WO1998023945A1

WO1998023945A1 - Perimeter light detection apparatus for enhanced collection of radiation

Info

Publication number: WO1998023945A1
Application number: PCT/US1997/022168
Authority: WO
Inventors: L. David Tomei; Fei Zhu
Original assignee: Optical Analytic Inc.
Priority date: 1996-11-27
Filing date: 1997-11-26
Publication date: 1998-06-04
Also published as: AU5371098A

Abstract

This invention comprises a device for improved collection of light, including fluorescent, phosphorescent, chemiluminescent, bioluminescent, and scattered light. A sample (30) embedded in an optically clear medium (40) and optionally contained between two substrates (or placed on one substrate), is irradiated with a light source. A predominantly large portion of light emitted or scattered from the sample is trapped within the waveguide formed by the substrate(s) (21, 22) and/or the embedding layer due to total internal reflection and directed to the end faces of the waveguide, where the light is focused and collected. Detecting all the light thus guided to the perimeter of the waveguide can increase collection efficiency four- to twenty-fold and consequently improve the detection sensitivity, as opposed to dectecting light in a conventional lens based optics placed in a forward or backward or angular direction to the illuminating light beam.

Description

PERIMETER LIGHT DETECTION APPARATUS FOR ENHANCED COLLECTION OF RADIATION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of copending United States provisional patent application 60/031,517 filed November 27, 1996. The contents of said application is hereby incorporated by reference herein in its entirety.

TECHNICAL FIELD

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.

BACKGROUND ART

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.

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. A substantial amount of light is emitted simultaneously in other directions; this portion of the total light emission will not be collected by an optical setup oriented in the conventional manner. Light emitted from the sample at certain angles with the normal to the surfaces of the optical plates is trapped in those materials due to the phenomenon of total internal reflection. When passing from a medium of higher index of refraction (medium 2, with index of refraction n₂) to a medium with a lower index of refraction (medium 1, with index of refraction n,; n, < n₂), a ray with incidence angle α (α > 0) emerges at the interface with exit angle β, where β is larger than α. The relationship of α and β is governed by Snell's law, n, sin β = n₂ sin α. If the incidence angle α is larger than the critical angle defined by arc sin (n,/n₂), the ray is completely reflected back into medium 2 and total internal reflection has occurred. 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. For an emitting point source which is embedded in an optically clear and homogeneous medium and placed on a plate (or sandwiched between two plates) of optical material, 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).

When a focused excitation beam perpendicular to the plane of the embedding material and/or plates (i.e., the beam travels along the normal of the embedding material or plates) is brought onto the sample, the excited sample emits light (e.g., fluorescent light), becoming a point source of light. 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 α_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 α_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 = 4πR² cos α_c where α_c is the critical angle at the medium-air interface.

The fraction of the light with incidence angle larger than or equal to α_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

As the critical angle varies from 0 to 90°, the fraction of light trapped varies from one to zero. Assuming a critical angle α_c of 50°, 64% of the emission undergoes total internal reflection and comes out of the end faces of the waveguide, as compared to 18% each for the forward and backward escape zones.

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.

Another type of collection optics, which employs a microscope objective, is often used in the field of high resolution imaging, sometimes in conjunction with a microscope. An objective lens is employed to both focus the excitation beam onto the sample and collect the emission or scattering in the backward direction. 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.

Improvements in collection efficiency for small quantities of emitted light would find immediate practical applications. One area where such improvements would find utility is fluorescence-based DNA gel sequencing. Fluorescence-based sequencing of

DNA permits the automation of the sequencing procedure and eliminates the necessity of using radioactive materials. Despite these advantages, 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 ³²P 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 6xl0⁸ fluorescent molecules, a quantity often unavailable. For example, in single molecule DNA hybridization or colony hybridization, the number of fluorescent molecules involved is around a few hundred. This puts even greater demands on detection sensitivity.

In addition to DNA sequencing, assays using fluorophores for labeling or staining have found use in many biological fields. 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. General principles of total internal reflection are discussed in Harrick et al., Analytical Chemistry 45: 687-691 (1973). 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.

Other devices utilize total internal reflection for excitation as well as detection of emitted light. 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 contents of the above-mentioned U.S. patents, as well as the contents of all patents and references mentioned herein, are hereby incorporated by reference in their entirety.

However, the need for collecting substantial amounts of the light emitted from a sample embedded in a bulk medium, where the bulk medium also acts as a waveguide, and the potential for using shaped end faces of a waveguide in contact with the bulk medium for focusing the emitted light onto a light detector, in order to enhance overall light collection efficiency and detection sensitivity has not been addressed.

