Patent Publication Number: US-2003232427-A1

Title: Optically active substrates for examination of biological materials

Description:
[0001] This application claims priority from U.S. Provisional Application 60/389,554, filed on Jun. 18, 2002 and U.S. Provisional Application 60/463,392, filed on Apr. 16, 2003, which are incorporated by reference. 
    
    
     
       BACKGROUND OF THE INVENTION  
       [0002] The present invention relates to optically examining, detecting, or analyzing biological materials, and particularly relates to examining, detecting or imaging biological polymers located on or near optically active substrates.  
       [0003] Microarray technology enables studying complex biochemical reactions and systems at once instead of studying them individually. The technology provides a massively parallel form of analysis that increases data collection per unit time, decreases the overall time required for analysis, uses smaller sample volumes and reagent volumes and sometimes reduces disposable consumption. Although the initial cost may be high, overall the technology represents considerable savings in the time and costs of associated labor. Microarray technology became a fundamental tool for genomic research. The technology can also be utilized for routine analysis used in clinical diagnostics or for industrial analytical purposes.  
       [0004] Microarrays with extremely large number of features are manufactured by methods described in PCT Application WO 92/10092 or U.S. Pat. Nos. 5,143,854; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,445,934; 5,744,305; 5,800,992; 6,040,138; 6,040,193 all of which are incorporated by reference. The array features usually have dimensions from about 10 microns to one hundred microns. Each feature can include several million DNA molecules. The synthesis area of a wafer may be about 110 mm×110 mm and may include several individual microarray chips. To examine a microarray, an optical scanner, for example, may need to scan approximately 65,536×65,536 pixels, for microarrays having feature sizes of 10 μm to 12.5 μm, and approximately 32,768×32,768 pixels, for microarrays having feature sizes of 20 μm to 25 μm. Alternatively, microarrays can be fabricated by other techniques as described in PCT Application PCT/US99/18438 published as WO 00/09757, which is incorporated by reference.  
       [0005] Microarrays may also be created by microfluidic delivery, as described in PCT Application PCT/US99/00730, published as WO 99/36760, which is incorporated by reference. These microarrays can contain a wide range of biological materials including, plant, animal, human, fungal and bacteria cells; viruses, peptides, antibodies, receptors, and other proteins; cDNA clones, DNA probes, oligonucleotides, polymerase chain reactions (PCR) products, and chemicals. These biological materials are delivered in form of an array of spots to various microarray substrates including chemically treated microscope slides, coverslips, various wafer, plastics, membranes, or gels. The number of deposited spots may be in the range of 100 to 50,000 per microarray, and the diameter of an individual spot may be in the range of 50 μm to 1000 μm, and preferably 100 μm to 250 μm. The volume of each deposited spot is in the range of 10 pL to 10 nL, and preferably 50 pL to 500 pL, where it is difficult to precisely deliver and measure the liquid volumes.  
       [0006] In general, fluorescence microscopy is a relatively inefficient process, wherein the light source-to-detector efficiency is estimated in parts per trillions. There is usually a very low efficiency of the fluorescence conversion. Furthermore, among other limitations, the scanning microscope systems cannot increase the intensity of the illumination by the laser source, because the fluorescent sample would be destroyed; this is known as photo-bleaching. Also, before photo-bleaching takes place, most fluorophores behave in a non-linear and possibly unpredictable manner. Additionally, numerous non-optical constrains come into play such as acceptable scan duration, detector performance, and electronic and image manipulation processes. These factors also affect the signal to noise ratio.  
       [0007] Therefore, there is a need for optical examination, detection or imaging systems that are relatively inexpensive and exhibit a high source-to-detector efficiency.  
       SUMMARY OF THE INVENTION  
       [0008] The present invention relates to systems and methods for detecting, examining or analyzing biological materials by optical techniques using novel, optically active substrates or surfaces. The present invention also relates to optically active substrates or surfaces capable of accommodating various probes or microarrays for optically detecting, examining or analyzing biological materials.  
       [0009] An optical system, for use with an optically active substrate or surface, may include a light source, a light detector, and an optical light path system. The use of optically active surface improves detection and examination efficiency of different biological materials. The biological materials include biological polymers such as oligonucleotides to which fluorescently labeled DNA or RNA is bound, polypeptides or other polymer arrays, electrophoresis gels, or other biological specimens such as agonists and antiagonists for cell membrane receptor, toxins and venoms, viral epitopes, hormones (e.g. opioid peptides, steroids, etc.), hormones receptors, peptides, enzimes, enzimes substrates, cofactors, drugs lectins, sugars, oliginucleoteds, nucleic acids, oligosaccharides, proteins, antibodies, cell receptors, monoclonal antibodies and antisera active with specific antigenic determinants (such as viruses, cells and other materials), drugs polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, celular membranes and organellesas well as other similar materials.  
