JP4064855B2 - Multi-wavelength observation optical probe microscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an apparatus for analyzing gene sequence information.
[0002]
[Prior art]
As a conventional technique, a method for analyzing a gene sequence will be described as an example. In gene analysis, genomic DNA is fragmented with a restriction enzyme or the like, and a fluorescently labeled base is incorporated with an enzyme such as DNA polymerase using the cloned DNA as a template. A method of preparing a DNA chain and obtaining an electrophoresis pattern for each base of A, T, G, and C by electrophoresis and analyzing the base sequence is widely used.
[0003]
Recently, many nucleotides of known sequences, such as oligonucleotides and cDNAs, are immobilized on a substrate, reacted with sample DNA that has been cloned, and detected that specific adsorption has occurred. A method called a DNA chip for estimating the sequence has also been developed.
[0004]
In addition, as a technique for analyzing DNA, an attempt has been made to observe a state in which a probe DNA is hybridized to a sample DNA with a fluorescence microscope by a fluorescence in situ hybridization method (for example, Non-Patent Document 1). reference.).
[0005]
Problems in the gene analysis by electrophoresis and the gene analysis by DNA chip include the complexity of the analysis operation. That is, according to this method, it is necessary to perform the work of fragmenting genomic DNA, cloning it through a number of steps, and analyzing the base sequences of the enormous number of clones obtained. Furthermore, since the base sequence data obtained from the analysis does not have position information on the chromosome, in order to specify the position of the target base sequence, clones are connected by various algorithms based on each base sequence. Therefore, a lot of time and information processing ability are required. This has the problem that preparation of sample DNA takes time and cannot be applied to analysis of DNA samples that cannot be cloned, such as damaged DNA. Another problem is that the analysis is complicated.
[0006]
In contrast, the Fluorescence in situ Hybridization method is a very effective method as a method for analyzing sequence information of a non-fragmented DNA sample, which requires few steps and does not require an operation for joining information. However, the conventional observation methods using a fluorescence microscope have a problem that the resolution is at most about 500 nm and the analysis can be performed only with a resolution of about 1.5 kbp.
[0007]
The construction of a genetic map occupies a very important part in the genetic manipulation technique in that it specifies a target gene site. Therefore, development of a method for constructing a genetic map quickly, efficiently and with high accuracy is strongly desired.
[0008]
As a method for solving the problem of the Fluorescence in situ Hybridization method, a method using a near-field optical microscope probe, a tunnel microscope probe, or optical tweezers is disclosed (for example, see Patent Document 1). A method for determining the position of a specific gene sequence contained in a DNA strand to be measured, using a DNA strand to be measured and a probe molecule, a DNA analysis method comprising a DNA-specific binding operation, a fixation / extension operation, and a detection operation using a probe; and Resolution and accuracy by a gene analysis method consisting of DNA fixation operation, specific binding operation, extension operation, probe detection operation, and gene analysis method consisting of DNA fixation / extension operation, specific binding operation, probe detection operation It is possible to realize a gene sequence analysis with a high level.
[0009]
[Non-Patent Document 1]
Holmes-Davis, "Trends in Plant Science" 3 (1999) 91
[0010]
[Patent Document 1]
JP 2001-165840 A (FIG. 9, paragraph 0019)
[0011]
[Problems to be solved by the invention]
In such a gene analysis method, a technique for clearly identifying the relative position of a gene by enabling identification of a plurality of markers is required, and the biotechnology field such as analysis of membrane proteins in biological membranes is required. In the analysis of other samples, it is necessary to simultaneously analyze a plurality of fluorescent labels.
[0012]
[Means for Solving the Problems]
In the present invention, in such a method for analyzing a fluorescently labeled sample, in order to enable identification of a plurality of labels and to clearly determine the relative position of the fluorescent label,
An optical probe capable of generating a light field localized at the tip portion, an optical probe position detection means for controlling the distance between the tip of the optical probe and the sample, a fine movement means and a control means, A scanning means for the optical probe to scan the sample surface two-dimensionally, a light source for generating the localized light field, and light emitted from the sample surface close to the tip of the optical probe are collected. A condensing optical system for emitting light, a light detection means, and a data collection means;
The light source can generate excitation light of at least two or more selectable wavelengths, and further has a dynamic optical path changing means, and controls the optical path of the excitation light, thereby allowing a plurality of fluorescence to the same sample. We have devised a multi-wavelength simultaneous observation optical probe microscope that can identify multiple types of fluorescent labels and analyze relative positional information by acquiring images.
