JP5470007B2 - Measuring device and total reflection fluorescence measuring device - Google Patents

Description

  The present invention relates to a measuring apparatus and a total reflection fluorescence measuring apparatus.

  Total reflection microscopy is an observation technique with a high signal-to-noise ratio that enables nano-scale local excitation. The greatest feature of this method is that it uses an evanescent wave generated by total reflection of light at the interface between two materials having different refractive indexes. When light is incident on the interface between the material 1 having the refractive index n1 and the material 2 having the refractive index n2 from the side of the material 2 having a large refractive index at a critical angle or more, the light is totally reflected at the interface, but the refractive index is small from the interface. An evanescent wave that decays exponentially is generated on the material 1 side. Since evanescent waves are light that slightly bleeds into the region of several tens to several hundreds of nanometers from the total reflection interface, in total reflection microscopy, an evanescent wave is generated at the interface between the fluorescently stained sample and the slide glass. Fluorescence observation at a high S / N ratio limited to a very small part of the sample in the vicinity of the slide becomes possible, and can be applied to single molecule observation.

  As applications using a total reflection microscope, Non-Patent Document 1 describes the observation of cell membrane activity and monomolecular events in the field of cell biology, and Non-Patent Document 2 describes the electrical characteristics of colloidal particles in the electrochemical field. In addition, the total reflection microscope has greatly contributed in many fields, such as the experimental elucidation of Brownian motion described in Non-Patent Document 3, and in recent years, application to nucleic acid sequence analysis (DNA sequence) has been attempted. This will be described in detail below.

  In the current DNA sequencing method, a capillary sequencing method, which is a combination of a DNA fragment preparation method called Sanger method and an electrophoresis method, is mainly used, and it has been used greatly for human genome analysis and the like. However, when considering individual genome analysis from the viewpoint of tailor-made medicine, there is a strong demand for a technology that can quickly, easily and inexpensively analyze fragments that are much longer than the length of DNA fragments that can be analyzed at once with a capillary sequence. . Conventional human genome analysis required about $ 10 million to decode one person's human genome, but in the future, human genome analysis will be performed at a cost of $ 10,000, which is 1/10000, and the application of the sequence to the medical field will be dramatic. Therefore, it is impossible to meet these requirements only by improving the conventional capillary method. If the nucleic acid to be ultimately decoded can be sequenced at the single molecule level without performing nucleic acid amplification such as PCR, the cost of the reagent can be reduced because of the absence of nucleic acid amplification, and rapid and simple sequencing becomes possible. A method that allows detection at the single molecule level is total reflection microscopy. Furthermore, since there is no difference in amplification efficiency due to nucleic acid amplification in a single molecule sequence, the number of mRNAs and the like expressed in cells can be quantified with higher accuracy than in the conventional method. Therefore, a single molecule DNA sequence based on a new system is desired.

  Currently, a massively parallel analysis method using optical technology has been proposed, and devices based on the principles of chemiluminescence and fluorescence have already been marketed by several companies. The feature of these methods is that it enables massively parallel analysis by dividing the reaction field using microbeads and microfabrication technology. In the conventional capillary sequencing method, the analysis efficiency is improved by increasing the number of channels (up to 384). However, the massively parallel analysis method using optical technology can perform more than 100 million massively parallel analyses. Compared to the capillary sequencing method, it is overwhelmingly large. Therefore, although the reading base length is inferior to a capillary sequence that can decode 100 bases or less and close to 1000 bases, the throughput is, for example, 100 bases × 100 million (108), which is 10 giga (1010) bases per day, compared with the capillary method. 1000 times or more of the throughput can be achieved. Moreover, since the amount of reagent per sample is reduced by the massively parallel analysis, the reagent cost is consequently reduced. Therefore, the analysis cost is currently about $ 100,000 per human genome, which is about 1/100 to 1/1000 of the capillary sequencing method. However, in these methods, since the nucleic acid to be decoded is amplified and sequenced, it is difficult to further reduce the analysis cost.

  In order to achieve further reduction in analysis cost, Non-Patent Document 4 proposes a method of sequencing a single molecule DNA by a massively parallel analysis method using optical technology. In this method, a total reflection microscope is used. In addition, as a single-molecule DNA sequencing method with higher throughput than the above-mentioned document, a real-time single molecule sequence is reported in Non-Patent Document 5, and a total reflection microscope is used. Many conventional DNA sequences use a DNA polymerase as an enzyme. However, the method of performing an extension reaction and a sequence for each base as in the above-mentioned document has the ability to continuously incorporate bases provided in the enzyme. I'm wasting it. The ability of one molecule of DNA polymerase to take in bases is about 1000 bases per second, can be read over a length of 100,000 bases or more, and the accuracy of bases to be taken is very high.

Japanese Patent Laying-Open No. 2005-083800 JP 2007-085915 A

Alelrod, D. et al vol.2, pp. 764-774, (2001) Prieve, D. C. and Frej, N. A., Langmuir, 6, pp. 396-403 Kihm, K. D. et al., In Fluids, 37, pp.811-824, (2004) PNAS 2003, Vol. 100, pp. 3960-3964 PNAS 105 (4): 1176-1181. (2008)

  The protocol assumed in the above-mentioned single molecule sequence in real time is as follows. Enzyme, template DNA, and primer are placed in a tube to form a complex. In order to prevent a decrease in enzyme activity, it is generally performed at 4 ° C. Next, the enzyme / template DNA / primer complex is fed to the substrate to fix the complex to the substrate. Also at this time, it is common to carry out at 4 ° C. in order to prevent a decrease in enzyme activity. Next, the substrate on which the complex of enzyme, template DNA, and primer is fixed is placed in a microscope, and after focusing, microscopic photography is started. Next, a fluorescently labeled dNTP is flowed to initiate the base extension reaction. Since it is common to use an enzyme having the maximum enzyme activity at 37 ° C., the temperature is adjusted to 37 ° C. The fluorescently labeled site of the fluorescently labeled dNTP is a mechanism that separates the fluorescent dye in the process of incorporating the base by the enzyme by attaching a phospholink nucleotide to the terminal phosphate instead of the base. Therefore, since the extension reaction proceeds continuously, it is necessary to fix the visual field until one sequence is completed. The sequence time required per field of view is assumed to be about 0 to 60 minutes from the time until the enzyme activity is lost. When the measurement is completed, the visual field is moved to another place where the base extension reaction has not occurred. After moving the field of view, fluorescence-labeled dNTPs are flowed to initiate the base extension reaction, and real-time sequencing is performed again. Therefore, it is necessary to control liquid feeding for each visual field. This operation is performed for the number of visual fields existing per substrate. In the case of one field of view per substrate, the next substrate is set after one measurement. However, since the enzyme begins to decrease in activity immediately after being immobilized on the substrate, the measurable time of the real-time reaction is long in the first visual field of measurement but tends to become shorter as it approaches the visual field at the end of measurement. In order to make liquid feeding control for each field of view and real-time reaction time as long as possible, one substrate may be used as one field of view.

