WO2000079250A1

WO2000079250A1 - System for eliminating background in gene chip array analysis

Info

Publication number: WO2000079250A1
Application number: PCT/US2000/016696
Authority: WO
Inventors: Allison C. Bliton
Original assignee: Genomic Solutions Inc.
Priority date: 1999-06-18
Filing date: 2000-06-16
Publication date: 2000-12-28
Also published as: AU5876700A

Abstract

A system to eliminate background in optically scanned samples is disclosed which selectively blocks excitation (11) or emission (12) light through the use of filters (10).

Description

SYSTEM FOR ELIMINATING BACKGROUND IN GENE CHIP ARRAY

ANALYSIS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority based on U.S. provisional patent application no. 60/140,063 filed June 18, 1999.

FIELD OF THE INVENTION

The Invention relates to the field of imaging. In particular, the Invention relates to that part of imaging that permits the selective transmittal or rejection of light through an objective. BACKGROUND OF THE INVENTION

This Invention is a system to increase the sensitivity of imaging low-level fluorescent samples, for example, "gene chip array" or "microarray" images found in the field of genomics research. As only one example, that field uses fluorescent dyes or markers to assess hybridization of deoxyribonucleic acid ("DNA") "probes" to DNA samples "spotted" or bond to a substrate, such as a glass slide. The degree of comparative hybridization is assessed by optical scanning of the sample microarray upon excitation from a light source.

However, depending on the level of fluorescence found among tested DNA "spots" in the microarray, certain factors, such as backscatter, may impair the accuracy of the acquired data. For example, the sensitivity of low-level fluorescence gene chip images can be degraded by excessive fluorescent background from planes above and below a sample surface that contains the fluorescent-labeled genetic material. The fluorescent background can emanate from the substrate (e.g., slide, coverslip, gel, plastic, etc.) that the genetic material is attached to, or from solutions that the material is immersed in. It can also emanate from the silination or other materials attached to the underside of the slide or substrate. Confocal microscopy can be used to reject some of this background, however, on-axis, out-of-focus fluorescence can still pass through the confocal pinhole, and thus contributes to the fluorescent background in the image. The present Invention incorporates novel features to reduce or eliminate such background. The Invention will eliminate background from some portions of the out- of-focus material, whereas standard confocal systems only partially eliminate the background fluorescence. The Invention allows for an extended depth of field while maintaining optical-sectioning properties. The extended depth of field allows for more options of automated slide handling techniques that can be utilized with this Invention. Although analogous techniques may be found in light microscopy, see Charles J. Koester, "Scanning mirror microscope with optical sectioning characteristics: applications in ophthalmology" APPLIED OPTICS, Vol. 19, No. II, 6/1/1980, the present Invention incorporates novel features to permit use with the large area samples (approximately >lxlmm) found in gene chip arrays, as well as to allow a larger depth of field and to optimize the fluorescence collection efficiency.

SUMMARY OF THE INVENTION This Invention is a system for eliminating background from out-of-focus fluorescent material, when imaging samples treated with labeled compounds, for example, gene chip arrays treated with fluorescent probes. The Invention is particularly novel in its application to gene chip arrays, its use of a larger depth of field than standard confocal techniques, and the technique used to maximize fluorescent light collection. The Invention eliminates background in optically scanned samples by selectively blocking excitation or emission light through the use of filters. BRIEF DESCRIPTION OF DRAWINGS The features and inventive aspects of the present Invention will become more apparent upon reading the following detailed description, claims, and drawings, of which the following is a brief description:

