Optical: systems and elements – Deflection using a moving element – Using a periodically moving element
Reexamination Certificate
2000-04-18
2002-05-07
Spyrou, Cassandra (Department: 2872)
Optical: systems and elements
Deflection using a moving element
Using a periodically moving element
C359S216100, C250S584000, C250S585000, C250S586000, C250S591000, C356S417000
Reexamination Certificate
active
06384951
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to optical scanning of substrates, and in particular to efficient and uniform collection of emitted or scattered light. The invention includes the application of phase sensitive detection to scanned images to improve the discrimination of multiple signal sources and reduce noise. It is particularly useful for automated, rapid and sensitive fluorescent gel scanning.
BACKGROUND OF THE INVENTION
Large-scale genome and proteome projects involve assay techniques that are significantly hindered by current substrate imaging techniques. For example, gel electrophoresis is a critical but slow step in analyses of nucleic acid sequences and proteins. DNA or protein samples are often electrophoresed in a gel, which separates sample components, and this is followed by formation of an image for quantitation. A gel image can be produced by using a radio-labeled sample and exposing the gel to an x-ray film or storage phosphor plate followed by film development or phosphorescence quantitation. Radioactive labeling, while sensitive, is hazardous and expensive. Fluorescent dyes which bind to the sample and thereupon fluoresce brightly are a preferred method of sample labeling. Application of fluorescence labeling to slab gel electrophoresis and other two-dimensional analysis substrates creates a need for fluorescence imaging instrumentation. Fluorescent intensity images can be created by photographing the substrate or imaging it with a charge coupled device (CCD) areal detector chip. Photography is non-quantitative due to the film's highly non-linear exposure/density function. A CCD detector is capable of quantifying the fluorescence intensity of an image but the spatial resolution is limited by the number of pixels on the chip itself. Both forms of detection involve the use of cameras which have relatively poor collection efficiency, rely on broad and even illumination, and require optics which minimize geometric distortion. These constraints become more acute when imaging large substrates such as electrophoresis gels. Further, cameras are not configured for the rejection of background light, resulting in high background levels.
One alternative for fluorescence quantitation of substrates is image scanning. The fluorescence is measured sequentially at each point in a substrate, creating an image based upon millions of individual pixel measurements. Scanning systems quantify only one point at a time and do not require imaging optics, allowing the optical system to be optimized for collection efficiency and to satisfy other constraints. Fluorescence is typically excited by a laser, which is far brighter and more uniform than other forms of illumination. There are two types of designs for scanning systems and in both types the time required to produce a scanned image increases with the area of the substrate. Scanning-head designs physically translate the excitation and collection optics over the substrate area, resulting in high collection efficiency and noise rejection at the expense of speed. Scanning-beam designs, also commonly termed scanning-spot and flying-spot, accomplish rapid movement of an illumination spot separate from a stationary or slow-moving collection system, imaging rapidly at the expense of collection efficiency.
The designs of existing fluorescence scanning devices provide a trade-off between scanning optic detection systems with high sensitivity and scanning beam detection systems with speed but low sensitivity. Neither system architecture achieves maximum image noise rejection because neither system architecture can eliminate non-random background noise. Background noise consists, in part, of excitation light scattered from the substrate itself as well as from surface and bulk contaminants. Scatter intensity varies with the characteristics of the substrate. Membranes are generally opaque and therefore scatter nearly all the excitation light. Agarose gels are translucent, scattering a fraction of the incident excitation light. Polyacrylamide gels are very clear and exhibit the least scatter. The scattered light component of background noise can consist of elastic scatter at the same wavelength as the incident light and Raman (inelastic) scatter that is red-shifted due to interaction between incident light and vibrating hydrogen-oxygen bonds of water in the substrate. Elastically-scattered light is largely blocked by an emission filter, but it can induce fluorescence in the filter itself which cannot be distinguished from signal fluorescence. Raman scatter often overlaps the emission spectrum of the fluorescent dye, thereby making it past the emission filter. Other significant components of background noise include fluorescence from unbound dyes and autofluorescence of the spectral filter and glass or plastic substrate materials. The foregoing components of background noise are considered to be non-random signals and cannot be removed by conventional signal-averaging techniques.
Therefore, there is a need in the art to provide a high-throughput image scanning device that produces high sensitivity and low background noise fluorescent images, thereby increasing the information content in an image for a given amount of nucleic acid or protein material. Ideally, the device would suppress undesired non-random signals to such a degree that faint fluorescence signals could be imaged even on opaque, scattering, or highly auto-fluorescent substrates.
SUMMARY OF THE INVENTION
The present invention provides a scanning apparatus and methods to obtain automated, rapid and sensitive scanning of substrate luminescence (fluorescence, phosphorescence, chemiluminescence, nano-particle emission, etc.), optical density or reflectance. The scanning apparatus employs moving-beam excitation to rapidly measure samples on or in a variety of substrates, including fluorescently-stained gels, silver-stained gels, developed x-ray films, storage phosphor screens, membranes, multi-well plates, petri dishes, glass and plastic surfaces, silicon chips and other emitting, reflecting or scattering substrates. The scanning apparatus employs a constant path length optical train to achieve highly uniform images with a minimum of optical complexity and no need for focus adjustment for a variety of substrates with widely-varying shapes, sizes, thicknesses, and optical characteristics. The constant optical path length also facilitates the use of phase sensitive signal processing. A method is provided for the use of phase nulling, either electronically or by a combination of electronics and software, to eliminate non-random baseline components and thereby enable the use of signal averaging to improve the signal-to-noise ratio. A method is further provided to allow the use of phase-sensitive detection for the improved discrimination of multiple dye fluorescence signals in the same substrate on the basis of their excited-state lifetimes.
The constant path length optical train of this invention is used to direct light onto and collect light from a substrate. It comprises a light source, a scanning mirror to receive light from the light source and sweep it across a steering mirror, a steering mirror to receive light from the scanning mirror and direct it to the substrate, whereby it is swept across the substrate along a scan arc, a waveguide or mirror to collect the optical signal, and a photodetector to receive emitted or scattered light from the substrate, wherein the optical pathlength from the light source to the photodetector is substantially constant throughout the sweep across the substrate.
The scan mirror preferably has one or more flat surfaces and is mounted on a scanning motor to rotate the mirror and thereby scan the reflected beam. Although the rotation can be oscillating, it is preferably unidirectional and at constant speed and therefore produces a constant angular sweep and constant incident optical power on the substrate per unit time. The scanning mirror can alternatively be curved, thereby simultaneously providing beam movement and focusing. In a first
Basiji David A.
Esposito Richard J.
van den Engh Gerrit J.
Boutsikaris Leo
Greenlee Winner and Sullivan P.C.
Spyrou Cassandra
University of Washington
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