  When all fields of view existing on one substrate have been detected, the substrate is removed and a new substrate is set. When exchanging the substrate with the prism type evanescent microscope, the position of the prism and the objective lens is separated from the substrate and the substrate is attached and detached. After that, a new substrate is set, and matching oil is filled between the prism and the substrate, and then the objective lens is brought close to focus. However, there is a risk that the laser will be diffusely reflected by the bubbles due to bubbles in the matching oil, and the substrate cannot be measured during time-consuming focusing, so the throughput will be reduced. It is. Although it is possible to pull out only the substrate with the position of the prism and objective lens fixed, the distance between the prism and the objective lens is generally only a few mm, so when the substrate is pulled out, the matching oil between the prism and the substrate Will come into contact with the objective lens. In this case, if the matching oil adhering to the objective lens is not wiped off, the next substrate cannot be measured, which is an obstacle to automation of the apparatus. Therefore, in devices such as objective / prism total reflection microscope devices and surface plasmon resonance measuring devices that require matching, liquids such as matching oil are automatically filled without bubbles, and focusing time is reduced. Replacing a small number of substrates is a major issue for device automation.

  Although a defect inspection method and a defect inspection apparatus are disclosed in Patent Document 1, the surface to be immersed is the upper surface of the wafer. When the lower surface of the wafer is immersed, liquid adheres to the detection system and cannot be detected. When the inspection of one wafer is completed, the liquid immersion portion is removed and the wafer is dried before the next wafer is inserted. Therefore, the inspection cannot be performed while the wafer is dried and replaced.

  As a result of diligent investigation on the above problems, by sliding the measurement target substrate onto which a liquid such as matching oil has been dropped onto the target object to be matched, such as a prism, matching can be performed without generating bubbles. The invention has been completed. More specifically, the measurement target substrate on which a liquid such as matching oil is dropped is placed on a turntable or the like and slid against the matching target such as a prism to perform matching without generating bubbles. The present invention relates to an automated measuring apparatus characterized by

  By automating the liquid matching process performed by a total reflection microscope, surface plasmon resonance device, etc., a series of processes including measurement target substrate installation, reagent dispensing, liquid matching, temperature adjustment, reaction, detection, and measurement target substrate replacement Can be fully automated, improving the throughput of the apparatus.

Turntable view of the prism type evanescent microscope (on the prism). Turntable top view (on prism). Turntable view of prism type evanescent microscope (under prism). Prism shape and operation mechanism diagram (on the prism). Prism shape and operation mechanism diagram (on the prism). Prism shape and operation mechanism (under the prism). Flow cell diagram. Board installation table of prism type evanescent microscope (on prism). Substrate mounting table for prism type evanescent microscope (under prism). Top view of the turntable (under the prism). Measurement board installation diagram on the turntable (on the prism). Device configuration diagram of surface plasmon resonance (under prism). The apparatus block diagram of a prism type evanescent microscope (the prism side). Top view of the turntable with matching oil sump (on the prism).

  As a result of intensive studies on the above problems, the measurement target substrate to which the matching oil has been dropped is slid onto a prism having a structure in which bubbles are not easily generated, thereby matching without generating bubbles. More specifically, the measurement target substrate on which the matching oil is dropped is placed on the turntable and rotated with respect to the prism having a taper or a curved surface so that the matching oil can easily enter between the prism and the measurement target substrate. In addition, the present invention relates to an automation device that performs matching without generating bubbles by slowing the rotation speed of the turntable in the vicinity of the prism contacting the matching oil.

  The present invention can be applied not only to the above-described single-molecule sequence in real time but also to a method (sequential method) in which a base is extended by one base to perform a sequence. It can be easily inferred from the following examples that the present invention can be applied to all protocols for detection using a microscope. Examples of protocols assumed for the sequential method and the real-time method are shown below.

  As a sequential method using a total reflection microscope, laser wavelengths of 532 nm and 635 nm are used for fluorescence detection of the phosphor Cy3 and phosphor Cy5, respectively. After sandwiching the sample solution between two glass slides, a single target DNA molecule is immobilized on the solution layer side on the refractive index boundary plane between the glass slide and the sample solution using biotin-avidin binding. . Next, when a Cy3-labeled primer is introduced into the solution at a constant concentration by solution exchange, a single Cy3-labeled primer molecule hybridizes to the target DNA molecule to form a nucleic acid duplex. Thereafter, unreacted Cy3-labeled primer molecules are removed by a washing operation. Since the Cy3-labeled primer molecule hybridized to the target DNA molecule is present at a specific position in the evanescent field, the binding position of the target DNA molecule can be confirmed by fluorescence detection. When there are multiple Cy3-labeled primer molecules that hybridize to the target DNA molecule in one field of evanescent, the subsequent sequences can be performed in parallel by grasping all the positions of the Cy3-labeled primer molecules. it can. Furthermore, when multiple Cy3-labeled primer molecules that hybridize to the target DNA molecule exist within one field of evanescent and straddle multiple fields of view, the stage holding the slide glass is moved to move the field of view. By grasping all the positions of the Cy3-labeled primer molecules while moving the sequence, the subsequent sequence can be performed in a super parallel manner. In order to increase the throughput of sequence analysis, it is better to enlarge the field of view by reducing the magnification of the microscope. In addition, it is better to increase the moving speed of the stage and shorten the moving time between the visual fields where fluorescence observation is not possible. After confirming the positions of all the primer molecules, fluorescent light fading (quenching) is performed by irradiating Cy3 with high-power excitation light for a certain period of time, thereby suppressing subsequent fluorescence emission. The purpose of this is to prevent the detection of Cy3 in the previous process when using Cy3 in the subsequent process. When a fluorescent dye different from Cy3 is used in the next step and after, it is not always necessary to quench the fluorescence. However, the fluorescence wavelength region of Cy3 may overlap with the fluorescence wavelength region of other fluorescent pigments. It is desirable to keep it. Next, it contains an enzyme that adds a base to a double-stranded nucleic acid and dNTP fluorescently labeled with Cy5 (N is one of A (adenine), C (cytosine), G (guanine), and T (thymine)). The solution is introduced to a constant concentration by solution exchange. Only when the target DNA molecule has a complementary strand relationship (A and T, C and G), the fluorescently labeled Cy5-dNTP molecule is incorporated into the extended strand of the primer molecule of the double-stranded nucleic acid. Normally, when dNTP fluorescently labeled with Cy5 is incorporated into the extended strand of the primer molecule, the enzyme tries to incorporate the next base. For example, by binding a specific molecule to the base portion of the Cy5-dNTP molecule, The structure is such that two or more bases are not taken in continuously. Thereafter, unreacted Cy5-dNTP molecules are removed by a washing operation. Since Cy5-dNTP incorporated into the extended chain is present at a specific position in the evanescent field, the binding position of Cy5-dNTP can be confirmed by fluorescence detection. Furthermore, by specifying the position where the binding position of Cy5-dNTP and the binding position of the target DNA molecule coincide with each other, the sequence of the target DNA molecule fixed at the specific position of the evanescent field can be decoded. If there are multiple Cy5-dNTPs that are incorporated into the extended strand of the primer molecule within one field of evanescent light, the sequence of the target DNA molecule can be parallelized by grasping all the positions of the bound Cy5-dNTPs. Can be deciphered.

