WO1998023945A1 - Dispositif de photodetection perimetrique permettant un captage accru du rayonnement - Google Patents

Dispositif de photodetection perimetrique permettant un captage accru du rayonnement Download PDF

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Publication number
WO1998023945A1
WO1998023945A1 PCT/US1997/022168 US9722168W WO9823945A1 WO 1998023945 A1 WO1998023945 A1 WO 1998023945A1 US 9722168 W US9722168 W US 9722168W WO 9823945 A1 WO9823945 A1 WO 9823945A1
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Prior art keywords
light
waveguide
sample
face
scattered
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PCT/US1997/022168
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English (en)
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WO1998023945A9 (fr
Inventor
L. David Tomei
Fei Zhu
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Optical Analytic Inc.
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Priority to AU53710/98A priority Critical patent/AU5371098A/en
Publication of WO1998023945A1 publication Critical patent/WO1998023945A1/fr
Publication of WO1998023945A9 publication Critical patent/WO1998023945A9/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/4738—Diffuse reflection, e.g. also for testing fluids, fibrous materials
    • G01N21/474—Details of optical heads therefor, e.g. using optical fibres
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6463—Optics

Definitions

  • This invention relates to a device for improved collection of light, including fluorescent light and scattered light.
  • the invention further relates to the collection of light by total internal reflection within a planar optical waveguide, and methods for improving the efficiency of collection thereof.
  • a large number of optical applications depend on the collection of very low levels of light. Examples of such applications are biological assays which depend on detection of specific substances labeled with fluorophores. Often these assays seek to detect the presence of exceedingly small amounts of the substance of interest (on the order of picomoles to femtomoles or less) in a biological sample. The very low level of fluorescence emitted by the fluorophores during the assay places stringent demands on the efficiency of light collection by the device employed for the assay. This can lead to difficulties in detecting and/or quantitating accurately the sample undergoing analysis.
  • a sample emitting fluorescent light When a sample emitting fluorescent light is embedded within a homogeneous and optically clear medium, and/or contained between two plates of an optical material, the emitted fluorescence is conventionally detected in either the forward or backward direction, or at an angle (generally 15° to 90°) away from the excitation beam.
  • Such samples usually fluoresce isotropically on a macroscopic scale; that is, equal intensities of light are emitted in all directions from the sample.
  • Forward detection involves collecting the light within a cone along the axis of the excitation beam (e.g., for a light source, sample, and light detector which are collinear, the source and detector are on opposite sides of the sample, generally along the same optical axis).
  • Backward detection involves collecting the light emitted in a cone opposite from the direction of the excitation beam (e.g., for a light source, sample, and light detector which are collinear, the source and detector are on the same side of the sample).
  • An angular configuration involves collecting the light emitted in a cone with its axis at a fixed angle to the excitation beam. Whichever orientation is used, the conventional method allows the collection of only a small portion of the emitted light within the collecting cone of the optics. Collection by the conventional method is limited by the proportion of the total isotropic light emission propagated along the optical axis of the lens (or similar optical device used to collect fluorescence), not by the optical efficiency of the light collecting device as represented by the numerical aperture.
  • This light is not available for conventional forward- or backward- collection setups; instead, the optical plate(s), together with the embedding medium, act as a waveguide and direct the light towards the end faces of the plate(s). This light emerges from the end faces of the waveguide at a distance from the emitting sample, rather than being transmitted through the upper and lower surfaces of the waveguide in the vicinity of the sample.
  • the amount of light directed to the perimeter of the waveguide depends on the indices of refraction of both the optical substrate material and the embedding medium containing the sample. In many instances, the total intensity of this light confined by the waveguide may be several times that of light collected in the forward or backward directions.
  • Conventional efforts to increase the light-gathering efficiency of the setup are directed toward optimization of the numerical aperture of the forward, angular, or backward detection optics alone.
  • the inability of standard detection methods to capture the light directed to the perimeter results in a loss of sensitivity and/or the inability to analyze many types of samples at low concentration. This disadvantage of conventional forward, angular, or backward detection optics for applications requiring high light collection efficiency can be demonstrated by considering the emission sphere of a point source.
  • an example of such a source would be fluorescently labeled DNA molecules.
  • Conventional collection optics positioned for forward, backward, or angular detection can collect only a very small portion of the emission sphere, typically 2-5%. The major portion of the emission is not collected.
  • both the embedding medium and optical substrates can act as a waveguide as long as their indices of refraction are significantly greater than one (for example, at an index of refraction of 1.4).
  • the emission sphere of this point source can be divided into three zones.
  • the first zone is forward emission, which comprises light emitted from the point source in the forward direction (i.e., along the direction of the original illuminating beam) at an incidence angle less than the critical angle ⁇ c formed at the medium-air interface.
  • the second zone contains backward emission, which comprises light emitted from the point source in the backward direction (i.e., away from the direction of the original illuminating beam) at an angle less than the critical angle ⁇ c formed at the medium-air interface. Both forward and backward emission escape from the embedded emitting point source via direct transmission.