       [0010] The optical light path system may include an objective lens. The objective lens and the optically active substrate or surface may form an optical system arranged for efficient delivery of photons to, and efficient collection of photons from, the examined biological material. The optical properties of the objective lens and the optically active substrate or surface may be cooperatively designed for a range of wavelength or a specific geometrical arrangement. The optical lens has the numerical aperture, focal length, size and other parameters, which at least some are co-operatively fabricated with the optical properties of an array of micro-optical elements located on the optically active surface. The objective lens delivers photons to the optically active surface, and collects photons after being scattered, reflected or re-emitted from the examined biological material and/or after interaction with the optically active surface.  
       [0011] According to one aspect, an optical apparatus for examination of biological material includes a surface comprising a dense array of micro-optical elements located in close proximity to biological material being examined. The apparatus also includes a light source, a light detector and optical elements for providing light from the light source to the biological material and from the biological material to the light detector. The optical elements may include or be associated with a lens (or a lens system or an “immersed lens-like” structure) arranged to provide light to and collect light from the dense array of micro-optical elements and the biological material.  
       [0012] According to another aspect, an optically active substrate is constructed for use with an optical system for examination of biological materials. The optically active substrate includes an optical substrate having a first surface and a second surface opposite to the first surface; and a dense array of micro-optical elements associated with the optical substrate and located in close proximity to biological material being examined. The dense array of micro-optical elements is constructed to have optical properties that increase the number of irradiation photons interacting with the biological material or increase the number of photons provided to an external optical system after interaction of the irradiation photons with the biological material.  
       [0013] According to yet another aspect, an optical method for examination of biological material includes using a surface comprising a dense array of micro-optical elements located in close proximity to biological material being examined. The method also includes emitting light from a light source, and detecting light by a light detector, wherein the light has interacted with optical elements for providing light from the light source to the biological material and from the biological material to the light detector.  
       [0014] Preferred embodiments of the above aspects include one or more of the following:  
       [0015] The micro-optical elements may form an integral part of an optically active substrate or just an optically active surface. The micro-optical elements may have at least one dimension comparable to the wavelength of light emitted from the light source. The micro-optical elements may include micro-structures formed at the substrate&#39;s surface or inside the substrate. The micro-structures may include micro-lenses. The micro-lenses may be formed by micro-cavities formed inside the substrate.  
       [0016] The micro-lenses may be formed by micro-cavities having parallel or semi-parallel groves in the form of half cylinders or quarter cylinders. The micro-cavities may be formed inside the substrate by spherical indentations about one radius in depth or indentations having a depth less than one radius. Alternatively, the micro-cavities may be formed inside the substrate by indentations having a hyperbolic shape. The micro-cavities may include a surface covered by a layer of a high index medium transparent at a wavelength of the light. The high index medium may substantially fill the micro-cavities.  
       [0017] The micro-optical elements may be micro-lenses formed by micro-cavities inside the substrate, wherein the micro-cavities have a radius in the range of 0.1 μm to 10 μm. In general, the micro-cavities have a radius less than 100 μm.  
       [0018] The micro-optical elements may include micro-lenses formed by micro-cavities inside a substrate, wherein the diameter and depth of the micro-cavities define the thickness of a high index coating deposited on the surface. The substrate may be transparent to fluorescent light emitted from fluorophores excited at their emission wavelength. The high index coating may have a thickness of about 10 angstrom to 1000 angstrom depending on the material so that a relatively low coefficient of transmission of the material causes acceptable optical losses.  
       [0019] The substrate may be optically transparent and can be made of various materials including polycarbonate, Mylar®), PMMA®, Plexiglas®, or a plastic having an index of refraction about 1.57. Alternatively, the substrate may include functionalized glass or other material and the optically active surface may be fabricated, deposited or attached to the substrate.  
       [0020] Additional aspects of the systems and methods for detecting, examining or analyzing biological materials are described in the related PCT Application PCT/US01/20177 filed on Jun. 25, 2001, which is incorporated by reference.  
       [0021] Additional aspects of the systems and methods for detecting, examining or analyzing biological materials are described below. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0022]FIG. 1 is a schematic illustration of a confocal optical scanning and imaging system for examination of biological material.  
     [0023]FIG. 2 is a schematic illustration of a CCD wide field optical imaging system for examination of biological material.  
     [0024]FIG. 2A is a simplified graphical representation of an optical scanning system.  
     [0025]FIG. 2B is a perspective view of a scanning arm portion of the scanner shown in FIG. 2A.  
     [0026]FIG. 2C is a top planar view of the scanning arm shown in FIG. 2B.  
     [0027]FIG. 3 illustrates a cross-sectional view of a reflective, optically active substrate.  
     [0028]FIG. 3A is an enlarged view of biological material located on a surface of the optically active substrate shown in FIG. 3.  
     [0029]FIG. 3B illustrates a cross-sectional view of another embodiment a reflective, optically active substrate.  
     [0030]FIG. 4 illustrates a cross-sectional view of yet another embodiment a reflective, optically active substrate.  
     [0031]FIG. 4A is an enlarged view of a micro-optical element created in the optically active substrate shown in FIG. 4.  
     [0032]FIG. 4B is a top view of the optically active substrate shown in FIG. 4 including semi cylindrical grooves.  