  In addition, when multiple Cy5-dNTPs incorporated into the extended strand of the primer molecule are present in one field of evanescent and exist across multiple fields of view, the stage holding the slide glass is moved to move the field of view. By grasping all the positions of Cy5-dNTP while moving the sequence, the sequence of the target DNA molecule can be decoded in a massively parallel manner. In order to increase the throughput of sequence analysis, it is better to enlarge the field of view by reducing the magnification of the microscope. In addition, it is better to increase the moving speed of the stage and shorten the moving time between the visual fields where fluorescence observation is not possible. After confirming all the Cy5-dNTP sequences (one base), Cy5 is irradiated with high-power excitation light for a certain period of time to cause fluorescence fading (quenching), thereby suppressing subsequent fluorescence emission. When a fluorescent dye different from Cy5 is used in the subsequent steps, it is not always necessary to quench the fluorescence. However, since the fluorescence wavelength region of Cy5 may overlap with the fluorescence wavelength region of other fluorescent pigments, it is quenched as much as possible. It is desirable to keep it. After Cy5 quenching, a specific molecule bonded to the Cy5-dNTP molecule so as not to continuously take in more than 2 bases is cleaved using means such as a catalyst or photodissociation. This can extend the next base. The base sequence of the immobilized target DNA molecule is determined by repeating the above Cy5-dNTP extension reaction process, for example, four types of bases in the order of dATP → dCTP → dGTP → dTTP → dATP. Is possible. A plurality of target DNA molecules can be sequenced in a massively parallel manner by processing the dNTP elongation reaction process in a massively parallel manner. The principle of the single molecule sequence has been described by taking the two-color fluorescent dyes Cy3 and Cy5 as an example, but is not limited to these two dyes, and can be realized by other fluorescent dyes and methods. For example, by labeling dNTPs with four different fluorescent dyes, it is not necessary to repeat the four types of bases dATP → dCTP → dGTP → dTTP → dATP in order, so that throughput is simple. The calculation is 4 times faster. It is also possible to label the primer molecule and dNTP all with the same fluorescent dye (one color).

  Next, an example of a real-time method using a total reflection microscope is shown. Many conventional DNA sequences use a DNA polymerase as an enzyme. However, in the sequential method, the ability to continuously take in bases provided in the enzyme is wasted. The ability of one molecule of DNA polymerase to take in bases is about 1000 bases per second, can be read over a length of 100,000 bases or more, and the accuracy of bases to be taken is very high. Therefore, using two techniques, sequencing is performed in real time while continuously extending nucleic acids. The first technique is a mechanism that separates the fluorescent dye in the process of incorporating the base by the enzyme by attaching the phosphoric acid nucleotide to the terminal phosphate instead of the base as the fluorescent label. This leaves a completely natural double stranded DNA after incorporation of the base. Continuous detection is possible by detecting in real time the fluorescence corresponding to the base when the enzyme takes in the base. However, the four types of bases must have different fluorescent labels. Note that the fluorescent dye is present at a specific location in the evanescent field only for a certain period of time until the enzyme is taken in, and can be sequenced by grasping the position at this time. In addition, after the fluorescent dye is separated by the second technique, the fluorescent dye drifts in the solution by Brownian motion, so that there is no influence on the sequence. Further, there is no need for a step of quenching the fluorescent dye by irradiating with a high-power laser as in the method of performing the extension reaction and sequencing for each base. The second technique is a zero-mode waveguide technique that enables single molecule detection. As a result, only the fluorescent dye inside the nanometer-sized hole can be measured, so that the fluorescent dye separated from the base and the unreacted fluorescent labeled base that does not contribute to the extension reaction can be measured without being removed by a washing operation. These technologies have suggested the feasibility of real-time DNA sequencing.

  In a single molecule sequence in real time, since the extension reaction proceeds continuously, it is necessary to fix the normal field of view until one sequence is completed. Therefore, in order to increase the throughput, it is effective to make the field of view as large as possible by reducing the magnification of the microscope. However, when an objective lens type total reflection microscope is used, the detection is limited to high magnification detection of 60 times or more. Accordingly, the field of view is narrowed. In the following, two types of total reflection microscopes used for single molecule sequencing will be described. Currently, an objective lens type total reflection microscope is generally used in the total reflection microscope. Inverted, the objective lens is positioned below the slide glass via oil immersion oil, and laser light for evanescent wave generation is incident obliquely from below the slide glass through the objective lens. An evanescent wave is generated near the placed interface. In this arrangement, the space above the objective lens (above the slide glass) can be handled freely, so that the operability and convenience are excellent, and a very bright fluorescent image is obtained. However, it is a drawback that it is limited to high-magnification observation with a magnification of 60 times or more due to the principle restriction of using a high numerical aperture oil immersion objective lens. As another type of total reflection microscope not limited to high-magnification observation, a prism type total reflection microscope in which a laser is incident through a prism is used. In this microscope, a sample is sandwiched between two slide glasses or between a slide glass and a cover glass, a prism is placed on the upper slide glass, and laser light for generating evanescent waves is passed through the prism to the upper slide. The light is incident obliquely above the glass, and an evanescent wave is generated in the vicinity of the interface in contact with the slide glass sample. In this arrangement, since laser light can be efficiently incident, observation at a higher S / N ratio than that of the objective lens type is possible. Unlike an objective lens type total reflection microscope, it is not necessary to use a high numerical aperture oil immersion objective lens, and an air immersion and water immersion type objective lens can be used.