  • the third zone is named belt emission, which comprises light emitted from the point source at an angle equal to or greater than the critical angle. This light is channeled out to the end faces of the plates via total internal reflection. If the radius of the emission sphere is R, then the surface area of the belt zone is given by:
  • a beltzone 4 ⁇ R 2 cos ⁇ c where ⁇ c is the critical angle at the medium-air interface.
  • the fraction of the light with incidence angle larger than or equal to ⁇ c over the whole emission sphere, which is trapped in the waveguide, is thus determined by the ratio of the surface area of the belt zone to the surface area of the sphere: trapping — -"-belt zone ' " ⁇ sphere ⁇ COS ®-c
  • a conventional lens based forward or backward fluorescence collection system may use F/1.5 optics, which collects light emission within a solid angle of 0.36 steradians. Such a system collects no more than 16% of the forward (or backward) escape zone, or 3% of the total emission.
  • the collection efficiency could be improved by using an optically clear index matching medium between the optical plate and the collection optics.
  • a 40X objective which may have a numerical aperture (NA) ranging from 0.5 to 0.75, may be used for fluorescence detection of a small sample area. This type of arrangement should capture about 40% to 90%) of the backward emission cone, or 7% to 17% at best of the total emission.
  • DNA permits the automation of the sequencing procedure and eliminates the necessity of using radioactive materials.
  • fluorescence-based sequencing technology has not yet been implemented in many biological laboratories.
  • One important limiting factor is the sensitivity of detection, which determines the minimum amount of DNA detectable.
  • the current optimum detection limit of fluorescence-based DNA sequencing technology is 1 femtomole per band of DNA for fluorescent-end-labeled DNA primer. Compared to 0.1 femtomole for autoradiography using radioactive 32 P end- labeling, this inability to detect weak bands limits sequencing accuracy. To address this concern, improvements in emission collection and detection are desirable.
  • a sensitivity of 1 femtomole requires detecting 6xl0 8 fluorescent molecules, a quantity often unavailable.
  • the number of fluorescent molecules involved is around a few hundred. This puts even greater demands on detection sensitivity.
  • Fluorescence analysis is used in gene mapping, RNA and DNA quantitation, cell identification and viability assays and many other applications. Fluorescence detection is also used to visualize various physiological phenomena due to changes in concentration of ions such as calcium, sodium, potassium, etc. Instruments used for all of these purposes and many other purposes would benefit from improvements in light collection efficiency and resultant detection sensitivity.
  • Several methods have been employed to detect fluorescence or other light emission from small amounts of sample, utilizing the fraction of light totally internally reflected in a waveguide rather than the light transmitted in the forward or backward direction.
  • U.S. Patent Nos. 4,810,658, 5,166,515 and 5,192,502 describe systems for analyzing a sample, part of which is bound to an optical waveguide and part of which is free in solution, to determine the proportion of bound and free material.
  • Total internal reflection within the waveguide channels light to the end faces of the waveguide. Light originating from bound and free material can be detected separately based on the angle of emission of the light from the waveguide.
  • U.S. Patent No. 5,072,382 describes a method to detect obliquely scattered light which is trapped in a specimen slide by placing a sensor at the edge of the slide.
  • U.S Patent No. 4,881,812 describes an apparatus for determining nucleic acid base sequences which utilizes total internal reflection.
  • U.S. Patent Nos. 5,492,674 and 5,469,264 describe devices for transmitting an excitation beam through a waveguide, and detecting fluorescence from a sample in contact with the waveguide.
  • the excitation beam is propagated down the waveguide via total internal reflection, and the evanescent wave external to the waveguide (produced by the electric field of the propagating excitation beam) produces fluorescence in substrates bound to the surface of the waveguide.
  • the emitted fluorescence which enters the waveguide at suitable angles can then be transmitted to a detector by total internal reflection in the waveguide.
  • the emitted or scattered light can be fluorescent, phosphorescent, chemiluminescent, bioluminescent, or scattered light.
  • the sample can emit light spontaneously or can be illuminated by a light source.
  • the waveguide can take the form of an embedding medium completely containing the sample. It is another object of the present invention to provide such an apparatus, where the waveguide can take the form of an embedding medium completely containing the sample in proximity to one or more layers of optical material; or if the sample is not contained in an embedding layer, the waveguide can take the form of one or more layers of optical material in proximity to the sample. These layers of optical material can be in the form of plates, and can be made from appropriate glasses or plastics. The index of refraction of the optical material can vary along the normal to the surface of the optical material.