SUMMARY OF THE INVENTION

It is one object of the present invention to provide an apparatus for improved light collection efficiency and detection sensitivity, where a sample in proximity to at least one waveguide emits or scatters light; part of the emitted or scattered light is totally internally reflected in the waveguide until it reaches an end face of the waveguide, where the light exits the waveguide; and the exiting light is detected by a light detection assembly. 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.

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. 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. It is another object of the present invention to provide such an apparatus, where the emitted or scattered light exiting the waveguide is focused by a focusing means such as a lens, a mirror, or an assembly of one or more lenses and/or one or more mirrors; or where the light exiting the waveguide is focused by a holographic lens and separated into at least two color components or wavelength regions; or where 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 index of refraction varies along the normal to the surface of the material.

It is another object of the present invention to provide such an apparatus, where the light detection assembly utilizes fiber optics or a light conduit to transfer light to a bulk detector, or where a photodiode array or other detector is used to detect light. It is another object of the present invention to provide such an apparatus for use with 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.

It is yet another object of the present invention to provide a method for adjusting the amount of light directed towards the perimeter of the apparatus by adjusting the indices of refraction of the sample and of the optical substrates acting as the waveguide. It is yet another object of the present invention to provide a highly sensitive detection method for DNA sequencing by gel electrophoresis using fluorescent labels and stains.

It is yet another object of the present invention to provide a highly sensitive detection method for DNA sequencing, genetic mapping and genetic diagnosis by hybridization using fluorescent labels and stains.

It is yet another object of the present invention to provide a highly sensitive detection method for cellular analysis and diagnosis by immunochemistry and hybridization using fluorescent labels and stains.

BRIEF DESCRIPTION OF THE DRA WINGS

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. DISCLOSURE OF THE INVENTION

The present invention encompasses methods to exploit the phenomenon of total internal reflection to efficiently capture light emitted from an embedded sample. For instance, 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). As drawn in Figure 1, 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. That is, the sample assembly (20) as a whole acts as a waveguide for the belt emission (60). In Figure 1 , 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. For clarity, in Figure 1 , the belt emission is shown exiting one part of one end face (25 and 26) of each of the upper and lower plates. However, it is again stressed that 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.

Light which is totally internally reflected in the sample assembly (20) can also exit from the end face of the embedding layer, although typically the end faces of the plates (25 and 26) will provide a greater area for the light to exit than the end face of the embedding layer, and hence most light will exit through the end faces of the plates. (As with the end faces of the plates, the light can impinge on any end face of the embedding layer; the belt emission (60) in Figure 1 is not depicted in its entirety for clarity.)

A lens (70) is employed to focus the belt emission (60) exiting the end face of the waveguide onto a light detection assembly (80). In Figure 1, 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). For clarity, the lens and fibers are only drawn along one end face of the waveguide. It is understood that 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. Preferably 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. In this embodiment, 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). As in Figure 1, 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. In this embodiment, 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. As in Figure 1, forward and backward emission and most of the belt radiation are not depicted for clarity.

It will be appreciated by those skilled in the art that many variations of the invention are possible. 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.

5,037,207, 4,887,966 and 4,758,727. 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. In certain applications, 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. For perimeter detection using fiber arrays, not only should the width of the image line be no greater than the height (or diameter) of each fiber face, the image space numeric aperture (NA) of the focusing end face should also match that of the fiber array. Thus the curvature of the cylindrical end face is determined by factors including the index of refraction of the embedding medium (i.e., α_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. One example is 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

-5.16 mm (note that the negative sign on the radius dimension indicates that the curved convex surface points towards the detector) and 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.

However, this type of embodiment is not desirable for applications where the image reduction is too small and/or the NA of the fibers is too small. 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. In this type of material, 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. Preferably, the embedding medium should have an index of refraction greater than about 1.3; more preferably, greater than about 1.4. In certain instances, 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 μm to 400 μm thick, more preferably approximately

200 μm to 350 μm thick, sandwiched between two flat glass plates of 1-4 mm, preferably 1-3 mm thickness each. The length and width of the glass plates can vary, for example, from 20 cm to 65 cm. Figure 4 depicts a cross-section of the glass (420)-gel (440)-glass (422) structure, where n,, n₂, and n₃ 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. All DNA molecules in a band are oriented randomly; thus their emissions are assumed to be isotropic, and can be represented as a point source. The emitted light exits the gel by various paths, depending on the incident angles at the various interfaces. Lines (470) and (480) indicate norms to the glass surface, from which the critical angle is measured.

As illustrated in Figure 4, rays with angle α larger than the critical angle α_c, as is the case for ray (460), undergo total internal reflection at the glass-air interface, since both I -_JEL and n_GLASS are greater than n_AIR(=^::l). Only the rays within the cone defined by α_c escape by direct transmission. These directly-transmitted rays emerge out of the glass-air interface with much larger exit angles, as is the case for ray (450). Using Snell's law, α_c is determined by arcsin (n._MRl' _G-_Ei) ■ For example, with nQ_EL(and n_GLASS) of 1.30, measured for a DNA sequencing gel made from a 6% solution of acrylamide, the critical angle α_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 α_c is varied.