  Therefore, since there is no restriction on magnification, low-magnification observation is easy. Since one field of view becomes large by low magnification observation, for example, the throughput in Non-Patent Document 5 is improved. Therefore, it can be said that the prism type total reflection microscope is more suitable than the objective lens type from the viewpoint of throughput improvement. However, in the prism type total reflection microscope, the space above the objective lens is blocked by the prism, so that the operability of the sample and the degree of freedom of the specimen are extremely low. The development of a prism-type total reflection microscope system that excels in sample operability and specimen flexibility, is easy to use in combination with other optical observation techniques, and enables low-magnification observation. In order to improve the throughput of the DNA sequence, it is important to widen the visual field that can be detected at one time. Although the objective lens type total reflection microscope is limited to high magnification observation with a magnification of 60 times or more, the prism type evanescent method has no magnification limitation, and thus throughput is improved by observing at low magnification. For example, the field of view of a 40 × objective lens is more than twice that of a 60 × objective lens. Therefore, when the prism type total reflection microscope is employed, the throughput is expected to be improved by 2 times or more.

  It is also important to increase the reaction rate of the enzyme in order to improve the throughput. As a method for increasing the reaction rate of the enzyme, it is conceivable to increase the substrate concentration and the optimum pH, as well as the optimum temperature of the enzyme. Among these, the substrate concentration and pH can be realized by changing the composition in the solution. However, for the control of the optimum temperature (reaction temperature) of the enzyme, a device for adjusting the temperature is required separately. The reaction rate of the enzyme increases with temperature, but denatures at high temperature because the enzyme is a protein. Therefore, the enzyme activity is decreased. Generally, it is 40-50 ° C. for animal enzymes and 50-60 ° C. for plant enzymes. However, some of them are 80 to 90 ° C. (90 ° C. or more for hyperthermophilic bacteria) such as thermophilic bacteria. From the above, the throughput of the apparatus can be maximized by using a prism type total reflection microscope with a temperature control function. Details of the examples are described below.

  The above and other novel features and effects of the present invention will be described with reference to the drawings. The drawings are used for explanation and do not limit the scope of rights. Placing the substrate on a turntable, which is a prism-type evanescent microscope stage, reagent dispensing, temperature adjustment, matching oil dispensing, reaction on the substrate, and microscope detection are performed fully automatically and continuously. It came to be completed. The configuration of the present invention will be described with reference to FIG. FIG. 1 is a turntable diagram of a prism type evanescent microscope. The laser beam oscillated from the laser 101 becomes circularly polarized light by the λ / 4 wavelength plate 102, passes through the condenser lens 103, and then enters the prism 104 perpendicularly. Since optical glass that can be manufactured with extremely high homogeneity is required for the glass for prisms, synthetic quartz or BK7 having high transmittance and high homogeneity is generally used.

  The laser beam is applied to the refractive index boundary plane of the measurement substrate 105b, that is, the measurement substrate 105b and the solution (solution in contact with the lower surface of the measurement substrate 105b. See Example 7. In all the examples, on the surface opposite to the matching object. A mechanism for holding or feeding a reagent can be provided.) The incident light is incident at an incident angle of about 68 ° (solution: water) to totally reflect the laser beam and form an evanescent wave. The types of the measurement substrate 105b include a quartz substrate, a gold thin film substrate coated with a metal film on the substrate, a substrate in which a functional group is introduced by chemically modifying the substrate, a substrate in which a structure is produced by a semiconductor process, fine particles In addition to a substrate on which a foreign substance or the like is immobilized, a substrate in which a nucleic acid, protein, antigen, antibody, small molecule, aptamer, or similar biomolecule is bound to the above-mentioned substrate can be used, but is not limited thereto. It is not a thing. In FIG. 1, the measurement substrates 105a and 105b are set on the turntable 107 while being fixed to the measurement substrate holder 106b. By fixing the measurement substrate 105 and the measurement substrate holder 106b with high parallelism, the turntable 107 can be set with high parallelism. Each parallelism is preferably 1 ° or less, but is not limited thereto. If the measurement substrates 105a and 105b are large, they can be installed directly on the turntable 107 without using the measurement substrate holder 106. The laser beam is prevented from being totally reflected at the boundary between the prism 104 and the measurement substrate 105b by filling the space between the prism 104 and the measurement substrate 105b with a matching oil 120b having a close refractive index. Since there is no measurement substrate holder 106 in the optical path of the laser, only the matching oil 120b exists between the prism 104 and the measurement substrate 105b. The distance between the prism 104 and the measurement substrate 105b is not limited, but if the distance is too large, the matching oil 120b cannot be held between the prism 104 and the measurement substrate 105b. If an air layer enters between the prism 104 and the measurement substrate 105b even if the distance is small, the laser beam is totally reflected at that portion. Therefore, it is essential that the structure does not contain bubbles. The turntable 107 can be rotated by rotating the turntable rotating shaft 108 with the XYZ stage. The turntable rotating shaft 108 with the XYZ stage is fixed to the XYZ stage, and the rotation center can be moved to an arbitrary position. Further, by holding the turntable 107 from above and below using the turntable holders 109a and 109b, the height of the measurement substrate 105b when the turntable rotating shaft 108 with the XYZ stage is rotated can be always constant. There is no limitation on the position and number of the turntable holders 109a and 109b, and the turntable 107 and the measurement substrates 105a and 105b can be held. Note that the positions of the prism 104 and the objective lens 110 are fixed except for fine adjustment of focusing in the microscopic measurement.

  In the evanescent field, the excitation light intensity is exponentially attenuated with increasing distance from the refractive index boundary plane, and the excitation light intensity becomes 1 / e (e is a natural logarithm) at a distance of about 50 to 150 nm. Compared with epifluorescence detection, the excitation light irradiation volume can be greatly reduced, dramatically reducing the fluorescence emission of free fluorescent substances floating in the solution and background light such as Raman scattering of water. It becomes possible. The fluorescence due to the evanescent wave is removed from the objective lens 110 focused by the objective lens Z-axis stage 111 through the filter unit 112, and then passes through the imaging lens 113 on the CCD 114 which is a two-dimensional detector. To form an image. The imaged signal is processed by the control PC 115 and the result is displayed on the monitor 116. Further, the reagent sucked from the reagent container 117 by the dispensing unit 118 is sent to the measurement substrate 105a through the liquid feeding tube 119. As the objective lens 110, an immersion lens, a water immersion lens, and an oil immersion lens are known, but an immersion lens is preferably used. Of course, in the case of a water immersion or oil immersion lens, air bubbles are mixed in, and in the case of a water immersion lens, there is a water evaporation, so a mechanism for preventing this is required.