  • a focusing means such as a lens, a mirror, or an assembly of one or more lenses and/or one or more mirrors
  • the light exiting the waveguide is focused by a holographic lens and separated into at least two color components or wavelength regions
  • the waveguide itself can focus the emitted, exiting light onto a light detection apparatus, either by shaping the end faces of the waveguide, or by using a material whose
  • samples such as gels (including polyacrylamide and agarose gels) containing proteins, DNA, RNA, nucleotides, peptides, and carbohydrates; solutions; cells; tissues; or particulates. It is another object of the present invention to provide a method for analyzing samples by means of such an apparatus.
  • Figure 1 shows a perspective view of one embodiment of the invention.
  • Figure 2 shows a perspective view of another embodiment of the invention.
  • Figure 3 shows a perspective view of yet another embodiment of the invention.
  • Figure 4 depicts various light paths in one configuration of optical media.
  • the present invention encompasses methods to exploit the phenomenon of total internal reflection to efficiently capture light emitted from an embedded sample.
  • the embedded sample can be contained between two layers of optical material; the layers can be plates or other shapes.
  • Using the perimeter capture scheme described herein can give an improvement of approximately twenty-fold in light gathering efficiency over an F/1.5 lens based collection system, and four-to eight- fold over a 40x microscope objective based collection system.
  • An additional advantage of this perimeter light detection scheme is that there is much less interference by excitation light.
  • Parallelepiped as used below is defined to mean a 6-faced polyhedron, all of whose faces are parallelograms lying in pairs of parallel planes.
  • a right rectangular parallelepiped is a parallelepiped whose faces are all rectangles and whose faces all meet at right angles.
  • One embodiment of the invention is (1), depicted in Figure 1.
  • the sample assembly (20) is comprised of an embedding medium (40), upper (21) and lower (22) optical plates as the top and bottom layers of optical material, and a sample (30).
  • the embedding medium (40) containing the sample (30) is sandwiched between the upper optical plate (21) and the lower optical plate (22).
  • the plates can be right rectangular parallelepipeds, having length, width, and thickness.
  • the top and bottom faces of the plate are circumscribed by the length and width.
  • Two of the four "end faces” are circumscribed by the length and thickness and the other two of the four end faces are circumscribed by the width and thickness.
  • One of the end faces of the upper plate (21) is indicated by (25); one of the end faces of the lower plate (22) is indicated by (26).
  • a light source (10) produces a light beam (12); the beam (12) passes through the upper optical plate (21) and is absorbed by the sample (30). The sample then emits light (which can be fluorescent, phosphorescent, or scattered light); a portion of this light, the "belt emission” (60) is trapped by both the plates and the embedding sample layer acting together as a waveguide.
  • the sample assembly (20) as a whole acts as a waveguide for the belt emission (60).
  • forward and backward emission is not depicted and only a portion of the belt emission (60) is drawn for clarity; however, forward and backward emission does occur, and belt emission is emitted in any direction from the sample satisfying the conditions for trapping in the waveguide.
  • the belt emission is shown exiting one part of one end face (25 and 26) of each of the upper and lower plates.
  • the sample can emit light in any direction, and that belt emission can impinge onto any region of the end faces of the plates, not merely those regions drawn in Figure 1.
  • a lens (70) is employed to focus the belt emission (60) exiting the end face of the waveguide onto a light detection assembly (80).
  • the light detection assembly (80) is composed of a fiber optic array (91), a housing (90), the fibers collected into a bundle (92), a detection means (94), and a device for receiving, converting, recording, and/or processing the output of the bulk detector (96).
  • the lens (70) focuses the belt emission (60) onto the end faces of the fibers (91).
  • the fibers are gathered into a bundle (92) and transfer the light to the detection means (94).
  • the lens and fibers are only drawn along one end face of the waveguide.
  • the lens and fibers can be arrayed along the entire perimeter of the waveguide, portions of the perimeter of the waveguide, or placed at discrete locations along the perimeter of the waveguide.
  • the detection means is at least one bulk detector.
  • Several bulk detectors can be used, for instance, for simultaneous multicolor detection.
  • the bulk detector sends a signal to a device (96) which can comprise an amplifier, an analog-to-digital converter and a computer for storing the data or further data processing.
  • Figure 2 shows another embodiment (200) of the invention.
  • the light detection assembly (290) is comprised of a diode array (294) containing individual diodes (295); the output from the diode array is fed to the device (296) for receiving, converting, recording, and/or processing the output of the diode array.
  • the belt emission (260) exiting the end faces (225, 226) is focused by the lens (270) onto the diode array (294).
  • forward and backward emission and most of the belt radiation are not depicted for clarity.
  • Figures 1 and 2 show the focusing lens as a simple cylindrical meniscus lens. It is understood that mirrors can be employed instead of lenses. A complex assembly of lens elements can also be employed, as well as other types of focusing devices.
  • Figure 3 shows another embodiment (300) of the invention.
  • the light detection assembly (380) is composed of a light conduit (370) adjacent to the end faces of the plates.
  • the light is conveyed to a detecting means (394); the output from the detecting means is fed to the device (396) for receiving, converting, recording, and/or processing the output of the bulk detector.