     [0033]FIG. 4C is a top view the optically active substrate shown in FIG. 4 including spherical elements. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
     [0034]FIG. 1 illustrates one embodiment of a confocal optical scanning and imaging system for examination of biological material located on, or near, an optically active substrate shown in FIG. 3. The optically active substrate forms an important part of the optical system for increasing the detected optical signal.  
     [0035] Referring to FIG. 1, optical system  10  includes a light source  12 , an entrance _aperture  14 , a lens  16 , a dichroic mirror  20 , an objective lens  24 , a two or three axis translation table  28 , a lens  32 , an exit pinhole  34 , a band pass filter or a rejection filter  31  and a detector  36 . In the following embodiment, optical system  10  is arranged for the detection of fluorescent light; however, optical system  10  may also be arranged for the detection of scattered or transmitted light at the irradiation wavelength.  
     [0036] Light source  12  emits an excitation light beam  15 , and dichroic mirror  20  directs the excitation light toward objective lens  24 . Objective lens  24  focuses light onto a pixel (A) located on or near an optically active substrate  26 . The irradiation beam is in the range of about 2 μm to about 10 μm. Fluorescent light emitted from pixel A is collected by objective lens  24  and transmitted through dichroic mirror  20 , over a light path  30 , toward and trough band pass or rejection filter  31  and to light detector  36 . The arrangement of a aperture and pinholes  14  and  34 , and lenses  16  and  32  provides fluorescent light from a selected depth (in the Z direction) of pixel A to detector  36 , while light emitted from other depths in the Z direction is substantially blocked and doesn&#39;t pass through pinhole  34 . This spatial filter improves the signal-to-noise ratio. The size of exit pinhole  34  is optimized to improve the resolving power of optical system  10 .  
     [0037] Light source  12  is constructed to emit light of a wavelength capable of exciting fluorophores associated with the examined biological tissue. For example, light source  12  includes a gas lasers or diodes lasers or LEDs that emits simultaneously or sequentially  — 473 or 488 or 490 nm as well as diodes that emit at 532 nm as well as diodes that emit at 638 nm or at 745 light directed to epi-optically active substrate  26  by dichroic mirror  20 . For example, the excitation light of 488 nm irradiates a pixel on the surface of substrate  26 , and excites fluorophores that emit fluorescent light, for example, in the range 515 nm to 595 nm. Various types of fluorophores (and their corresponding absorption maxima) are Fluorescein (488 nm), Dichloro-fluorescein (525 nm), Hexachloro-fluorescein (529 nm), Tetramethylrhodamine (550 nm), Rhodamine X (575 nm), Cy3™ (550 nm), Cy5™ (650 nm), Cy7™ (750 nm), and IRD40 (785 nm). Detector  36  with suitable band pass or rejection filters, detects the fluorescent light emitted from the pixel of optically active substrate  26 . Preferably, objective lens  24  has a relatively large numerical aperture (at least a numerical aperture of 0.1, but preferably, a numerical aperture above 0.5, and more preferably 0.6 or 0.7). Optical system  10  can collect optical data over an array of pixels by displacing epi-optically active substrate  26  in the X and Y directions using a translation table  28 .  
     [0038] In general, light source  12  may be a light emitting diode, or a lamp with a filter, but preferably a laser (such as an argon laser, a helium-neon laser, a diode laser, a dye laser, a titanium sapphire laser, a frequency-doubled diode pumped Nd:YAG laser, or a krypton laser). Typically, the excitation source illuminates the sample with an excitation wavelength that is within the visible spectrum, but other wavelengths (i.e., near ultraviolet or near infrared spectrum) may be used depending on the type of markers and/or samples or detection methods. Preferably, the angle of the irradiation is well controlled with respect to the irradiated surface, wherein the alignment provides an aligned beam within the anglular range of a few hundred microradians, and preferably within 100 microradian, in order to launch an evanescent wave as described below. Light detector  36  may be a photomultiplier (PMT), a diode, or another photodetector.  
     [0039]FIG. 3 illustrates one embodiment of an epi-optically active substrate  26  with biological material on a proximal side relative to irradiation beam  25 . Epi-optically active substrate  26  includes an optical plate  80  having an optically active surface  83  with a dense array of micro-optical elements  84 . The dense array of micro-optical elements  84  has optical properties designed and fabricated to increase the number of photons interacting with the biological material and increase the number of photons collected by lens  24  after interaction with biological material  82 . Specifically, micro-optical elements  84  are designed to excite evanescent field used for probing biological material  82  located near the optically active surface, as shown in FIG. 3A. The evanescent field excites fluorescent emission from flourophores, associated with biological material  82 , and the fluorescent light is collected by lens  24  and provided to detector  36 , as described above.  
     [0040]FIG. 2 schematically illustrates another embodiment of an optical imaging system constructed and arranged to examine of biological material located on an optically active substrate. Optical system 50 includes a light source array  55 , a lens  57 , band pass filter  59 , a dichroic mirror  60 , a lens  68 , band pass or rejection filter  67  and a detector array  70 . Light source array  55  emits light directed by lens  57  to filter  59 , to dichroic mirror  60 , which in turn directs the emitted light beam toward optically active substrate  26 , shown in FIG. 3.  
