1. Field of the Invention
The present invention relates to a fluorescence detection method and a fluorescence detection system in which a fluorescence-labeled biological specimen is irradiated with excitation light, and fluorescence emitted from the biological specimen by the irradiation is detected.
2. Description of the Related Art
Conventionally, the fluorescence detection systems which use fluorescent material as a labeling material are known in the field of biochemistry. It is possible to investigate gene arrangement or gene expression levels, isolate or identify protein, or evaluate molecular weight or characteristics of protein, by reading fluorescent images obtained by use of a fluorescence detection system.
For example, a fluorescent dye is added to a solution containing DNA fragments which are to be electrophoresed, and thereafter the DNA fragments are electrophoresed on a gel support. Alternatively, DNA fragments are electrophoresed on a gel support which contains a fluorescent dye. After the electrophoresis, the electrophoresed DNA fragments are labeled, for example, by soaking the gel support in a solution containing a fluorescent dye. Then, the DNA distribution on the gel support can be detected by exciting the fluorescent dye with excitation light, detecting fluorescence emitted from the excited fluorescent dye, and producing an image of the detected fluorescence.
Alternatively, it is possible to detect a DNA distribution by using the Southern blotting technique as follows.
(a) DNA fragments are electrophoresed on a gel support.
(b) DNA fragments are denaturated.
(c) At least a portion of the DNA fragments is transcribed onto another support for transcription, which is made of, for example, nitrocellulose, in accordance with the Southern blotting technique.
(d) The denaturated DNA fragments are hybridized with a probe which is prepared by labeling a DNA or RNA complementary to an objective DNA with a fluorescent dye, so that only DNA fragments which are complementary to the probe DNA or probe RNA are selectively labeled.
(e) The fluorescent dye is excited with excitation light, fluorescence emitted from the fluorescent dye is detected, and an image of the detected fluorescence is produced, so that the distribution of the objective DNA on the support for transcription is detected.
In recent years, the microarray analysis system as a biochemical analysis system has been receiving attention. For example, in the microarray analysis system in which a fluorescent material is used as a labeling material, a number of separate spots of specifically binding materials are formed on different positions on a support such as a slide glass or membrane filter by putting drops of the specifically binding materials on the support by use of a spotter device, where each of the specifically binding materials can be specifically bound to a substance of biological origin such as a hormone, tumor marker, enzyme, immune body, antigen, abzyme, other protein, or nucleic acid (e.g., cDNA, DNA, RNA), and the nucleotide sequences, and the nucleotide lengths, compositions, and the like of the specifically binding materials are known. Subsequently, a substance of biological origin labeled with a fluorescent labeling material such as a (fluorescent) dye is specifically bound to (hybridized with) the spots of the specifically binding materials on the support, where the substance of biological origin is obtained in advance from an organism by extraction, isolation, and the like, and may be chemically processed or chemically modified. Thereafter, analysis of the substance of biological origin is performed by irradiating the above microarray with excitation light, and photoelectrically detecting fluorescence or the like emitted from the labeling material.
In the conventional microarray analysis systems, devices which produce a two-dimensional image of fluorescence emitted from the spots (which are two-dimensionally arrayed in a grid pattern) for reading are mainly used. For example, U.S. Pat. No. 6,504,167 discloses a device which reads a two-dimensional distribution of fluorescence by use of a line scanning system, and produces an image of the two-dimensional distribution of fluorescence. However, in the line scanning system, it is necessary to repetitively move an excitation-light irradiation unit and a fluorescence reception unit, so that the inertia management in the mechanical units is required, and it is difficult to downsize the above device.
On the other hand, for example, Japanese Unexamined Patent Publication No. 2004-333333 and U.S. Patent Application Publication No. 20050048595 disclose other systems for reading fluorescence emitted from the spots on a substrate disk. In the disclosed systems, spots of the hybridized specifically binding materials are concentrically or spirally arrayed on the substrate disk, and reference marks enabling determination of the positions of the spots are arranged on the substrate disk. Then, information on the fluorescence is read from the spots by one-dimensionally (concentrically or spirally) scanning the spots on the substrate disk.
Specifically, U.S. Patent Application Publication No. 20050048595 discloses a method for analyzing the strength of bonding between a target substance and materials for detection. In the disclosed method, drops of solutions containing the materials for detection are put on predetermined positions on the substrate disk while rotating the substrate disk, and the drops of solutions containing the materials for detection are solidified on the substrate disk. Then, drops of a solution containing the target substance labeled with a fluorescent labeling material are put on the solidified materials for detection so as to cause reaction between the target substance and the materials for detection, and portions of the target substance which are not used in the reaction are washed off. Thereafter, portions of the target substance which have reacted with the materials for detection are irradiated with excitation light while the substrate disk is rotated, fluorescence emitted from the fluorescent label is detected with a detector, and the detected intensity of the fluorescence is analyzed for obtaining the strength of bonding between the target substance and the materials for detection.
In the systems for fluorescence detection by use of the substrate disk which are disclosed in Japanese Unexamined Patent Publication No. 2004-333333 and U.S. Patent Application Publication No. 20050048595, the fluorescence is detected at the same time as the irradiation of the excitation light, the lens which makes the excitation light converge on predetermined spots also collect the fluorescence emitted from the spots, and the optical axis of the excitation light is identical to the optical axis of the fluorescence emitted from the spots. However, when the excitation light enters the fluorescence detector, the excitation light produces noise, and lowers the signal-to-noise ratio in the signal indicating the detected fluorescence. Therefore, an optical filter for cutting off the excitation light is arranged in front of the light reception surface of the fluorescence detector in order to prevent entrance of the excitation light into the fluorescence detector. Nevertheless, the above optical filter cannot completely prevent entrance of the excitation light into the fluorescence detector. Therefore, conventionally, it is difficult to achieve sufficient signal-to-noise ratio in the signal indicating the detected fluorescence.