Classifications

• • 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
  • G01N21/55—Specular reflectivity
  • G01N21/552—Attenuated total reflection
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1429—Signal processing
  • G01N15/1433—Signal processing using image recognition
• • 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
  • G01N21/645—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
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/47—Scattering, i.e. diffuse reflection
  • G01N2021/4704—Angular selective
  • G01N2021/4707—Forward scatter; Low angle scatter
• • 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
  • G01N2021/4704—Angular selective
  • G01N2021/4728—Optical definition of scattering volume
• • 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
  • G01N2021/4735—Solid samples, e.g. paper, glass

Definitions

• the present invention relates in general to a method and apparatus for the detection of light scattered through a range of wide angles and, in particular, to the detection of light scattered by microscopic biological samples through wide angles.
• FIG. 1 is a block diagram of a conventional laser imaging system as shown in Tomei et al.
• a primary laser 10 provides a beam 12 to a beam expander 14 composed of an objective lens and a spatial filter.
• the beam 12 exits the beam expander 14 as an input collimated beam 16.
• a three dimensional beam position controller 18 receives collimated beam 16.
• the beam controller 18 includes an imaging lens and galvanometrically driven mirrors to provide control of the spot focus on sample target 20.
• Detector assembly 22 comprises an optical fiber faceplate 24, diffusion elements 26 and a photomultiplier tube 32.
• the image signal produced by tube 32 is subsequently sent to a support computer system which further processes the image signal for display on a high resolution monitor or for storage in an image storage unit. Further details concerning the overall construction and operation of the laser imaging system are provided in Tomei et al.'s above-incorporated patent.
• One novel aspect of Tomei et al.'s laser scanning system is its means of image capture. Laser light is directed to a particular location on a sample target, such as a glass slide.
• Forward-scattered light that passes through the sample target is collected by an optical fiber faceplate and thereafter transmitted to a photomultiplier tube for detection.
• the optical fiber faceplate is combined with an interference filter that is matched to the emission cone of the optical fibers to eliminate unscattered primary laser light.
• the optical fiber faceplate may be designed to image both forward light scatter and fluorescence emission by employing an optical fiber faceplate that is bias-cut, as opposed to square-cut which is designed to capture both scattered and unscattered light. Both square-cut and bias-cut faceplates can be used to collect scattered and fluorescence emission.
• the bias-cut one is devised to reduce greatly the background due to the unscattered and slightly scattered excitation laser beam.
• FIGs 2 A and 2B are schematic diagrams of the square-cut and bias-cut optical fiber faceplates respectively. Both types of faceplates comprise an internal reflectance tube 28 and flashed opal diffusion filters 30 that comprise the diffusion elements 26 in Figure 1.
• the bias-cut optical fiber faceplate in Figure 2B additionally employs a dielectric interference filter 34. The operation of these particular elements are further described in Tomei et al.
• the orientation of both faceplates in Figures 2 A and 2B show that only forward scattered light is collected by these faceplates. Light that is widely scattered to angles approaching 90 degrees, however, is not collected by either faceplate. Although forward scattered light gives a good deal of image information about the sample, light that is widely scattered provides important information that is not necessarily found in forward scattered light.
• high-angle scattered light provides important information needed to differentiate events marked by other modes such as absorption and fluorescence.
• Some debris in cell preparations may bind stain materials to the same degree as do target cells; however, the cells have very different light scattering properties compared with the debris and therefore, can be differentiated from the debris during the course of the analysis.
• Both forward and high angle scattering data contain important information regarding to the size, shape and others of scatters. It is widely accepted that small angle (within 10 to 15 degree) forward scattering intensity provides information on size of the scatters such as cells, while the wide-angle scattering intensity is relevant to the surface property (granularity, shape) of scattering particles.
• small angle (within 10 to 15 degree) forward scattering intensity provides information on size of the scatters such as cells
• the wide-angle scattering intensity is relevant to the surface property (granularity, shape) of scattering particles.
• cells of interest and cellular debris may be of similar size, but they may have quite distinctive surface properties. For example, cells are somewhat round and smooth and have membranes enclosures - resulting in a number of intracellular dielectric interfaces. On the other hand, cellular debris tend to lack these properties. Therefore, the wide-angle scattering signals are helpful in discriminating between cells and debris. In U.S.
• Patent Number 5,072,382 entitled “METHODS AND APPARATUS FOR MEASURING MULTIPLE OPTICAL PROPERTIES OF BIOLOGICAL SPECIMENS", issued on December 10, 1991, Kamentsky describes a way to detect obliquely scattered light that is trapped in a specimen slide by placing a sensor at the edge of the slide.
• Kamentsky describes a way to detect obliquely scattered light that is trapped in a specimen slide by placing a sensor at the edge of the slide.
• the wide angle scattering detection, by one given sensor at one edge of a slide is prone to the artifacts due to variations in distance and orientation of cells relative to the detector, and therefore may not give accurate event correlation.