     [0041] Preferably, light source array  55  includes an array of laser diodes or micro-lasers that are aligned to provide a parallel and collimated array of beams. The angle of the beams is controlled within a range of a few hundred microradians and preferably within 100 microradian in order to launch (or excite) evanescent radiation as described below.  
     [0042] Preferably, the optical system is arranged to detect fluorescent light emitted from the examined biological material in response to the evanescent wave. For example, light source array  55  includes a two-dimensional array of elements cooperatively arranged to emit a two-dimensional beam of 490 nm light. This excitation light irradiates a large area of optically active substrate  26  and excites fluorescent light over the irradiated area. Specifically, the two-dimensional beam is directed to dichroic mirror  60 , which reflects the light beam to an optical path  62  and enables irradiation of a large area of optically active substrate  26 . As described below, the optical elements provide a highly parallel and collimated array of irradiation beams.  
     [0043] A fluorescently labeled biological material, located over the irradiated area, emits fluorescent light, for example, in the range 515 nm to 595 nm. Lens  68  receives, from dichroic mirror  60 , fluorescent light emitted from the irradiated area of optically active substrate  26 . Rejection filter or band pass filter  67  permits preferably fluorescent radiation to progress toward detector array  70 . Detector array  70  detects the fluorescent light over a two-dimensional detection area. The detected signal is digitized and stored individually for each pixel in a memory.  
     [0044] According to another embodiment, the optical system is constructed to probe a one-dimensional area of optically active substrate  26  at a time. Specifically, optical system 50 includes light source array  55  with a one-dimensional array of elements arranged to emit a one-dimensional beam of excitation light (i.e., a strip or ribbon of excitation light) and an excitation band pass filter  57 . Optically active substrate may be located on a linear stage and translated under linear light source and detector. The optical system may include a mirror, or other optical elements, arranged to provide the fluorescent light emitted from one line of the examined biological material to a one-dimensional detector array  70 . The optical system may optionally include a slit located before the one-dimensional detector array for confocal detection as well as suitable band pass or rejection filters.  
     [0045] In general, light source array  55  may include a one-dimensional or two-dimensional array of micro-lasers, laser diodes, or possibly light emitting diodes (LEDs) wherein each source element may include a separate lens or a similar optical element. Detector array  70  may include an array of photomultiplier tubes (PMTs) or avalanche photodiodes (APDs), or an integrating or non-integrating array of charge coupled devices (CCD). More expensive optical systems 50 may use, as an image detector, a scientific-grade cooled CCD camera or a cooled CCD with an image intensifier, which usually exhibits an excellent geometrical and photometric linearity, a wide dynamic range and good photon detection efficiency.  
     [0046] Optionally, detector array  70  can convert the collected light into video-level electrical signals for display on a video monitor. Optical system 50 may include video output peripheral devices, including a computer frame grabber for digitizing the image information and for storing it in a computer memory.  
     [0047] There are other optical systems suitable for use with epi-optically active substrates  26 . These systems may include various light-detectors employing photodiodes, charge-coupled devices, photomultiplier tubes, or similar devices to register the collected emission beams. For example, a scanning system for use with a fluorescently labeled target is described in U.S. Pat. No. 5,143,854, hereby incorporated by reference in its entirety for all purposes. Other embodiments of the scanners or scanning systems are described in U.S. Pat. Nos. 5,578,832, 5,631,734, 5,834,758, 5,936,324, 5,981,956, 6,025,601, 6,141,096, 6,185,030, 6,201,639, 6,218,803, and 6,252,236; in PCT Application PCT/US99/06097 (published as WO99/47964); and in from U.S. patent applications Ser. Nos. 09/682,071; 09/682,074; and 09/682,076 all of which were filed on Jul. 17, 2001, each of the above-listed patent documents is hereby incorporated by reference in their entireties for all purposes.  
     [0048]FIG. 2A is a simplified graphical representation of selected components of an illustrative type of a scanner  400  suitable for scanning hybridized spotted arrays deposited on epi-optically active substrates  26  (i.e., in this example, spotted arrays are after the hybridization process described in PCT Application PCT/US01/26297, which is incorporated by reference). These illustrative components, which will be understood to be non-limiting and not exhaustive, are referred to collectively for convenience as scanner optics and detectors  401 . Scanner optics and detectors  401  include excitation sources  420 A and  420 B (collectively referred to as excitation sources  420 ). Any number of one or more excitation sources  420  may be used in alternative embodiments. In the present example, sources  420  are lasers; in particular, source  420 A is a diode laser producing red laser light having a wavelength of 635 nanometers and, source  420 B is a doubled YAG laser producing green laser light having a wavelength of 532 nanometers. Further references herein to sources  420  generally will assume for illustrative purposes that they are lasers, but, as noted, other types of sources, e.g., x-ray sources, may be used in other implementations.  