  A procedure until the measurement substrate 105a is detected is shown. First, the matching oil 120a is dropped from the liquid feeding tube 119 onto the measurement substrate 105a. The liquid feeding tube 119 is merely an example of a mechanism for dropping the matching oil 120a. The amount of matching oil is preferably 5 to 50 μL, but is determined by the area of the portion of the prism 104 where the matching oil 105a is filled and the distance between the prism 104 and the measurement substrate 105b. Therefore, the amount is not limited. By dispensing the amount of liquid in which the liquid height of the matching oil 120a is higher than the distance between the prism 104 and the measurement substrate 105b, the prism 104 and the matching oil 120a come into contact with each other, and the prism 104 and the measurement substrate 105b are in contact with each other. Between them, the matching oil 120a can be filled. When the liquid height of the matching oil 120a is lower than the distance between the prism 104 and the measurement substrate 105b, the matching between the prism 104 and the measurement substrate 105b occurs because the prism 104 and the matching oil 120a do not contact each other. Oil 120a cannot be filled. Therefore, microscopic observation is impossible. Next, the turntable 107 is rotated 180 ° by rotating the turntable rotating shaft 108 with the XYZ stage 180 °. At this time, the matching oil 120a enters between the prism 104 and the measurement substrate 105b. When the turntable 107 is rotated, since the prism 104 and the matching oil 120b are in contact with each other, the matching oil 120b has a shape extending in the rotation direction of the turntable 107 due to its viscosity. Therefore, it is desirable that the position where the matching oil 120a is dropped from the liquid feeding tube 119 is on the concentric circle of the focal position of the objective lens 110 around the rotation table 108 with the XYZ stage on the front side in the rotation direction on the measurement substrate 105a. . As a result, the matching oil 120b can be filled in the central portion of the prism 104 and the measurement substrate 105b.

  Further, by reducing the rotation speed of the turntable rotating shaft 108 with the XYZ stage after the matching oil 120a comes into contact with the prism 104, bubbles are less likely to enter, and the central portion of the prism 104 and the measurement substrate 105b is filled with the matching oil 120b. be able to. When an image position shift or a focus shift occurs due to a height shift of the measurement substrate 105b or the like, the shift is corrected by moving the XYZ stage that fixes the turntable rotating shaft 108 with the XYZ stage. Further, if necessary, the objective lens 110 is moved up and down on the Z-axis stage 111 for objective lens to focus, or the position of the prism 104 is moved up and down. After the measurement, the measurement substrate 105b rotates the turntable 107 by 180 ° by rotating the turntable rotation shaft 108 by 180 °. Thereafter, the substrate autoloader 121 is used to remove the substrate holder 106a and install a new substrate holder 106a. The above process is performed fully automatically and continuously. FIG. 1 shows a prism type evanescent microscope, but in the case of an objective type evanescent microscope capable of detecting single-molecule fluorescence, the prism 104 is a component that is omitted from FIG. The laser is incident from the edge of the objective lens. Therefore, the present invention can be applied not only to a prism type evanescent microscope but also to a general microscope such as an objective type evanescent microscope.

  FIG. 2 is a top view of the turntable of FIG. The diagram is merely an example, and is not limited to this. The substrate autoloader arm can access the substrate autoloader 202 by rotating the dispensing arm / substrate autoloader arm rotating shaft 201. A dotted line centering on the dispensing arm / substrate autoloader arm rotating shaft 201 indicates a trajectory on which the dispensing arm or substrate autoloader arm rotates. In addition, the dispensing arm can attach the dispensing tip with the dispensing tip attachment unit 203, suck the reagent with the reagent container 204, and discard the used dispensing tip to the dispensing tip discarding unit 205. In addition, the dispensation arm / substrate autoloader arm rotating shaft 201 can be arranged in parallel, so that substrate installation and dispensing can be performed in parallel. Using the above mechanism, the matching oil 207 is dispensed by the dispensing tip 206 to the measurement substrate holder 209 set on the turntable 208. Thereafter, the turntable rotating shaft 210 with the XYZ stage is rotated by 180 °. At this time, by holding the turntable 208 from above and below with the turntable holder 211, the height of the measurement substrate 212 when the turntable rotating shaft 210 with the XYZ stage is rotated can be kept constant. There is no limitation on the position and number of the turntable holder 211, and the turntable 208 and the measurement substrate 212 can be held. The positions of the prism 213 and the objective lens 214 are fixed except for fine adjustment of focusing in the microscopic measurement. When the turntable rotating shaft 210 with the XYZ stage is rotated by 180 °, the matching oil 207 enters between the prism 213 and the measurement substrate 212. When the turntable 208 is rotated, since the prism 213 and the matching oil 207 are in contact with each other, the matching oil 207 has a shape extending in the rotation direction of the turntable 208 due to its viscosity. Therefore, the position where the matching oil 207 is dropped from the dispensing tip 206 is preferably on the concentric circle of the focal position of the objective lens 214 around the rotation table 210 with the XYZ stage on the front side in the rotation direction on the measurement substrate 212. . As a result, the matching oil 207 can be filled in the center position of the objective lens 214 between the prism 213 and the measurement substrate 212 as a result. Further, by reducing the rotation speed of the turntable rotating shaft 210 with the XYZ stage after the matching oil 207 comes into contact with the prism 213, bubbles are less likely to enter, and the center of the objective lens 214 is between the prism 213 and the measurement substrate 212. The matching oil 207 can be filled in the position. When an image position shift or a focus shift occurs due to a height shift of the measurement substrate 212 or the like, the shift is corrected by moving the XYZ stage that fixes the turntable rotating shaft 210 with the XYZ stage. After the measurement, the measurement substrate holder 209b is moved to the position of the measurement substrate holder 209a by rotating the turntable rotating shaft 210 with the XYZ stage 180 degrees. Thereafter, the substrate autoloader arm of the dispensing arm / substrate autoloader arm rotating shaft 201 removes the measurement substrate holder 209a from the turntable 208 and installs the unmeasured measurement substrate holder 209a. The above process is performed continuously and fully automatically.