• the present invention is a novel apparatus for the detection and analysis of widely scattered light that impinges on a sample target.
• the presently claimed apparatus operates in conjunction with a light imaging system that focuses light onto a sample target.
• the sample target comprises a translucent material that allows total internal reflection of light at some given critical angle (i.e. a wide angle).
• the presently claimed apparatus comprises: a supporting structure, said structure supporting said sample target; at least one optical fiber, each fiber having a first and a second end, the first end being placed proximate to the side surface of the sample target such that light scattered and transmitted through the side surface proximate to the side surface is collected by the first end; a photomultiplier tube coupled to at least one second end of said optical fibers.
• a plurality of optical fibers around the side surface of the sample target may be extended to cover a substantial portion of the side surface periphery in order to capture an even greater percentage of the widely scattered light through the sample target medium.
• the optical fibers can optionally be flexibly formed into a bundle. Each bundle of fibers can then become the input into a detector; thereby reducing the number of detectors needed to capture widely scattered light.
• One advantage of the present invention is that light scattered through a wide angle is accurately detected and analyzed simultaneous with forward scatter image data.
• Another advantage of the present invention is cost.
• the use of a plurality of comparatively low cost optical fibers that can funnel light into a small number of comparatively more expensive detectors reduces manufacturing cost over systems that might employ a great number of detectors arrayed around the periphery of the target sample.
• FIG. 1 is high level block diagram of a conventional laser imaging system.
• Figures 2 A and 2B are schematic illustrations of two types of optical fiber faceplates used in the convention laser imaging system depicted in Figure 1.
• Figure 3 is a side, cut-away view of a wide angle scatter detector made in accordance with the principles of the present invention.
• Figure 4 is a perspective view of the wide angle scatter detector depicted in Figure 3.
• Figure 5 is a top view of an embodiment based on the design of Figures 3 and 4.
• Figure 6 is a side view of one embodiment of the present invention whereby the sample target comprises an electrophoretic gel sandwiched by two translucent plates.
• Figures 7 A and 7B are top views of two separate embodiments of the present invention using the electrophoretic gel sample target as depicted in Figure 6.
• FIG. 3 a side, cut-away view of a wide angle scatter detector made in accordance with the principles of the present invention is depicted.
• Laser beam 40 is focused onto a specific area of the sample target 42.
• Sample target 42 is supported at its bottom surface by frame 44.
• Frame 44 may be constructed out of aluminum or some other suitable material.
• Sample target 42 may comprise either a single light transmissive substrate (such as glass, plexiglass fused silica and polymers of optically clear or the like) or a multiplicity of such substrates.
• cells or particles or a thin layer of tissue 46 are placed for image analysis.
• these rays reflect off the bottom surface instead of being transmitted through the target. Through a series of such internal reflections, a subset of these rays are eventually transmitted laterally through the side surface of the target. These rays are collected by optical fibers 50. Once collected by optical fibers 50, these rays are subsequently sent to light detector 52 and an output digital image is sent for analysis to an image processor. It will be appreciated that any suitable light detector is sufficient for the purposes of the present invention, such as a photomultiplier tube or any sensitive solid state detector or the like.
• the amount of transmitted widely scattered light depends on the index of refraction of the target substrate and the surface characteristics.
• a different surface interface may be presented.
• cells may be embedded in a thin layer of optically clear polymer coating resting on the top surface of a glass slide - thus providing for an enhanced coupling of wide scattered light into the glass slide.
• the material at the interface might be a gel for DNA sequencing applications or other transmissive materials such as immersion oil or wax.
• Figure 4 gives a perspective view of one embodiment of the wide angle detector made in accordance with the principles of the present invention.
• the optical fibers are arrayed substantially around the perimeter of the sample target. Having substantial coverage around the perimeter improves the accuracy of detecting the actual amount of widely scattered light.
• the individual optical fibers themselves may be made of any suitable transmissive material, such as silica, glass, translucent polymer.
• suitable transmissive material such as silica, glass, translucent polymer.
• the choice of material depends on a number of factors, including the wavelength of the scattered light, the required numerical aperture and cost. Additionally, it may be advantageous to select a material that can be easily bent while maintaining the maximal transmission.
• Figure 5 is a top view of a current embodiment where individual fibers are connected and formed into a bundle.
• the optical fibers of Figure 5 are abutted end-to- end such that the fibers are routed incoherently into a bundle 60 of a certain size and shape.
• the shape of bundle 60 may be round, rectangular, or square depending upon the detector in use.
• the size of the bundle is a function of individual fiber sizes (typically
• the total number of fibers in turn depends upon the amount of the perimeter that is desired to cover.
• Bundle 60 is then coupled to a detector 64. If, however, the light transmitting area of the bundle is larger than the sensitive area of a detector, lenses 62 may optionally be used in between to make the coupling more efficient.