     [0049] There are other optical systems suitable for use with epi-optically active substrates  26 . A typical optical employs optical and other elements to provide an excitation beam, such as from a laser, and to selectively collect the emission beams. These systems may include various light-detectors employing photodiodes, charge-coupled devices, photomultiplier tubes, or similar devices to register the collected emission beams. For example, a scanning system for use with a fluorescently labeled target is described in U.S. Pat. No. 5,143,854, hereby incorporated by reference in its entirety for all purposes. Other scanners or scanning systems are described in U.S. Pat. Nos. 5,578,832, 5,631,734, 5,834,758, 5,936,324, 5,981,956, 6,025,601, 6,141,096, 6,185,030, 6,201,639, 6,218,803, and 6,252,236; in PCT Application PCT/US99/06097 (published as WO99/47964); and in from U.S. patent applications Ser. Nos. 09/682,071; 09/682,074; and 09/682,076 all of which were filed on Jul. 17, 2001, each of these patent documents is hereby incorporated by reference in their entireties for all purposes.  
     [0050] Sources  420 A and  420 B may alternate in generating their respective excitation beams  435 A and  435 B between successive scans, groups of successive scans, or between full scans of an array. Alternatively, both of sources  420  may be operational at the same time. For clarity, excitation beams  435 A and  435 B are shown as distinct from each other in FIG. 4. However, in practice, turning mirror  424  and/or other optical elements (not shown) typically are adjusted to provide that these beams follow the same path.  
     [0051] Scanner optics and detectors  400  also includes excitation filters  425 A and  425 B that optically filter beams from excitation sources  420 A and  420 B, respectively. The filtered excitation beams from sources  420 A and  420 B may be combined in accordance with any of a variety of known techniques. For example, one or more mirrors, such as turning mirror  424 , may be used to direct filtered beam from source  420 A through beam combiner  430 . The filtered beam from source  420 B is directed at an angle incident upon beam combiner  430  such that the beams combine in accordance with optical properties techniques well known to those of ordinary skill in the relevant art. Most of combined excitation beams  435  are reflected by dichroic mirror  436  and thence directed to periscope mirror  438  of the illustrative example. However, dichroic mirror  436  has characteristics selected so that portions of beams  435 A and  435 B, referred to respectively as partial excitation beams  437 A and  437 B and collectively as beams  437 , pass through it so that they may be detected by excitation detector  410 , thereby producing excitation signal  494 .  
     [0052] In the illustrated example, excitation beams  435  are directed via periscope mirror  438  and arm end turning mirror  442  to an objective lens  445 . As shown in FIGS. 5A and 5B, lens  445  in the illustrated implementation is a small, light-weight lens located on the end of an arm that is driven by a galvanometer around an axis perpendicular to the plane represented by galvo rotation  449  shown in FIG. 4. Objective lens  445  thus, in the present example, moves in arcs over hybridized spotted arrays  132  disposed on slide  379 . Flourophores in hybridized probe-target pairs of the arrays that have been excited by beams  435  emit emission beams  452  (beam  452 A in response to excitation beam  435 A, and beam  452 B in response to excitation beam  435 B) at characteristic wavelengths in accordance with well-known principles. Emission beams  452  in the illustrated example follows the reverse path as described with respect to excitation beams  435  until reaching dichroic mirror  436 . In accordance with well-known techniques and principles, the characteristics of mirror  436  are selected so that beams  452  (or portions of them) pass through the mirror rather than being reflected.  
     [0053] In the illustrated implementation, filter wheel  460  is provided to filter out spectral components of emission beams  452  that are outside of the emission band of the fluorophore, thereby providing filtered beams  454 . The emission band is determined by the characteristic emission frequencies of those fluorophores that are responsive to the frequencies of excitation beams  435 . In accordance with techniques well known to those of ordinary skill in the relevant arts, including that of confocal microscopy, filtered beams  454  may be focused by various optical elements such as lens  465  and also passed through illustrative pinhole  467  or other element to limit the depth of field, and thence impinges upon emission detector  415 .  
     [0054] Emission detector  415  may be a silicon detector for providing an electrical signal representative of detected light, or it may be a photodiode, a charge-coupled device, a photomultiplier tube, or any other detection device that is now available or that may be developed in the future for providing a signal indicative of detected light. For convenience of illustration, detector  415  will hereafter be assumed to be a photomultiplier tube (PMT). Detector  415  thus generates emission signal  492  that represents numbers of photons detected from filtered emission beam  454 .  
     [0055]FIG. 2B is a perspective view of a simplified representation of the scanning arm portion of scanner optics and detectors  401 . Arm  500  moves in arcs around axis  510 , which is perpendicular to the plane of galvo rotation  449 . A position transducer  515  is associated with galvanometer  515  that, in the illustrated implementation, moves arm  500  in bi-directional arcs. Transducer  515 , in accordance with any of a variety of known techniques, provides an electrical signal indicative of the radial position of arm  500 . Certain non-limiting implementations of position transducers for galvanometer-driven scanners are described in U.S. Pat. No. 6,218,803, which is hereby incorporated by reference in its entirety for all purposes. The signal from transducer  515  is provided in the illustrated implementation to a user computer so that clock pulses may be provided for digital sampling of emission signal  492  when arm  500  is in certain positions along its scanning arc, as is described in detail in PCT Application PCT/US01/26297, which is incorporated by reference.  