[0013]
Further, in the present invention, an optical probe capable of generating a light field localized at the tip, a light source that generates excitation light having a plurality of selectable wavelengths on the optical probe, and a scanning means for scanning the optical probe are provided. It was set as the optical probe microscope which has. Here, a calculation means for comparing and calculating fluorescence images observed with excitation light having a plurality of wavelengths can be provided.
[0014]
In the present invention, a fluorescence-labeled DNA probe or a DNA to which a fluorescence-labeled PNA probe is bound is irradiated with excitation light having a plurality of wavelengths from an optical probe capable of generating a light field localized at the tip, and a plurality of fluorescence images Was used as a DNA sequence mapping apparatus.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0016]
FIG. 1 shows a configuration diagram of a multi-wavelength simultaneous observation optical probe microscope of the present invention. In FIG. 1, a probe 11 having a light field localized at the tip portion, and probe position detection means 12, fine movement means 13 and control means 14 for controlling the distance between the tip of the probe 11 and the sample to be close to each other. Scanning means 15 for scanning the probe two-dimensionally on the sample surface; a light source 16 for generating the localized light field; and light emitted from the surface of the sample 20 adjacent to the tip of the probe 11 The optical system 17 for condensing light and the photodetector 18 are included. In the present invention, by switching the optical path of excitation light and switching data collection with the dynamic optical path changing unit 21 and the data collecting unit 19 with respect to the signal of the scanning unit 15, a plurality of fluorescent images are obtained for the same sample. Can be obtained. In this way, for example, by observing the DNA to be inspected to which a fluorescently labeled DNA probe or PNA probe is bound by high-resolution fluorescence observation, the binding position of the DNA probe or PNA probe is detected at the molecular level, and a specific DNA sequence is mapped. When performing two-dimensional scanning once, a plurality of fluorescent images are acquired for the same sample so that a plurality of types of fluorescent labels can be identified and relative positional information can be analyzed. It is a thing.
[0017]
Wherein either the light source generates excitation light of at least two or more wavelengths simultaneously, or be a combination of a light source for generating excitation light of at least different wavelengths two or more, the dynamic optical path changing means, either The wavelength can be selected. FIG. 2A is a schematic diagram of the light source and the dynamic optical path changing means, and shows an example of a light source that generates two wavelengths. Light from the light source 161 can be selected by the movable filters 31 and 32 in the dynamic optical path changing means 201. FIG. 2B shows an example in which one wavelength is selected from two light sources in the schematic diagram of the light source and the dynamic optical path changing means. The light from the light sources 162 and 163 can be switched by the movable mirror 33 in the dynamic optical path changing means 202, and the wavelength can be selected.
[0018]
In addition to the switching method using a filter or a mirror, the optical path can be switched using an acousto-optic element.
[0019]
By using such a dynamic optical path changing unit, the excitation wavelength introduced into the optical probe is switched by the dynamic optical path changing unit in the forward path and the return path during each line scan in the two-dimensional scanning of the optical probe. Two fluorescent images can be acquired simultaneously in one two-dimensional scan. FIG. 3 is an explanatory diagram of a state of switching of excitation light in two-dimensional scanning, in which fluorescence is acquired by the first excitation light in the forward path 301 and fluorescence is acquired by the second excitation light in the return path 302. Can do.
[0020]
Meanwhile, the one light source for generating excitation light of at least two or more wavelengths simultaneously, or be a combination of a light source for generating excitation light of at least different wavelengths two or more lines in the two-dimensional scanning optical probe In scanning, two fluorescent images can be simultaneously acquired in one two-dimensional scanning by switching the excitation wavelength introduced into the optical probe alternately by the dynamic optical path changing means for each line scanning. FIG. 4 is an explanatory diagram of the state of switching of the excitation light in the two-dimensional scanning. In the odd-numbered scan 401, the fluorescence is acquired by the first excitation light, and in the even-numbered scan 402, the second excitation light is used. Fluorescence can be acquired.
[0021]
Further, by combining the data acquisition methods described with reference to FIGS. 3 and 4, at least four fluorescent images can be acquired simultaneously in one two-dimensional scan. In addition, a fluorescent filter switching means can be provided and switched simultaneously.
