the response to the analyte is optical, it can be identified by a detector configuration in the disc drive. The response to the analyte provides a positive or negative use to the disc usage map. The chemistry may also, according to another embodiment of the present invention, react over a period of time to identify that the mapped locations usage has expired, which provides the viability of that position for disc usage.

Disc Program Logic : According to another embodiment of the present invention, a micro-controller performs a target inquiry of the drive using a program run from the bio-disc. Fig. 23 illustrates a disc program logic that includes the above mentioned target inquiry. At step 200, the disc drive performs a target inquiry of the drive using a program run from the disc with the help of a micro-controller. At step 210, a check is made to see if the drive is a Binary Coded Decimal (BCD) drive. If, at step 210 it is found that the drive is a non-BCD drive, then at step 220 there is a video disclaimer and at step 230, the disc is ejected. If, on the other hand, step 210 reveals that the drive is a BCD drive, then at step 240, the program logic performs a command like BTl. inf. At step 250, the pseudo code variables are read from the disc into the system, which is on board (refer to the pseudo-code and mapping section above). At step 260, mapping described in the pseudo-code and mapping section above is performed. At step 270, the scanning results are compared with the pseudo-code to produce a usage map for the disc. Finally, at step 280 an instructional video is displayed to the user. Fig. 24 is an illustration of a bio-disc with an analyte (300) placed at a location on its mapping area (310). The arrows illustrate the use of a program disc logic, like the one explained above to analyze the analyte.

Concluding Statements Thus, methods and an apparatus for an assay device for multi-use mapping of an optical bio-disc is described in conjunction with one or more specific embodiments.

All patents, provisional applications, patent applications, and other publications mentioned in this specification are incorporated herein in their entireties by reference.

While this invention has been described in detail with reference to a certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present optical bio- disclosure that describes the current best mode for practicing the invention, many

modifications and variations would present themselves to those of skill in the art without departing from the scope and spirit of this invention. The scope of the invention is, therefore, indicated by the following claims rather than by the foregoing description. All changes, modifications, and variations coming within the meaning and range of equivalency of the claims are to be considered within their scope.

Furthermore, those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the following claims.