     [0056] Arm  500  is shown in alternative positions  500 ′ and  500 ″ as it moves back and forth in scanning arcs about axis  510 . Excitation beams  435  pass through objective lens  445  on the end of arm  500  and excite fluorophore labels on targets hybridized to certain of probes  370  in arrays  132  disposed on slide  333 , as described above. The arcuate path of excitation beams  435  is schematically shown for illustrative purposes as path  550 . Emission beams  452  pass up through objective lens  445  as noted above. Slide  333  of this example is disposed on translation stage  542  that is moved in what is referred to herein as the “y” direction  544  so that arcuate path  550  repeatedly crosses the plane of arrays  132 .  
     [0057]FIG. 2C is a top planar view of arm  500  with objective lens  445  scanning arrays  132  as translation stage  542  is moved under path  550 . As shown in FIG. 5B, arcuate path  550  of this example is such that arm  500  has a radial displacement of θ in each direction from an axis parallel to direction  544 . What is referred to herein as the “x” direction, perpendicular to y-direction  544 , is shown in FIG. 2C as direction  543 . Further details of confocal, galvanometer-driven, arcuate, laser scanning instruments suitable for detecting fluorescent emissions are provided in PCT Application PCT/US99/06097 (published as WO99/47964) and in U.S. Pat. Nos. 6,185,030 and 6,201,639, all of which have been incorporated by reference above. It will be understood that although a galvanometer-driven, arcuate, scanner is described in this illustrative implementation, many other designs are possible, such as the voice-coil-driven scanner described in U.S. patent application, Ser. No. 09/383,986, hereby incorporated herein by reference in its entirety for all purposes.  
     [0058] Referring again to FIG. 3, epi-optically active substrate  26  includes a unique array of micro-optical elements  84  with optical properties designed to increase the number of photons interacting with biological material  82  and increase the number of photons collected by lens  24  after interaction with biological material  82 . As shown in FIG. 3, micro-optical elements  84  may be formed by microelement structures  86 , coated with thin, highly reflective layer  87 , and an optical layer  88  having a refractive index greater than the refractive index of surface layers  82  or  90 . Optical layer  88  has a surface  92 , which includes a surface chemistry binding layer or a ligand  90  arranged to immobilize biological material  82 . Reflective layer  87  is made of, for example, a single metal layer, such as gold or aluminum, or several metal layers such, as chromium and gold. Reflective layer  87  is relatively thin, having less than 1000 Å, and preferably less than 200 Å. Optical layer  88  may be created by vapor deposition, sputtering, chemical deposition, or electrochemical deposition. Various methods can be used to form a substantially planar surface  92 .  
     [0059] Microelement structures  86  may be formed by microgrooves (having, for example, a triangular cross-section) or cavities having micro-cylindrical, micro-spherical shape, or micro conical section formed, for example, by ellipsoids, paraboloids or hyperboloids. (Alternatively, the microgrooves may have a partially triangular cross-section and partially cylindrical or similar cross-section.) In general, microelement structures  86  and reflecting coating  87  have a shape and surface conditions selected for directing light to a surface  90  at critical angle such that an evanescence wave is created and possibly sustained in resonant mode such as to couple with fluorescent labels attached to biological material  82 .  
     [0060] Referring still to FIG. 3, microelement structures  86  include triangularly shaped features designed to reflect light beam  25  toward surface  92  to that most of the reflected light arrives at surface  92  generally at the critical angle. In this embodiment, the triangular features are designed to have approximately symmetrical sides  86  (i.e., approximately the same angle and length). Surface  92  forms a boundary between a material with a high index of refraction (n 2 ) and another material with a lower index (n 1 ). At the critical angle θc (sin θc=n 2 /n 1 ) total internal reflection occurs, which excites evanescent field across the interface, having most of the energy contained within less than the wavelength of light. The penetration of the excited evanescent field depends on the interface and the incident angle of the reflected radiation. The evanescent field interacts with biological material  82  located close to the interface. As shown in FIG. 3A, for example, if biological material  82  includes fluorophores, the evanescent field excites fluorescent radiation that is collected by lens  24 .  
     [0061] In short, light beam  25  first interacts with semi transparent biological layer  82 , and enters high index region  88  at near normal incidence, with little diffraction before arriving at reflective layer  87 . Reflective layer  87  is arranged to direct the reflected light generally at an angle to launch evanescent wave  83 . Microelements  84  are shaped to reflect light beam  25  away from its incident path and such that preferably, only fluorescent emission from biological material  82  shall be directed toward lens  24 . Furthermore, triangularly shaped features  84  (shown in FIG. 3) are achromatic in their ability to achieve total internal reflection.  
     [0062]FIG. 3B illustrates another embodiment of the epi-optically active substrate. Epi-optically active substrate  26 A includes an array of micro-optical elements  84 A having non-symmetrical shapes designed to generate the evanescent field (at surface  92 ) more optimally for two or more wavelengths. Similarly as optically active substrate  26 , optically active substrate  26 A includes micro-optical elements  84 A formed by microelement structures and optical layer  88  having a high refractive index. Optical layer  88  has surface  92 , which includes binding layer  90  arranged to immobilize biological material  82 , as shown in FIG. 3A.  