[0022]
On the other hand, in the multi-wavelength simultaneous observation optical probe microscope of the present invention, in order to discriminate the labeling dye, it is possible to provide an arithmetic means for performing a comparison process on the fluorescence images observed with the two excitation lights.
[0023]
【The invention's effect】
By identifying multiple types of fluorescent labels and enabling analysis of relative positional information, for example, as a technique for constructing a genetic map quickly, efficiently, and highly accurately, which is useful in genetic manipulation techniques, In the method for determining the position of a specific gene sequence contained in a DNA strand to be measured, a gene analysis method further comprising a DNA-specific binding operation, a fixation / extension operation, and a detection operation using a probe using the DNA strand to be measured and a probe molecule It has become possible to provide a device for unambiguously determining the relative position of a gene, allowing the identification of multiple labels.
[Brief description of the drawings]
FIG. 1 is a diagram showing a configuration of a multi-wavelength simultaneous observation optical probe microscope of the present invention.
FIG. 2 is a schematic diagram of a light source and dynamic optical path changing means in the multi-wavelength simultaneous observation optical probe microscope of the present invention.
FIG. 3 is an explanatory diagram of a switching state of excitation light in two-dimensional scanning. FIG. 4 is an explanatory diagram of a switching state of excitation light in two-dimensional scanning.
11 Probe 12 Position detection means,
13 Fine movement means 14 Control means 15 Scanning means 16 Light source 17 Condensing optical system 18 Photo detector 19 Data collection means 20 Sample 21 Dynamic optical path changing means 22 Image arithmetic processing means 31, 32 Movable filter 33 Movable mirror 161 Light sources 162, 163 Light source 201 Dynamic optical path changing means 202 Dynamic optical path changing means 301 Outward path 302 Return path 401 Odd-numbered scan 402 Even-numbered scan

Claims (5)

An optical probe arranged opposite to the sample, a light source for introducing excitation light into the optical probe to generate a light field localized at the tip of the optical probe, and the optical probe on the sample surface in two dimensions to the scanning means for scanning, made by fluorescent staining or labeled sample fluorescence observation, the light probe microscope for performing Imaging fluorescence image, the light source, generates excitation light of at least two or more wavelengths either, or is a combination of a light source for generating excitation light of at least different wavelengths two or more, with dynamic light path changing means for selecting a wavelength to be introduced into the optical probe from the excitation light, the dynamic specific optical path change means, when said reciprocal scanning, switches the excitation light having a wavelength to be introduced to the optical probe in the forward and backward by controlling the optical path of the excitation light, the same sample A plurality of fluorescent images by acquiring the optical probe microscope characterized in that to be able to analyze the relative position information identifies the fluorescent label of a plurality of types and. An optical probe disposed opposite to the sample, a light source for introducing excitation light into the optical probe to generate a light field localized at the tip of the optical probe, and the optical probe on the surface of the sample in two dimensions to the scanning means for scanning, made by fluorescent staining or labeled sample fluorescence observation, the light probe microscope for performing Ime Jingu fluorescent image, the light source, generates excitation light of at least two or more wavelengths either, or is a combination of a light source for generating excitation light of at least different wavelengths two or more, with dynamic light path changing means for selecting a wavelength to be introduced into the optical probe from the excitation light, the dynamic specific optical path change means, wherein when the two-dimensional scanning, switches the excitation light having a wavelength that be introduced into the optical probe alternately every line, by controlling the optical path of the excitation light, the same A plurality of fluorescent images by acquiring the light probe microscope is characterized in that as the fluorescent labeling of plural kinds can parse identify Betsushite relative position information to the fee. The dynamic optical path changing means, wherein when the two-dimensional scanning, switches the excitation light having a wavelength to be introduced to the optical probe in the forward and backward, and switches the excitation light having a wavelength to be introduced to the optical probe for each in the same direction line The optical probe microscope according to claim 1 or 2 . 3. The optical probe microscope according to claim 1 , further comprising a calculation unit that compares the fluorescence images observed with the plurality of excitation lights, and performs label discrimination. Using the optical probe microscope according to any one of claims 1 to 4, from an optical probe capable of generating a light field localized at the tip of a fluorescently labeled DNA probe or a DNA bound with a fluorescently labeled PNA probe. A DNA sequence mapping apparatus characterized by irradiating excitation light of a plurality of wavelengths and acquiring a plurality of fluorescent images.

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