  However, the matching oil 207 accumulates on the turntable 208 as the number of substrates to be measured increases. This is because the matching oil 207 remaining on the prism 213 is applied onto the turntable 208 other than the measurement substrate holder 209 as the turntable rotating shaft 210 with the XYZ stage rotates. As a method for preventing this, the matching oil wipe 215 can be installed on the turntable 208. The height of the matching oil wipe 215 is a height that can reach the lower surface of the prism 213 to which the matching oil 207 is attached. As the turntable rotating shaft 210 with the XYZ stage rotates, the matching oil 215 wipes the lower surface of the prism 213 to which the matching oil 207 is attached, so that the matching oil 207 can be wiped off. If the matching oil wiping 215 has passed through the prism 213 and the XYZ stage-equipped turntable rotating shaft 210 is rotated in the opposite direction when the wiping operation cannot be performed once, the matching oil 207 can be wiped a plurality of times. Can be wiped off completely. The amount of the matching oil 207 can always be kept constant by wiping off the matching oil 207 each time measurement of the measurement substrate holder 209 is completed. In the embodiment of FIG. 2, since the positions of the measurement substrate holder 209 and the matching oil wipe 215 are shifted by 90 °, the rotation of the turntable rotating shaft 210 with the XYZ stage causes the measurement substrate holder 209 and the matching oil wipe 215 to be located The matching oil 207 is applied to the area of the turntable 208. In order to prevent this, the region where the matching oil 207 is applied to the turntable 208 can be minimized by disposing the matching oil wipe 215 near the measurement substrate holder 209 or on the measurement substrate holder 209. The matching oil wipe 215 is preferably a material that does not damage the prism 213 and does not adhere residue, and includes felt, optical lens paper, and the like. The matching oil wipe 215 can be installed at any place on the turntable 208. However, when the amount of the matching oil 207 applied to the turntable 208 is small or does not affect the measurement, the matching oil wipe 215 is not necessarily installed. The matching oil wipe 215 need not be installed on the turntable, and may be installed independently of the turntable 208 as a component of the turntable holder 211. In addition, a reagent A port 216 and a reagent B port 217 can be provided as reagent ports at arbitrary locations on the measurement substrate holder 209. The number of reagent ports can be set arbitrarily. By rotating the dispensing arm / substrate autoloader arm rotating shaft 201, the dispensing arm or the substrate autoloader arm is placed on the center of the measurement substrate holder 209, the reagent A port 216, and the reagent B port 217 as shown in FIG. It can move and a reagent etc. can be dripped. As the objective lens 214, an immersion, water immersion, and oil immersion lens are known, but an immersion lens is preferably used. Of course, it is possible to use a water immersion or oil immersion lens, but a mechanism that prevents bubbles from being mixed and evaporation of liquid (particularly water) is required. As an example, a method of feeding water 219 to the objective lens 214 with the pump 220 through the liquid feeding tube 218 can be considered, but the present invention is not limited to this. 219 can be other liquids such as matching oil. FIG. 2 shows a prism-type evanescent microscope, but in the case of an objective-type evanescent microscope capable of detecting one molecule, the prism 213 is a component that is omitted from FIG. The laser is incident from the edge of the objective lens. Therefore, the present invention can be applied not only to a prism type evanescent microscope but also to a general microscope such as an objective type evanescent microscope.

  Further, by partially adjusting the temperature of the turntable 208, the measurement substrate holders 209a and 209b can be set to different temperatures. As temperature control methods, there are air temperature control and local temperature control. Air temperature control is a method of adjusting the air temperature of a box to be kept warm by using a heating or cooling device and a temperature control box. Although the temperature stability is high, there are problems that it takes a long time to reach the temperature and that a high temperature of 60 ° C. or higher is difficult. When an air temperature control box is used in a total reflection microscope, there is a high possibility of covering all the microscope stage, prism, and objective lens that hold the slide glass. In particular, regarding the objective lens, use at a high temperature is outside the manufacturer's specification range, so it is desirable to keep it at room temperature as much as possible. The local temperature control is a method of adjusting the temperature by bringing the heating or cooling device into direct contact with the portion where temperature control is desired. Therefore, thermal expansion of the objective lens, which is a concern for air temperature control, does not occur. Although local temperature control is inferior in temperature stability to air temperature control, it has a feature that it takes a short time to reach the temperature and can control a high temperature of 60 ° C. or higher. For example, when it is necessary to change the temperature by enzyme reaction and washing, or when a highly heat-resistant enzyme is used, the local temperature control method is more suitable than air temperature control. Therefore, as a method for adjusting the temperature, a method in which a Peltier or heater is brought into contact with the turntable 208, a method in which a heating wire is embedded in the turntable 208, a method using an infrared laser, or the like can be considered. Is not to be done. The turntable holder 211 may have a role of a heater. As a result, while the measurement substrate 212 sandwiched between the prism 213 and the objective lens 214 is being detected, the reagent is supplied to the other measurement substrate 212 and reacted at different temperatures for a certain time, and then detection is performed. Bioassays are possible. For example, in PCR, a nucleic acid can be doubled by generally passing through three temperature cycles (eg, thermal denaturation: 95 ° C., hybridization: 58 ° C., extension: 72 ° C.). Therefore, by installing three measurement substrate holders 209 on the turntable 208 and setting the temperature in three steps, the nucleic acid is doubled every time the turntable 208 makes one rotation. Quantitative PCR that measures the initial amount of DNA can be realized. In addition, as shown in the examples, there are many applications such as performing DNA sequencing by extending a fluorescently labeled base one base at a time for two strands of DNA and primer. The application of the present invention is not limited to these.

  FIG. 3 is a turntable diagram of a prism type evanescent microscope. Therefore, in FIG. 3, it is assumed that the matching oil 120 shown in FIG. 1 is on the lower side of the measurement substrate 105 and the objective lens 110 is a water immersion objective lens, but the present invention is not limited to this. The laser beam oscillated from the laser 301 becomes circularly polarized light by the λ / 4 wavelength plate 302, passes through the condenser lens 303, and then enters the prism 304 perpendicularly. Since optical glass that can be manufactured with extremely high homogeneity is required for the glass for prisms, synthetic quartz or BK7 having high transmittance and high homogeneity is generally used. The laser beam is incident on the refractive index boundary plane of the measurement substrate 305b, that is, the interface between the measurement substrate 305b and the solution (solution in contact with the upper surface of the measurement substrate 305b) at an incident angle of about 68 ° (solution: water). The laser beam is totally reflected to form an evanescent wave. In FIG. 3, the measurement substrates 305a and 305b are set on the turntable 307 while being fixed to the measurement substrate holder 306b. By fixing the measurement substrate 305 and the measurement substrate holder 306b with high parallelism, the turntable 307 can be set with high parallelism. Each parallelism is preferably 1 ° or less, but is not limited thereto. If the measurement substrates 305a and 305b are large, the measurement substrates 305a and 305b can be installed directly on the turntable 307 without using the measurement substrate holder 306. The space between the prism 304 and the measurement substrate 305b is filled with matching oil 320b having a close refractive index to prevent the laser beam from being totally reflected at the boundary between the prism 304 and the measurement substrate 305b. Since there is no measurement substrate holder 306 in the optical path of the laser, only matching oil exists between the prism 304 and the measurement substrate 305b. There is no limitation on the distance between the prism 304 and the measurement substrate 305b, but if the distance is too large, the matching oil 320b cannot be held between the prism 304 and the measurement substrate 305b. If an air layer enters between the prism 304 and the measurement substrate 305b even if the distance is small, the laser beam is totally reflected at that portion. Therefore, it is essential that the structure does not contain bubbles. The turntable 307 can be rotated by rotating the turntable rotating shaft 308 with an XYZ stage. The turntable rotating shaft 308 with the XYZ stage is fixed to the XYZ stage, and the rotation center can be moved to an arbitrary position.