Table 1

If n_GEL>n_GLASS>n_AIR, then a dual waveguide is formed. A portion of emitted light with angle α larger than α_c, (where α_cl = arcsin (^_LASS/Π_OEL)) undergoes total internal reflection first at the gel-glass interface. Of the light that is transmitted to the glass medium, another portion with angle α larger than α_c (where α_c = arcsin

and α_c < α_cl) undergoes total internal reflection at the glass-air interface. In other words, only the portion of the light coming from the sample with an angle of incidence (at the first interface) greater than α_c but less than α_cl is trapped in the glass-gel-glass waveguide; the portion with incident angle greater than α_cl is trapped only in the gel. If this dual- waveguide configuration is used, it is important that the light can propagate with little attenuation in the gel (e.g., that labeled DNA molecules do not absorb the emitted fluorescent light); the light can then be detected at the perimeter of the glass-gel-glass gel double waveguide. Clearly, by varying the index of refraction of the glass medium, the critical angle α_cl can be adjusted, and varying amounts of the fluorescent light can be directed to the gel medium as desired. Table 2 summarizes the conditions stated above. In Table 2, α is the angle of incidence of a light ray from the emitting source in the gel onto the gel-glass interface.

Table 2

In either case described in Table 2, the size of the cone of escaping light is entirely determined by the ratio of n^ to n^, or the index of refraction of the gel for n^ = 1. 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 α_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°.

In either case described in Table 2, the light confined in the waveguide travels towards the end faces, where different detection configurations can be implemented.

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. In addition, conventional filters (not shown in the figure) can be used for rejection of excitation light. However, using 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 300μm; 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%.

Table 3

In the embodiment depicted in Figure 2, 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. Examples of 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. For one-color detection, filter implementation (for rejection of excitation wavelengths) is straightforward, and output from all sensing elements can be combined at the analog level. This has the additional advantage of signal averaging, which reduces random noise. For multiple-color detection, however, a different arrangement is required. Multiple sensing elements responsive to different wavelengths can be used; the output of the same color sensing elements are combined for signal averaging. In an embodiment which places detectors in proximity to the sample assembly, the sample holder is preferably designed to avoid direct exposure of the sensor elements to ambient light during sample loading and unloading. In the embodiments depicted in Figure 1 and Figure 2, the number of sensor elements increases dramatically when the size of a sample doubles. In order to keep the number of sensors required at a manageable number with increased sample size, 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.

While 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.

To ensure efficient coupling, the light coming out of the end face of the waveguide is focused. However, 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 β and γ emissions and X-rays from the radioactive samples is stored in the active ingredient particles (BaFBr:Eu⁺² crystals) of the screen. 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.

Several of the applications above use fluorescent light emission to illustrate the mode of operation of the invention. Other types of light which can be detected include, but are not limited to, phosphorescence, chemiluminescence, bioluminescence, and scattered light, including Raman- and Rayleigh-scattered light. One such application is for angular- dependent light scattering (i.e., for light which is scattered non-isotropically by a sample). The angular dependence of scattering can be determined by measuring the amount of light channeled to the end faces of the waveguide as a function of the index of refraction of the embedding medium (since the critical angle will change with the change in the index of refraction). The following examples are given for illustration of the invention, and are not intended to limit the invention in any manner.

EXAMPLES

Example 1 Analysis Of Fluorescently-Stained Cells HT29 cells are fixed and fluorescently stained using propidium iodide for cell cycle analysis. The excess dye is washed off and the cells are then placed onto a pre-cleaned microscope slide (75 mm x 25 mm x 1 mm) without a frosted end, and a drop of immersion oil is applied to the cells as the embedding medium. The embedded cells are covered with a cover glass (22 x 22 mm). A blue illuminating beam is focused onto a cell by scanning the beam. The excited stain in the cell then emits red fluorescent light. In this sample configuration, the index of refraction of the immersion oil at the fluorescent wavelengths is very close to that of the slide (i.e., n_EM = 1.5150, and n_PLT = 1.5168; EM and

PLT stand for embedding medium and plate, respectively), and both are much larger than 1. As a result, 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.

Example 3

Use of Holographic Components with a Micro lens Array to Obviate Color Filtering 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. This permits different colors (i.e., different wavelength regions of the emitted light) to be selectively focused on different elements of 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

Optics 20: 1845-1847 (1981).

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. Thus, 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.

Although the forgoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practical. Therefore, the description and examples should not be construed as limiting the scope of the invention, which is delineated by the appended claims. All references, publications and patents mentioned herein are hereby incorporated by reference herein in their entirety.