• the fiber ends may be stacked into several layers. Typically, three layers are used for round fibers. If square fibers are used, a single layer of the right size may be sufficient. To increase the transmissivity of the fibers, both ends are polished.
• the numeric aperture (NA) of a single fiber preferably has a range that depends on the size of the fiber, the thickness of the sample plate and the gap from the edge of the plate to the entrance face of the fiber arrays.
• the first embodiment is suited to analyzing microscopic slides supporting cells, or particles, or a thin layer of tissue.
• microscopic slides are 2.5 cm by 7.5 cm in area.
• the fiber array might consist of 2600 plastic optical fibers of 0.25 mm in diameter in three layers.
• the embodiments of Figures 3, 4 and 5 are well suited to this type of application.
• Another embodiment of the present invention is suited to electrophoretic gel plates.
• the embodiment for this application is very similar to the one for microscopic slides. The main difference is size.
• Gel plates are typically 45cm X 35cm.
• the typical structure is a gel layer (usually comprising polyacrylamide or some such gel ⁇ like substance) approximately 300 microns thick and sandwiches between two glass plates, 2-3mm thick each.
• Figure 6 shows a side view of the embodiment 70 having the electrophoretic gel plates coupled to the optical fiber bundles.
• Plates 72 and 74 coupled to an optional light-transmissive side retainer 76, provide a top and bottom boundary for gel layer 80 to reside.
• Optical fiber 82 also coupled to optional side retainer 76, captures the widely scattered light or fluorescent light 88 that emanates as a result of the interaction between impinging laser light 84 and interaction sites 86, which may comprise DNA fragments or the like as discussed below.
• some of the fluorescent or widely scattered light (such as ray 88) is transmitted through the gel and directly into fiber 82; while other rays (such as ray 90) are channelled through the glass plate to fiber 82 at the edges of the gel plate.
• optical fibers can be placed at other locations around the gel plates, such as the bottom to capture forward scatter light or fluorescent light.
• the gel layer contains several columns of DNA fragment bands.
• the bands, tagged with fluorophores, are normally invisible; but fluorescent when a focused laser beam interacts with their labeling fluoro-molecules.
• the light is then passed through cutoff filters to block the excitation wavelengths before reaching the detector.
• the number of detectors in a given embodiment can be determined by the manner in which the DNA fragments are labeled.
• the gel can contain 4 lanes of (A), (C), (G), and (T), each tagged with a single fluorophore.
• the gel could contain a single lane with (A), (C), (G) and (T) fragments tagged with four different fluorophores.
• tags and lanes are possible.
• a single detector may be sufficient whereas, for the latter scheme, four detectors, having suitable filters, may be necessary to measure simultaneously the four color fluorescence.
• FIGS 7A and 7B are top views depicting two such embodiments - a first embodiment having a single detector and a second embodiment having multiple detectors respectively.
• Embodiment 100 comprises gel 102 having four lanes 104, 106, 108, 110 tagged with a single fluorophore. Light from the optical fibers, proximate to each of the lanes, are combined into a single bundle 112 and is passed through filter 114 to block transmission of the impinging laser light to detector 116.
• the second embodiment 120 also has gel 102, but instead of four lanes, it has one lane 122. Fluorescent light from this single lane 122 is captured by optical bundle 124 and is from there split into four separate channels 126, 128, 130, and 132. These channels are coupled to filters 134, 136, 138, and 140, which only pass a given wavelength corresponding to one of the fluorescent wavelengths. Thus, each fluorescent wavelength is detected by a given detector 142, 144, 146, or 148.
• the fluorescent light captured by optical bundle 124 could be input into a integrating sphere having multiple detectors thereto.
• An embodiment of such an alternative is shown in commonly assigned, co-pending application Serial Number PCT US96/XXXXX filed on 13 May 1996, entitled MULTI ⁇ CHANNEL ACQUISITION USING INTEGRATING SPHERE, by D. Tomei; and herein incorporated by reference.
• the waveguide plate to couple the fluorescence at any laser-fluorophore interaction sites to the edges of the plate.
• n gl and n ⁇ i.e. the refractive index of glass substrates 72 and 74, respectively
• the fluorescence is mostly confined within the gel layer, or the gel forms the waveguide.
• n gl and n g2 is large than n ge *, the fluorescence emission is mostly confined respectively in both the top and bottom glass plates. It will be appreciated that the present invention encompasses all such variations.
• the upshot of these principles is that it is possible to discriminate cells from cellular debris and fluoro-stained cells from color stained cells by either gating or thresholding on the intensities of scattered light.
• discrimination may be made by counting events with finite absorption or fluorescence but within certain range (or "gate") of side scattering intensity.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Engineering & Computer Science (AREA)
• Signal Processing (AREA)
• Dispersion Chemistry (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Priority Applications (3)

Applications Claiming Priority (2)

Publications (1)

Family

ID=23794759

Family Applications (1)

Country Status (4)

Cited By (9)

Citations (1)

• 1996
  • 1996-05-21 WO PCT/US1996/007374 patent/WO1996037767A1/en not_active Application Discontinuation
  • 1996-05-21 EP EP96920356A patent/EP0830586A1/de not_active Ceased
  • 1996-05-21 AU AU58692/96A patent/AU5869296A/en not_active Abandoned
  • 1996-05-21 JP JP8535820A patent/JPH11505928A/ja active Pending

Patent Citations (1)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events