  Further, by holding the turntable 307 from above and below using the turntable holders 309a and 309b, the height of the measurement substrate 305b when the turntable rotating shaft 308 with the XYZ stage is rotated can be always constant. There is no limitation on the position and number of the turntable holders 309a and 309b, and the turntable 307 and the measurement substrates 305a and 305b can be held. The positions of the prism 304 and the objective lens 310 are fixed, except for fine adjustment of focusing in the microscopic measurement. In the evanescent field, the excitation light intensity is exponentially attenuated with increasing distance from the refractive index boundary plane, and the excitation light intensity becomes 1 / e (e is a natural logarithm) at a distance of about 50 to 150 nm. Compared with epifluorescence detection, the excitation light irradiation volume can be greatly reduced, dramatically reducing the fluorescence emission of free fluorescent substances floating in the solution and background light such as Raman scattering of water. It becomes possible. The fluorescence due to the evanescent wave is removed from the objective lens 310 focused by the Z-axis stage 311 for the objective lens through the filter unit 312 and then passes through the imaging lens 313 and then on the CCD 314 which is a two-dimensional detector. To form an image. The imaged signal is processed by the control PC 315 and the result is displayed on the monitor 316. Further, the reagent sucked by the dispensing unit 318 from the reagent container 317 is fed to the measurement substrate 305 a through the liquid feeding tube 319. Although the objective lens 310 shows an example using a water immersion objective lens, an air immersion or oil immersion lens can be used and is not limited. When using the objective lens 310 using a liquid, a mechanism that prevents bubbles from being mixed and the liquid (particularly water) from evaporating is required.

  A procedure until the measurement substrate 305a is detected will be described. First, water 320a for the objective lens is dropped from the liquid feeding tube 319 onto the measurement substrate 305a. The amount of water is preferably 50 μL or more in order to prevent evaporation. This amount is determined by the influence of the measurement time of the measurement substrate 305, the evaporation time of water, the measurement temperature, and the like. By dispensing a liquid amount in which the liquid height of the water 320a is higher than the distance between the objective lens 310 and the measurement substrate 305b, the objective lens 310 and the water 320a come into contact with each other, and the objective lens 310 and the measurement substrate are in contact with each other. Water 320a can be filled between 305b. When the liquid height of the water 320a is lower than the distance between the objective lens 310 and the measurement substrate 305b, the objective lens 310 and the water 320a are not in contact with each other. The water 320a cannot be filled. Therefore, microscopic observation is impossible. Next, the turntable 307 is rotated 180 degrees by rotating the turntable rotating shaft 308 with an XYZ stage 180 degrees. At this time, the water 320a enters between the objective lens 310 and the measurement substrate 305b. Unlike the matching oil 120 shown in FIG. 1, in the case of water, bubbles do not enter between the objective lens 310 and the measurement substrate 305b. When an image position shift or a focus shift occurs due to a height shift of the measurement substrate 305b or the like, the shift is corrected by moving the XYZ stage that fixes the turntable rotating shaft 308 with the XYZ stage. Further, if necessary, the objective lens 310 is moved up and down by the objective lens Z-axis stage 311 or the prism 304 is moved up and down.

  After the measurement, the measurement substrate 305b rotates the turntable 307 by 180 ° by rotating the turntable rotation shaft 308 by 180 °. Thereafter, the substrate autoloader 321 is used to remove the substrate holder 306a and install a new substrate holder 306a. The above process is performed fully automatically and continuously. As for the matching oil between the prism 304 and the measurement substrate 305, a method of feeding the matching oil 323 to the prism 304 by the pump 324 through the liquid feeding tube 322 can be considered, but the present invention is not limited to this. 323 can be another liquid. Further, a thing corresponding to the matching oil wipe 215 shown in FIG. 2 can be installed on the lower surface of the turntable 307. Further, the matching oil reservoir 325 collects the matching oil spilled from the prism 304. FIG. 3 shows a prism-type evanescent microscope, but in the case of an objective-type evanescent microscope capable of detecting one molecule, the prism 304 is a component that is omitted from FIG. The laser is incident from the edge of the objective lens. Therefore, the present invention can be applied not only to a prism type evanescent microscope but also to a general microscope such as an objective type evanescent microscope.

  When the prism surface in contact with the matching oil is perpendicular to the rotation surface of the turntable, bubbles may be mixed between the prism and the measurement substrate, or the matching oil may not enter the center of the measurement field. In this case, it is possible to fill the matching oil between the prism and the measurement substrate by spreading the matching oil by putting time after the matching oil contacts the prism and rotating the turntable again. However, this method has a problem that it takes time, a large amount of matching oil is used, and bubbles cannot be prevented from being mixed. Therefore, as a result of intensive studies by the authors, by setting the prism surface in contact with the matching oil at an acute angle with respect to the rotation surface of the turntable, even if the matching oil is blocked by the prism, the rotational force of the turntable As a result, the inertial force of the matching oil overcomes the frictional force generated between the matching oil and the prism, leading to the idea that the matching oil is drawn between the prism and the measurement substrate. This method is characterized in that bubbles are not mixed and the matching oil can be filled between the prism and the measurement substrate with a short amount of matching oil in a short time. FIG. 4 shows a prism shape and an operation mechanism diagram.

  This is an enlarged view of the prism portion of FIGS. The prism shape is given as an example and is not limited thereto. 4A to 4D show a tapered prism. FIG. 4A is a three-dimensional view of the prism. The matching oil 402 dropped on the measurement substrate holder 401 moves in the direction of the arrow and contacts the tapered prism 403. As shown in the figure, the laser 404 is preferably irradiated from a direction perpendicular to the traveling direction of the matching oil to the prism. This is because, when the laser 404 is irradiated from the surface where the matching oil 402 and the tapered prism 403 are in contact with each other, if the matching oil 402 is present at the incident portion of the laser 404, measurement cannot be performed due to irregular reflection. Therefore, it is preferable to irradiate the laser 404 from the surface where the matching oil 402 and the tapered prism 403 do not contact. FIG. 4B is a perspective view of the prism. The matching oil 402 dropped on the measurement substrate holder 401 moves in the direction of the tapered prism 403. The laser 404 is irradiated perpendicularly to the paper surface. The measurement substrate 405 is fixed to the measurement substrate holder 401, and the objective lens 406 is below the measurement substrate 405.