  • a detecting means 394
  • the output from the detecting means is fed to the device (396) for receiving, converting, recording, and/or processing the output of the bulk detector.
  • forward and backward emission and most of the belt radiation are not depicted for clarity.
  • the light beam (10, 210, 310) can originate from a source including, but not limited to, a laser, a lamp, a light emitting diode, or any other suitable light source. Examples of suitable light sources are described in U.S. Patent Nos.
  • the light beam can be monochromatic or polychromatic; the wavelength or wavelengths of the beam are between about 200 nm and 1 micron, preferably between about 250 nm and 1 micron, more preferably between about 250 nm and 860 nm. Wavelengths in the 600-900 nm range can be preferable in some applications, as there will be less background fluorescence from glass plates and less expensive glasses can be used (e.g., soda glass).
  • the incident angle of the light beam (10, 210, 310) on the upper plate (21, 221, 321) is drawn perpendicularly; this incident angle can be about +/- 15 degrees from the normal to the surface, preferably about +/- 10 degrees, more preferably about +/- 5 degrees.
  • Many applications of the device involve samples which are non-uniformly distributed over the length and/or width of the embedding medium, or several different samples contained in different spatial locations in the embedding medium; in these cases, means for illuminating different parts of the embedding medium are necessary.
  • the beam can be scanned across the sample by varying the angle of the beam from the source, as described in the patents cited above, or by moving the sample assembly while keeping the beam position fixed, or by both scanning the beam across the sample and moving the sample assembly.
  • the waveguide assembly can be moved in small increments by using a stepping motor and a platform; the increments can be greater or smaller than the spot size of the illuminating beam.
  • the plates (21 and 22; 221 and 222; 321 and 322) can be glass, such as borosilicate, flint, or soda glass, or plastics such as polycarbonate, or any other material suitable for use in the waveguide assembly for the wavelength of the detected light.
  • the material used should have minimal fluorescence background under the illuminating light source, so as not to interfere with the measurement of the sample fluorescence.
  • either the upper (21, 221, 321) or lower (22, 222, 322) plate, or both plates can be omitted, and the embedding sample layer alone acts as the waveguide.
  • the plates are drawn in Figure 1 as right rectangular parallelepipeds (i.e., with all faces at right angles and substantially flat).
  • the shape of the end faces can vary with the application, however.
  • the plates of Figure 1 illustrate the invention by using an upper (21) and a lower (22) plate as upper and lower layers of optical materials; other shapes can be used for the optical materials, including, but not limited to, those described below.
  • One preferred embodiment of the plates is with flat upper and lower faces, having cylindrically curved convex end faces.
  • the cylindrical end face focuses the guided light into a line.
  • the thickness of this line of focused light referred to as the image line, is equal to the image height, and should be no greater than the height of the sensor elements of the perimeter detector.
  • the image space numeric aperture (NA) of the focusing end face should also match that of the fiber array.
  • the curvature of the cylindrical end face is determined by factors including the index of refraction of the embedding medium (i.e., ⁇ c ), the index of refraction of the optical substrate(s), the thickness of the waveguide, the height of the sensor elements, and the NA of the fiber array.
  • ⁇ c the index of refraction of the embedding medium
  • the index of refraction of the optical substrate(s) the thickness of the waveguide
  • the height of the sensor elements the NA of the fiber array.
  • a sample assembly comprising fluorescently labeled HT29 cells embedded in a thin layer of advanced acrylic resin (CytoSeal-60 from Edmund Scientific).
  • the embedding layer is contained between two pieces of fused silica plates, each with dimensions of 10 x 10 x 1 mm.
  • the embedding medium has a index of refraction of 1.418 for the Helium d line; the fused silica has an index of refraction of 1.458.
  • the curvature of the cylindrical end face has a radius of about
  • N-MOS linear image sensors sized at 12.28 mm x 2.5 mm (S5930-256S from Hamamatsu) are used for detection.
  • the image sensors are placed 10 mm away from the curved end surface. If fiber arrays (FT-500- EMT, from 3M Specialty Optical Fibers) are used instead of the perimeter detector arrays, and are placed 10 mm away from the curved end surface, the radius of the curvature would be set at about -10.68 mm.
  • This embodiment of the waveguide incorporates a refracting cylindrical surface as an integral part of the waveguide itself.
  • the length of the image line is limited by the length of the end face, and can be controlled by another external cylindrical lens oriented at 90° to the axis of the cylindrical lens curved end face.
  • This second cylindrical lens perpendicularly oriented, reduces the number of sensor elements necessary in the perimeter detector, or the number of optical fibers necessary in the perimeter fiber arrays.
  • Another preferred embodiment of the waveguide is to use plates with an axial gradient of the index of refraction. That is, along the normal to the surface of the plates, the index of refraction of the optical material used for the plates is high at the sample-glass interface, and gradually decreases towards the glass-air interface.