     [0063] Micro-optical elements  84 A includes non-symmetrically shaped triangular features designed to have side  86 A shorter than side  86 B, i.e., the two sides have different angles relative to the incoming radiation. Reflective layer  87  includes reflective parts  87 A and  87 B (deposited on respective sides  86 A and  86 B) designed or optimized to reflect two different wavelengths to achieve total internal reflection and form evanescent field at surface  92 .  
     [0064] According to yet another embodiment, epi-optically active substrate  26 A includes an array of micro-optical elements having a distribution of not completely symmetrical shapes having somewhat different angles designed to generate the evanescent field at surface  92  for a range of irradiation wavelengths. This distribution of shapes may also be somewhat random due to the fabrication process.  
     [0065]FIG. 4 illustrates another embodiment of an epi-optically active substrate. Epi-optically active substrate  26 B includes an array of micro-optical elements  100  having cylindrical or spherical shapes distributed over a region or layer  102 . Micro-optical elements  100  include a reflective layer  105  designed to reflect irradiation light toward surface  104  and fluorescent light toward lens  24 . Similarly as optically active substrate  26 , optically active substrate  26 B includes micro-optical elements  100  formed by microelement cavities  106  and an optical layer  108  having a high refractive index. Optical layer  108  has surface  104 , which includes binding layer  110  arranged to immobilize biological material. In this embodiment, the biological material is located on the proximal (near) side with respect to the irradiation beam  25 .  
     [0066] Reflective layer  105  is relatively thin, having less than 1000 Å, and preferably less than 200 Å. Reflective layer  105  is made of, for example, a single metal layer, such as gold or aluminum, or several metal layers such, as chromium and gold. Optical layer  108  may be created by vapor deposition, sputtering, chemical deposition, or electrochemical deposition. The shape of micro-element  100  and shape of outer surface  104  of high index coating  108  cooperate to capture and redirect fluorescence emission from the biological material (as shown in FIG. 4A) such that it may be captured in totality or in part by lens  24  and transmitted to detector  36  (or detector  70 ). Optical features  106  can be manufactured in support  101  by embossing, injection molding, photolithography, or other conventional means. Optical layer  108  is made of a high index material such as TiO 2  or TeO 2  or others.  
     [0067] The size and period of micro-optical elements  100  are preferably such that micro-optical elements  100  within the area illuminated at any defined pixel by beam  25  do not cause excessive artifact, wherein the illuminated area has a radius of about 2 μm to about 10 μm. The radius of the having cylindrical or spherical features  100  is in the range of about 2,000 Å to about 5,000 Å, and optical layer  104  is located at least about 100 Å above the features. Therefore, the period is between about 0.2 μm and about 5.0 μm, and preferably between about 0.35 μm and about 2.0 μm. This may cause diffraction effect as well as scatter effects to induce surface wave creation. The periodic distance between micro elements or micro structure preferably enhances diffraction effects that launches light incident onto it.  
     [0068] Referring to FIGS. 4B and 4C, because the diffraction effect is correlated to the wavelength and angle of incident light, grooves or optical features may incorporate a suitable selection of distances so as to accommodate a selected number of wavelengths. Instead of spherical cavities, micro-optical elements  100  may be formed by parallel or semi-parallel half cylinders, parallel or semi-parallel hyperbolic structures, or other similar microstructures. FIG. 4B illustrates a top view of micro-optical elements  100  formed by semi-parallel half cylindrical features having variable spacing. FIG. 4C illustrates a top view of micro-optical elements  100  formed by of spherical cavities. Furthermore, instead of spherical cavities, micro-optical elements  100  may be formed to have conical shapes, or any conic sections such as formed by ellipses, paraboloids or hyperboloids.  
     [0069] Referring again to FIG. 3, micro-optical elements  84  may be formed by microlens structures  86 , such as spherical cavities with a radius in the range of 100 nm to 100 μm. The diameter and depth of the spherical microlens cavities defines the thickness of the high index layer  88 . Layer  88  may partially or completely fill the cavities. High index coating  88  includes titanium dioxide with an index of refraction of 2.4, gallium phosphate with an index of refraction 3.4, or other medium with suitable index of refraction. High index layer  88  is designed to have a thickness depending on its transmission coefficient so that a relatively low transmission coefficient will cause acceptable optical losses.  
     [0070] The microstructures can again be created in the substrate by forced embossing at a proper temperature, or by casting against a suitably formed negative master (as used when creating CD and DVD discs) or combination of both. As described above, the substrate may include a PMMA®, Plexiglas® or a similar plastic with an index of refraction of about 1.57. The high index coating may include titanium dioxide, Tellurium dioxide, gallium phosphate or other medium with suitable index of refraction in the range of 2 to 4 and preferably an index of refraction between about 2 and 3.5.  
     [0071] According to one embodiment, optical plate  80  (or  101 ) is made of an optically transparent material such as PMMA®, Plexiglas® or a similar plastic. Microelements  84  (or  100 ) can be created in plate  80  (or  101 ) by forced embossing at a proper temperature, or by casting the plate against a suitably formed negative master or a combination of both (as used when creating CD and DVD discs). Alternatively, plate  80  and micro-optical elements  84  could be made of etched glass, quartz or another material. Alternatively, plate  80  includes an optically opaque material such as aluminum or brass and micro-optical elements may be diamond machined on air bearing lathe or milling machines such as Pneumo Precision machines.  