Figure 1 is an explanatory diagram of light transmission characteristics using confocal microscopy. Figure 2 is a diagram of light transmission characteristics and relative position and characteristics of filters used in the present Invention.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT A system to eliminate background in optically scanned samples is disclosed which selectively blocks excitation or emission light through the use of filters The Invention can be applied optimally for rejecting background when imaging the gene chip array samples, which are typically much larger than microscope samples. In confocal microscopy (Figure la), the excitation light, such as laser light, excites fluorophores throughout the region shaded in gray 1. Diffraction spreads the focus of the laser beam out to the white oval spot-width 2. The "optical section" is defined as the length of the oval from top to bottom. However, light is also collected in the on-axis triangular regions 3, at decreasing intensity as the distance from the focal plane increases. In the Koester light microscopy technique, excitation light is passed through only one half of the objective, shown in the gray region 4 of Figure lb, and fluorescence light is collected only through the other half of the objective, shown in the black region 5; a small central region 6 is blocked altogether, shown in the center white region. Diffraction still spreads the laser focus out to an oval 7 (Figure lc), exciting the full central black region. The confocal pinhole limits light collection to the same region as before. However, because the on-axis fluors above and below the focal plane 8 (white triangular regions) are not excited at all, no fluorescence is collected from them. Because only one half of the objective numerical aperture is used in the

Koester technique, the lateral spot size 9 (Figure lc) in the direction shown is twice as wide as in the orthogonal direction (e.g., into the page). The Koester technique collects only approximately one half of the light as confocal, using the same objective, but rejects the background above or below the focal plane. If there is a lot of background from solution on the sample substrate, or from a glass or plastic slide or coverslip, the technique could perform significantly better than confocal due to the background suppression.

The Invention improves on this technique through novel means for use with stage scanning or objective scanning techniques in the analysis of gene chip arrays. If the pupil plane (back aperture) is external to the large-field objective, it can be adapted to beam scanning techniques. It can also be adapted either in a line or point- scanning mode. The Invention allows a deeper depth of field, while maintaining complete rejection of background from the back of the slide or from other out-of- focus regions. As shown in Figure 2a, the Invention positions a filter 10 specific for wavelengths of the operator's choice which passes the excitation light through one section 11, and passes emitted fluorescence light 12 through another, while blocking all light through the remaining area 13 of the filter.

Figure 2b shows a filter analogous to the Koester technique described above and shown in Figure 1 used in the Invention. The horizontally-striped area 14 is where the excitation light passes through, the blank area 15 is where all light is blocked, and the vertically striped area 16 is where the emitted fluorescence passes thorough.

Figure 2c shows a filter 10 that is part of the Invention. In order to have a deeper depth-of-field, the excitation laser beam is condensed to the area shown with horizontal striping 17. The excitation laser beam is placed off-center in the objective so that fluorescence from the triangular region 3 is not excited. Light in the blank area 18 is blocked, and light in the vertically striped area 19 is collected. This allows most of the fluorescence from the sample to be collected, in contrast to one half of it being blocked as in Figure 2b, while still blocking high backgrounds from the back of the slide or other sources in the sample. For ease of manufacture, a filter 10 such as that shown in Figure 2d may be preferable, with select excitation transmission area 20, emission light transmission area 21, and complete light blockage area 22 of the indicated configuration.

Preferred embodiments of the present Invention have been disclosed. A person of ordinary skill in the art would realize, however, that certain modifications would come within the teachings of this Invention, and the following claims should be studied to determine the true scope and content of the Invention. In addition, the methods and structures of the present Invention can be incorporated in the form of a variety of embodiments, only a few of which are described herein. It will be apparent to the artisan that other embodiments exist that do not depart from the spirit of the Invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.

Claims

CLAIMSWhat is claimed is:

1. A system for eliminating background in images of scanned samples, comprising: a. a source of excitation light; b. a test sample containing one or more chemical compounds reactive to one or more wavelengths in said excitation light; c. an objective lens; d. an optical filter; e. means for illuminating said sample with said excitation light by passing said light through said objective and filter; and f. means for directing emission light from said sample illuminated with said excitation light through said objective and filter.

2. The system of claim 1, wherein said filter is a wavelength-specific filter.

3. The system of claim 2, wherein said filter has separate and discrete regions for transmission of said excitation light, for transmission of said emission light, and for complete blockage of any and all light.

4. The system of claim 1, wherein said excitation light is laser light.

5. The system of claim 3, wherein said excitation light is laser light.

6. The system of claim 1, wherein said sample is a DNA microarray.

7. The system of claim 3, wherein said sample is a DNA microarray.

8. The system of claim 4, wherein said sample is a DNA microarray.

9. The system of claim 5, wherein said sample is a DNA microarray.