  • the path of the totally internally reflected ray through the waveguide is curved, not straight. Rays originating from the same point source but with different incidence angles are brought to a focus periodically. This characteristic offers better control of the end face focusing optics. It is also possible to focus the totally internally reflected light without using a curved end face or an external lens.
  • the embedding medium (40, 240, 340) can be a liquid, gel, glassy solid, or any other suitable material which provides a homogeneous environment in which to place or suspend the sample or samples.
  • the material used for the embedding medium should be selected so that, under conditions of operation, the embedding medium does not fluoresce to an extent that will interfere with the measurement of the light emitted by the sample.
  • the embedding medium should have an index of refraction greater than about 1.3; more preferably, greater than about 1.4.
  • the sample and the embedding medium are identical; that is, the sample will be of such a nature that it does not need an extrinsic embedding medium.
  • Preferred embedding media will vary with the application; some of the preferred media include polyacrylamide gels, polycarbonate, immersion oil, cytosealant and liquids (including water, organic solvents, and other liquids).
  • the embedding medium can range in thickness from 1 micron to 1 millimeter, preferably 50 microns to 400 microns for the polyacrylamide gel embodiment. While the sample itself can be uniformly or non-uniformly distributed within the embedding medium, the embedding medium itself is preferably of a homogeneous nature.
  • a preferred embodiment of the invention is for detection of fluorescently labeled DNA in a sequencing gel.
  • a slab gel for DNA sequencing can consist of a layer of polyacrylamide of approximately 100 ⁇ m to 400 ⁇ m thick, more preferably approximately
  • Figure 4 depicts a cross-section of the glass (420)-gel (440)-glass (422) structure, where n,, n 2 , and n 3 are the indices of refraction of air, glass and gel, respectively. These indices are referred to herein as n ArR , n GLASS , and n QEL , respectively.
  • An excitation beam is focused on fluorescently labeled DNA molecules in a band (430), which then emit fluorescent light of longer wavelength.
  • nQ EL and n GLASS ) of 1.30, measured for a DNA sequencing gel made from a 6% solution of acrylamide, the critical angle ⁇ c is 50°. A emission ray making an angle of 45° with the optical axis exits at an angle of 67° from the glass-air interface. It is preferred that nQ EL is very close to but smaller than no LASS in order to utilize the glass plates as the major portion of the waveguide with minimum loss at the gel-glass interface. Table 1 illustrates the approximate percentage of light emitted from the sample which is totally internally reflected within the waveguide assembly as the critical angle ⁇ c is varied.
  • n GEL >n GLASS >n AIR a dual waveguide is formed.
  • ⁇ c arcsin and ⁇ c ⁇ ⁇ cl ) undergoes total internal reflection at the glass-air interface.
  • a larger nQ EL leads to more emitted light being channeled into the waveguide, and less light escapes via direct forward or backward transmission.
  • a DNA sequence gel made of 6% acrylamide has an index of refraction of 1.30 ⁇ 0.02 for the HeNe laser emission wavelength of 632.8 nm, resulting in 50 ⁇ 1 ° for the critical angle ⁇ c .
  • Agarose gels have similar indices of refraction (1.29 +/- 0.02) for the HeNe 632.8 nm laser emission. An increase of 1/10 in the index of refraction of the gel would reduce the forward and backward escape zones down to a cone angle (critical angle) of 44°.
  • Figure 1 depicts an embodiment where an optical fiber array at the waveguide end face captures and transfers the light to a remote bulk detector.
  • Figure 2 depicts an embodiment where a detector is placed at the waveguide end face, while Figure 3 depicts an embodiment which uses a light conduit to channel light to a bulk detector.
  • the first configuration shown in Figure 1, uses a fiber optic array as a light guide and offers simplicity in design of the sample holder and flexibility in implementation of a detector or detectors.
  • Various end products can be developed conveniently as add-ons without requiring changes in light collection optics around a sample.
  • conventional filters (not shown in the figure) can be used for rejection of excitation light.
  • fibers to convey the light from the waveguide to the detector may introduce additional coupling losses at the input and output ends, and transmission losses mainly due to micro-bending of fibers and surface reflections.
  • Other factors that should be considered include, but are not limited to, core-cladding ratio and filling (packing) efficiency.
  • Square fibers offer higher filling efficiency but are more expensive.
  • Additional optical elements such as fiber optical taper can be employed to couple the output of a large size bundle to a detector which has a small sensing area.
  • Table 3 gives general guidance on the estimated number of optical fibers for optical plates of various sizes.
  • the number of fibers given in Table 3 is estimated assuming that FT-300-UMT fibers from 3M are used without buffer coating; the core diameter is 300 ⁇ m; and the fibers are arrayed in two rows. It is possible to use only a single row of fibers with good alignment, resulting a smaller fiber bundle for ease of handling.
  • the size (diameter) of the fiber bundle given in Table 3 is estimated assuming a packing efficiency of 78.5%.