Claims

CLAIMSWhat is claimed is:

1. An apparatus for light collection and detection comprising:

a sample which emits or scatters light;

at least one waveguide in proximity to or completely surrounding the sample, wherein the

waveguide has at least one end face; and

a light detection assembly optically coupled to the at least one end face of the waveguide;

such that light emitted or scattered by the sample and totally internally reflected within the

waveguide reaches the at least one end face of the waveguide where the emitted or

scattered, totally internally reflected light exits from the waveguide and is detected by the

light detection assembly.

2. An apparatus for light collection and detection comprising:

a light source;

a sample, wherein the sample emits or scatters light as a result of illumination by light

from the light source;

waveguide has at least one end face; and

light detection assembly.

3. The apparatus of claim 2, further comprising means for focusing light exiting from

the at least one end face of the waveguide.

4. The apparatus according to claim 1 , wherein the at least one waveguide in

proximity to the sample comprises an upper layer of optical material between the light

source and the sample, and a lower layer of optical material positioned such that the

sample is between the upper layer of optical material and the lower layer of optical

material.

5. The apparatus according to claim 1, wherein the at least one waveguide in

proximity to the sample comprises an embedding medium completely surrounding the

sample.

6. The apparatus according to claim 1 , wherein the at least one waveguide in

sample and at least one additional layer of optical material contacting the embedding

medium.

7. The apparatus according to claim 6, wherein the at least one waveguide contacting

the embedding medium comprises an upper layer of optical material between the light

source and the embedding medium, and a lower layer of optical material, positioned such

that the embedding medium is between the upper layer of optical material and the lower

layer of optical material.

8. The apparatus according to claim 7, wherein the optical material of the upper plate

and the optical material of the lower plate are independently selected from the group

consisting of glasses and plastics.

9. The apparatus according to claim 7, wherein the upper layer and lower layer of

optical materials are plates and the embedding medium is a gel.

10. The apparatus according to claim 9, wherein the gel is selected from the group

consisting of polyacrylamide gels and agarose gels.

11. The apparatus according to claim 9, wherein the sample contained in the gel is

selected from the group consisting of proteins, DNA, RNA, nucleotides, peptides, and

carbohydrates.

12. The apparatus according to claim 1, wherein the sample comprises cells, tissue

slices, or particulates.

13. The apparatus according to claim 3, wherein the means for focusing light exiting

from the at least one end face of the waveguide comprises a shaped lens integrated into the

end face of the waveguide.

14. The apparatus according to claim 3, wherein the means for focusing light exiting

from the at least one end face of the waveguide comprises a holographic lens assembly which separates the light exiting from the waveguide into at least two color components or

wavelength regions.

15. The apparatus according to claim 1 , wherein the at least one waveguide comprises

an optical material with an index of refraction which varies along the normal to the surface

of the waveguide.

16. The apparatus according to claim 15, wherein the optical material with an index of

refraction which varies along the normal to the surface of the waveguide also focuses the

light which exits from the at least one end face of the waveguide.

17. The apparatus according to claim 1, wherein the light detection assembly comprises

a fiber optic array.

18. The apparatus according to claim 1 , wherein the light detection assembly comprises

a photodiode array.

19. The apparatus according to claim 1 , wherein the light detection assembly comprises

a light conduit.

20. The apparatus according to claim 3, wherein the means for focusing light exiting

from the at least one end face of the waveguide comprises at least one lens.

21. The apparatus according to claim 3, wherein the means for focusing light exiting

from the at least one end face of the waveguide comprises at least one mirror.

22. The apparatus according to claim 3, wherein the means for focusing light exiting

from the at least one end face of the waveguide comprises at least one lens and at least one

mirror.

23. The apparatus according to claim 1, wherein the light emitted or scattered by the

sample is bioluminescent, chemiluminescent, or phosphorescent light.

24. The apparatus according to claim 1, wherein the light emitted or scattered by the

sample is scattered light.

25. The apparatus according to claim 1, wherein at least about 64% of the light emitted

or scattered by the sample is totally internally reflected within the waveguide.

26. The apparatus according to claim 1 , wherein at least about 77% of the light emitted

27. A method of detecting light, comprising

placing a sample in proximity to or completely within at least one waveguide, where the

waveguide has at least one end face;

illuminating the sample with a light source, such that the illuminated sample emits or scatters light; focusing light exiting from the at least one end face of the waveguide; and

detecting light exiting from the at least one end face of the waveguide;

wherein the light emitted or scattered by the sample is totally internally reflected within the

waveguide, and reaches the at least one end face of the waveguide where the emitted or

scattered, totally internally reflected light can exit from the waveguide and is detected by

the light detection assembly.