     [0072] Referring still to FIG. 4, after irradiation, optically active substrate  26 B receives, at a surface  110 , the excitation light over light path  25 . The excitation light first illuminates near transparent thin (approximately 20 to 200 Angstroms) biological material and is diffracted inside optically transparent high index layer  108 . The dense array of micro-optical elements  110  includes reflective coating  105  over all micro-features  106 . Reflective coating  105  reflects and concentrates the transmitted photons by increasing the number of photons interacting with fluorophores  90  in the biological material. Furthermore, the dense array of micro-optical elements  100  increases the number of photons of the excited radiation transmitted back to lens  24 , and thus the number of photons collected by lens  24 . Therefore, the dense array of micro-optical elements  100  enables an increase in the signal-to-noise ratio.  
     [0073] Referring to FIG. 1, optical system  10  collects the reflected and fluorescent light by lens  24  and delivers this light over light path  30  and light path  33  to detector  36 . Lens  32  receives fluorescent light transmitted through dichroic mirror  20  over a light path  30  and focuses the light onto pinhole  34  and possibly rejection or band pass filter  31 . Detector  36  detects fluorescent light emitted from a depth of pixel A corresponding to the location of the examined biological material on the proximal surface of optically active substrate  26 B.  
     [0074] In general, a probe is a surface-immobilized molecule that is recognized by a particular target. Examples of probes that can be investigated by this invention include, but are not restricted to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (e.g., opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.  
     [0075] A target is a molecule that has an affinity for a given probe. Targets may be naturally-occurring or manmade molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Targets may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of targets which can be employed by this invention include, but are not restricted to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles. Targets are sometimes referred to in the art as anti-probes. As the term targets is used herein, no difference in meaning is intended. A “probe target pair” is formed when two macromolecules have combined through molecular recognition to form a complex.  
     [0076] Generally, the sample nucleic acid for which sequence information is desired is contacted with the array. This “target” sequence is typically labeled with a detectable group such as a fluorescent moiety, i.e., fluorescein or rhodamine. Following hybridization of the target to the array, one can detect the position on the array to which the target sequence binds by scanning the surface of the array for fluorescence.  
     [0077] The surface is typically scanned by directing excitation radiation at the surface to activate the fluorescent labeling group in the target, which in turn emits a fluorescent response radiation. The fluorescent response radiation is detected and assigned to the region from which it originated. By knowing the position from which the fluorescence originates, one can identify the sequence to which the target binds.  
     [0078] Although generally used herein to define separate regions containing differing polymer sequences, the term “feature” generally refers to any element, e.g., region, structure or the like, on the surface of a substrate. Typically, substrates to be scanned using the scanning systems described herein, will have small feature sizes, and consequently, high feature densities on substrate surfaces. For example, individual features will typically have at least one of a length or width dimension that is no greater than 100 μm, and preferably, no greater than 50 μm, and more preferably no greater than about 20 μm. Thus, for embodiments employing substrates having a plurality of polymer sequences on their surfaces, each different polymer sequence will typically be substantially contained within a single feature.  
     [0079] The probe arrays will have a wide range of applications. For example, the probe arrays may be designed specifically to detect genetic diseases, either from acquired or inherited mutations in an individual DNA. These include genetic diseases such as cystic fibrosis, diabetes, and muscular dystrophy, as well as acquired diseases such as cancer.  
     [0080] Genetic mutations may be detected by a method known as sequencing by hybridization. In sequencing by hybridization, a solution containing one or more targets to be sequenced (i.e., samples from patients) contacts the probe array. The targets will bind or hybridize with complementary probe sequences. Generally, the targets are labeled with a fluorescent marker, radioactive isotopes, enzymes, or other types of markers. Accordingly, locations at which targets hybridize with complimentary probes can be identified by locating the markers. Based on the locations where hybridization occurs, information regarding the target sequences can be extracted. The existence of a mutation may be determined by comparing the target sequence with the wild type.  
     [0081] Substrate  26  may be fabricated of, or may include, a wide range of material, either biological, non-biological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, etc. The substrate may have any convenient shape, such as a disc, square, sphere, circle, etc. The substrate is preferably flat but may take on a variety of alternative surface configurations. For example, the substrate may contain raised or depressed regions on which a sample is located. The substrate and its surface preferably form a rigid support on which the sample can be formed. The substrate and its surface are also chosen to provide appropriate light-absorbing characteristics. For instance, the substrate may be a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO 2 , SiN 4 , modified silicon, or any one of a wide variety of gels-or polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinations thereof. Other materials with which the substrate can be composed of will be readily apparent to those skilled in the art upon review of this disclosure.  
     [0082] While the invention has been described with reference to the above embodiments, the present invention is by no means limited to the particular constructions described and/or shown in the drawings. The present invention also comprises any modifications or equivalents within the scope of the following claims.