  • a peripheral detector is placed at the sample perimeter to detect the light directly without additional loss due to intermediate optics, and consequently higher detection sensitivity is expected.
  • peripheral detectors which can be used include, but are not limited to, linear photodiodes, Avalanch photodiodes, or a charge-coupled device array, depending on the type of signal to be detected.
  • filter implementation for rejection of excitation wavelengths
  • output from all sensing elements can be combined at the analog level. This has the additional advantage of signal averaging, which reduces random noise.
  • multiple-color detection however, a different arrangement is required.
  • the sample holder is preferably designed to avoid direct exposure of the sensor elements to ambient light during sample loading and unloading.
  • the number of sensor elements increases dramatically when the size of a sample doubles.
  • a light transfer conduit fabricated as a 2D taper or lens plate can be used; for one example, see light conduit (370) as shown in Figure 3.
  • Figures 1, 2, and 3 depict detectors along the entirety of one end face, it is emphasized that detectors can be placed along one or more of the end faces of the waveguide, or at discrete locations along only a portion of one or more of the end faces of the waveguide, or any combination thereof, according to the particular application for which the perimeter light detector system is employed.
  • the light coming out of the end face of the waveguide is focused.
  • introducing a separate optical element may incur additional coupling loss.
  • the alternative embodiment of the waveguide described above, with cylindrical curved end faces incorporates the focusing lenses into the end faces of the waveguide so that the light coming out of the shaped end faces is focused automatically. This alternative embodiment reduces loss at the end face-air interface.
  • Other shaped end faces including, but not limited to, beveled end faces, can be employed. If a light transfer conduit is used, the conduit can be fabricated into a shape which matches that of the waveguide to ensure high light transfer efficiency. It is emphasized that application of the perimeter light collection is not limited to DNA sequencing. The following non-limiting examples illustrate the breadth of applications of the invention.
  • the sample contained in the embedding medium can be proteins; the embedding medium for proteins can be polymerized gels or natural or artificial membranes. Analysis of polynucleotides is not limited to sequencing by gel electrophoresis; other types of DNA gels can be analyzed, as well as DNA contained in microplates and membranes. Other biomolecules, such as RNA and proteins, can also be analyzed in microplates and membranes using the invention. Assays of biomolecules on chips of suitable size and properties, such as those described in U.S. Patent No. 5,556,752, can also be carried out with the perimeter light detection system.
  • Cells (such as labeled fixed cells or living cells) can be analyzed; a suitable microscope slide and an embedding medium like immersion oil or cytoseal resin can comprise the waveguide necessary for perimeter light detection. Thin slices of tissues can also be analyzed. Spectroscopic measurements on solutions or solids can also be performed using the invention.
  • Another application is for imaging of a storage phosphor screen.
  • a storage phosphor screen is used to store latent images of radioisotope labeled blots, gels, TLC plates or tissues, without the use of film. The energy of ⁇ and ⁇ emissions and X-rays from the radioactive samples is stored in the active ingredient particles (BaFBr:Eu +2 crystals) of the screen.
  • the energized particles Upon the stimulation of a red laser beam, typically, a HeNe laser (632 nm), the energized particles release energy as blue light. To improve the light collection efficiency, the blue emission can be collected using the perimeter detection method, by sandwiching the screen between two index-matched optical plates.
  • a red laser beam typically, a HeNe laser (632 nm)
  • the blue emission can be collected using the perimeter detection method, by sandwiching the screen between two index-matched optical plates.
  • PLT stand for embedding medium and plate, respectively), and both are much larger than 1.
  • the glass-immersion oil-glass acts naturally as the waveguide guiding most of the emission to exit from the end faces.
  • a glass lens focuses the emission exiting from the end faces onto a diode array.
  • the diode array output is fed to a computer for analysis.
  • the perimeter detection scheme of this example has the potential to catch 75% of the emission, an improvement of 7- to 25-fold as compared to conventional methods.
  • Example 2 DNA Sequencing By Gel Electrophoresis
  • a typical slab gel consists of a thin layer of gel around 300 mm, sandwiched between two glass plates of 3-4 mm thick. Gel plates from Genomyx Corp. are used to pour the gel. Spacers 0.34 mm thick are used to separate the two plexiglass plates (33 cm x 61 cm) used to contain the gel. A 46 tooth comb is used to create wells for loading fluorescently end-labeled DNA samples. A 6% acrylamide solution is prepared, and the solution is poured using the Lang method. The DNA sample is prepared according to protocols stated in Genomyx LRDNA Sequencing System Operating Manual. After electrophoresis is complete, the gel assembly is then used as the sample assembly for the perimeter light detection apparatus.
  • the gel sandwiched between the plates, is placed under a scanning beam. Two end faces along the direction of separation are used for detection. Along each end face, a molded cylindrical lens is placed to focus the light onto a fiber optic array.
  • the fiber optic array feeds light to a photomultiplier tube, and the output of the photomultiplier tube is sent to a computer for processing.
  • a known sample of a DNA gel is scanned beforehand to determine values to correct for the effects of variable distances between the source and detector; these correction data are used by the computer for processing the sample (unknown) data.
  • Color separation of polychromatic light can be accomplished at one or more end faces of one or both waveguides by employing holographic components with a microlens array. This eliminates the need for color filters to discriminate light output of various wavelengths.
  • a sample can be designed so that emission of different wavelengths of light occurs at the same spatial location in the sample, for example by labelling cells or tissue samples with multiple fluorophores, each emitting at different wavelengths.
  • the polychromatic light travels to the end face of the waveguide by total internal reflection, and is then dispersed by a holographic microlens array.
  • a detecting device for example, a linear charge-coupled device array or a binned linear charge-coupled device array.
  • the holographic microlens can be designed to reject the excitation wavelength. Examples of holographic lens arrays which can be used in the invention are described in U.S. Pat. No. 4,807,978. Other examples of holographic lens technology are found in Ming et al., Applied Optics 29: 5111-5114 (1990), Tang et al., IEEE Photonics Technology Letters 8:1498-1500 (1996), and Homer et al., Applied
  • the holographic microlens can be fabricated by a variety of methods known in the art.
  • the optimum configuration of the holographic lens will depend on the shape, orientation, and position of the light-emitting object contained in the waveguide assembly.
  • the holographic lens is fabricated and developed using the light from an object of as similar a size and shape as possible to the objects to be analyzed.
  • the objects to be analyzed are placed in as similar a position and orientation as the position of the object used for developing the holographic lens as possible.
  • a holographic microlens can be an integral part of the waveguide sample assembly; for example, it may be fabricated into the waveguide by microelectronic mechanical fabrication techniques.
  • the methods and devices employing holographic lens color separation are particularly well-suited to analysis of light coming from chips used for biomolecule assays, as producing chips of approximately the size and shape is relatively easy to accomplish, and positioning the individual chips used for analysis in a uniform location in the waveguide assembly is relatively straightforward.
  • This type of structure can be designed in a very compact manner, for use in such applications as a microstation bioanalysis device.

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Abstract

Dispositif permettant un captage amélioré de la lumière, y compris de la lumière fluorescente, phosphorescente, chimioluminescente, bioluminescente et diffuse. Un échantillon (30) inséré dans un milieu (40) optiquement vide et éventuellement placé entre deux substrats (ou sur un substrat) (21, 22) est irradié par une source lumineuse. Une portion importante de la lumière émise ou diffusé par l'échantillon est captée à l'intérieur d'un guide d'onde formé par le ou les substrat(s) et/ou la couche d'inclusion sous l'effet d'une réflexion interne totale, et dirigée vers les surfaces terminales du guide d'onde où la lumière est focalisée et captée. La détection de toute la lumière ainsi dirigée vers le périmètre du guide d'ondes permet d'augmenter de quatre à vingt fois l'efficacité du captage, et en conséquence d'améliorer la sensibilité de détection par rapport à la photodétection par système optique conventionnel à lentille disposé devant ou derrière le faisceau lumineux ou formant un angle avec ce dernier.
PCT/US1997/022168 1996-11-27 1997-11-26 Dispositif de photodetection perimetrique permettant un captage accru du rayonnement WO1998023945A1 (fr)

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Cited By (11)

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EP0909947A2 (fr) * 1997-10-14 1999-04-21 Bayer Ag Système de mesure optique pour la détection d'émissions luminescentes ou fluorescentes
EP1006355A2 (fr) * 1998-11-30 2000-06-07 The Institute of Physical and Chemical Research Dispositif d'electrophorese capillaire
GB2348018A (en) * 1999-03-16 2000-09-20 Cambridge Imaging Ltd Optical system with lens and tapered fibre optics
WO2001038844A2 (fr) * 1999-11-12 2001-05-31 Motorola, Inc. Dispositifs d"electrophorese capillaire comportant des guides d"ondes optiques
WO2002059583A1 (fr) * 2001-01-23 2002-08-01 Dublin City University Capteur dont le fonctionnement est fonde sur la luminescence
EP1322946A2 (fr) * 2000-08-01 2003-07-02 Sensis Ltd. Appareil d'electrophorese et plaque correspondante
EP1672355B1 (fr) * 2004-12-20 2010-05-19 Palo Alto Research Center Incorporated Procédé amélioré de balayage et de collection de lumière pour un détecteur de cellules rares
FR3004814A1 (fr) * 2013-04-19 2014-10-24 Commissariat Energie Atomique Systeme de detection optique comportant un dispositif de collection de lumiere
CN106066320A (zh) * 2016-07-26 2016-11-02 中国科学院南海海洋研究所 基于多波长激光诱导细菌内在荧光的海水细菌检测系统
CN106770316A (zh) * 2016-12-02 2017-05-31 东莞市天合机电开发有限公司 一种模板检测装置
EP2671105A4 (fr) * 2011-02-04 2018-01-10 The U.S.A. as represented by the Secretary, Department of Health and Human Services Guides de lumière pour simplifier une détection d'émission totale pour une microscopie multiphotons

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US4703182A (en) * 1984-09-17 1987-10-27 AVL Gesellschaft fur Verbrennungskraftmaschinen und Messtechnik m.b.H. Prof.Dr.Dr.h.c. Hans List Arrangement for fluorescence-optical measurement of concentrations of substances contained in a sample
US4881812A (en) * 1987-03-31 1989-11-21 Shimadzu Corporation Apparatus for determining base sequence
EP0793090A1 (fr) * 1996-02-29 1997-09-03 AVL Medical Instruments AG Système de mesure avec un élément de support transparent pour le faisceau d'exitation et de détection

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US4703182A (en) * 1984-09-17 1987-10-27 AVL Gesellschaft fur Verbrennungskraftmaschinen und Messtechnik m.b.H. Prof.Dr.Dr.h.c. Hans List Arrangement for fluorescence-optical measurement of concentrations of substances contained in a sample
US4881812A (en) * 1987-03-31 1989-11-21 Shimadzu Corporation Apparatus for determining base sequence
EP0793090A1 (fr) * 1996-02-29 1997-09-03 AVL Medical Instruments AG Système de mesure avec un élément de support transparent pour le faisceau d'exitation et de détection

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6191852B1 (en) 1997-10-14 2001-02-20 Bayer Aktiengesellschaft Optical measurement system for detecting luminescence or fluorescence signals
EP0909947A3 (fr) * 1997-10-14 1999-06-09 Bayer Ag Système de mesure optique pour la détection d'émissions luminescentes ou fluorescentes
EP0909947A2 (fr) * 1997-10-14 1999-04-21 Bayer Ag Système de mesure optique pour la détection d'émissions luminescentes ou fluorescentes
US6461492B1 (en) 1998-11-30 2002-10-08 The Institute Of Physical And Chemical Research Capillary electrophoretic apparatus
EP1006355A2 (fr) * 1998-11-30 2000-06-07 The Institute of Physical and Chemical Research Dispositif d'electrophorese capillaire
US6783650B2 (en) 1998-11-30 2004-08-31 The Institute Of Physical & Chemical Research Capillary electrophoretic apparatus
EP1006355A3 (fr) * 1998-11-30 2000-11-08 The Institute of Physical and Chemical Research Dispositif d'electrophorese capillaire
GB2348018A (en) * 1999-03-16 2000-09-20 Cambridge Imaging Ltd Optical system with lens and tapered fibre optics
GB2348018B (en) * 1999-03-16 2002-01-23 Cambridge Imaging Ltd Sample imaging
US6592733B1 (en) 1999-11-12 2003-07-15 Motorola, Inc. Capillary electrophoresis devices incorporating optical waveguides
WO2001038844A3 (fr) * 1999-11-12 2002-06-20 Motorola Inc Dispositifs d"electrophorese capillaire comportant des guides d"ondes optiques
WO2001038844A2 (fr) * 1999-11-12 2001-05-31 Motorola, Inc. Dispositifs d"electrophorese capillaire comportant des guides d"ondes optiques
EP1322946A4 (fr) * 2000-08-01 2008-09-24 Sensis Ltd Appareil d'electrophorese et plaque correspondante
EP1322946A2 (fr) * 2000-08-01 2003-07-02 Sensis Ltd. Appareil d'electrophorese et plaque correspondante
US7655475B2 (en) 2001-01-23 2010-02-02 Fluorocap Limited Luminescence based sensor using protuberances to redirect light
AU2002228308B2 (en) * 2001-01-23 2007-06-21 Dublin City University A luminescence based sensor
WO2002059583A1 (fr) * 2001-01-23 2002-08-01 Dublin City University Capteur dont le fonctionnement est fonde sur la luminescence
EP1672355B1 (fr) * 2004-12-20 2010-05-19 Palo Alto Research Center Incorporated Procédé amélioré de balayage et de collection de lumière pour un détecteur de cellules rares
EP2671105A4 (fr) * 2011-02-04 2018-01-10 The U.S.A. as represented by the Secretary, Department of Health and Human Services Guides de lumière pour simplifier une détection d'émission totale pour une microscopie multiphotons
FR3004814A1 (fr) * 2013-04-19 2014-10-24 Commissariat Energie Atomique Systeme de detection optique comportant un dispositif de collection de lumiere
CN106066320A (zh) * 2016-07-26 2016-11-02 中国科学院南海海洋研究所 基于多波长激光诱导细菌内在荧光的海水细菌检测系统
CN106770316A (zh) * 2016-12-02 2017-05-31 东莞市天合机电开发有限公司 一种模板检测装置
CN106770316B (zh) * 2016-12-02 2019-04-02 旺天凯精密机器(昆山)有限公司 一种模板检测装置

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