  FIG. 4C is a perspective view of the prism, and shows a state where the matching oil 402 is in contact with the tapered prism 403 and is filled between the tapered prism 403 and the measurement substrate 405. By setting the prism surface in contact with the matching oil at an acute angle with respect to the rotating surface of the turntable, even when the matching oil 402 is blocked by the tapered prism 403, the tapered prism 403 and the measurement substrate 405 In the middle, the matching oil 402 is drawn. Since there is no measurement substrate holder 401 in the optical path of the laser 404, only matching oil exists between the tapered prism 403 and the measurement substrate 405. FIG. 4D is a top view of the prism. As shown in the figure, the laser 404 is preferably irradiated from a direction perpendicular to the traveling direction of the matching oil to the prism. FIG. 4E shows a perspective view when R (Round) is added to the prism shape in order to fill the matching oil 402 between the prism 403 and the measurement substrate 405. By using the prism 407 with the R, the matching oil 402 is drawn between the prism 407 with the R and the measurement substrate 405, and the oil remaining by wiping the matching oil shown at 215 in FIG. 2 is tapered. Compared to the prism 403, the R-attached prism 407 reduces the number. This is because when the prism is curved, the angle between the measurement substrate surface and the prism surface always changes, so that a frictional force is always generated between the prism surface and the movement of the matching oil wipe. On the other hand, in the case of the tapered prism, the friction force is small compared to that of the prism with R even if the matching oil wiping moves at the part where the measurement substrate surface and the prism surface are parallel. It is.

  From this example, by setting the prism surface in contact with the matching oil to a taper or curved surface structure with an acute angle with respect to the rotating surface of the turntable, there is no mixing of bubbles, and a short amount of matching oil can be obtained. The matching oil can be filled between the prism and the measurement substrate.

  In FIG. 4, the laser irradiation surface is perpendicular to the traveling direction of the matching oil to the prism, thereby preventing the matching oil from adhering to the irradiation position of the laser to the prism. However, if the measurement is continued without wiping off the matching oil on the turntable, the matching oil will collect on the prism and the matching oil will wrap around the laser irradiation surface. As a result, there is a possibility that the matching oil adheres to the irradiation position of the laser prism. Therefore, the prism shape shown in FIG. 5 is conceivable as a shape for preventing the matching oil from entering the laser irradiation surface. FIG. 5 shows a prism shape and an operation mechanism diagram. FIG. 5A is a top view of the prism, and the matching oil 502 dropped on the measurement substrate holder 501 moves in the direction of the arrow and contacts the prism 503. The top view of the prism 503 has a bow tie shape, and the length of the central portion is shortened. As the measurement substrate holder 501 moves in the direction of the arrow, the matching oil 502 that has been dammed and spread by the prism 503 has an angle on the surface of the prism 503 with respect to the traveling direction of the measurement substrate holder 501. The matching oil 502 gathers at the center of the bow tie type. As a result, the possibility that the matching oil adheres to the irradiation position of the laser prism is reduced.

  FIG. 5B is a side view of the prism. The matching oil 502 dropped on the measurement substrate holder 501 moves in the direction of the prism 503. The laser 504 is irradiated perpendicularly to the paper surface. The measurement substrate 505 is fixed to the measurement substrate holder 501, and the objective lens 506 is below the measurement substrate 505.

  FIG. 5C is a side view of the prism, showing a state in which the matching oil 502 is in contact with the prism 503 and is filled between the prism 503 and the measurement substrate 505. By setting the prism surface in contact with the matching oil at an acute angle with respect to the rotation surface of the turntable, even when the matching oil 502 is blocked by the prism 503, the matching is performed between the prism 503 and the measurement substrate 505. Oil 502 is drawn. Since there is no measurement substrate holder 501 in the optical path of the laser 504, only matching oil exists between the tapered prism 503 and the measurement substrate 505. FIG. 5 (d) is a side view of the prism, which has the configuration of the prism with R shown in FIG. 4 (e). By making the prism curved, the differential value at the point on the prism changes continuously, so the angle between the measurement substrate surface and the inclination (differential value) of the prism surface always changes, and the matching oil wipe Along with the movement, a frictional force is always generated between the prism surface. On the other hand, in the tapered prism, in the portion where the measurement substrate surface and the prism surface are parallel, even if the matching oil wiping moves, the frictional force is smaller than that of the prism with R, so the force to wipe the matching oil is weak.

  As shown in FIG. 3, the prism shape when the prism is arranged under the turntable is shown in FIG. FIG. 6 is a modification of the shape of FIG. 5, which is a prism shape in which matching oil does not adhere to the laser irradiation position on the prism. FIG. 6A is a side view of the prism. Matching oil 602 is held between the measurement substrate holder 601 and the prism 603. The laser 604 irradiates the measurement substrate 605 with a surface perpendicular to the traveling direction of the measurement substrate holder 601 and is detected through the objective lens 606. The matching oil 602 that hangs down from the prism 603 is collected in the matching oil reservoir 607. The matching oil 602 is filled between the prism 603 and the measurement substrate 605 using the pump 610 from the matching oil bottle 609 through the liquid feeding tube 608. FIG. 6B is a top view of the prism. In order to drop the matching oil 602 from the prism 603, the central portion of the bow tie type is made shorter than that in FIG. FIG. 6C is a side view of the prism when FIG. 6B is viewed from the irradiation direction of the laser 604. The matching oil 602 does not adhere to the prism 603 surface at the laser incident / reflecting position 611. The matching oil 602 dripping from the prism 603 passes through one of the dotted lines 602b and 602c and reaches 602d. When the surface tension can no longer support the own weight, it falls into the matching oil reservoir 607 as 602e and is collected. Although the matching oil 602 does not adhere to the prism 603 surface at the laser incident / reflecting position 611, another problem is that the prism 603 surface at the laser incident / reflecting position 611 may be touched by hand. In this case, since organic substances in the hands adhere to the prism surface, the laser 604 is irregularly reflected on the surface of the prism 603 and the measurement substrate 605 cannot be measured. As a method for avoiding this, there is a method in which a recess 612 is provided so that only the laser incident / reflecting position 611 of the prism 603 cannot be touched by hand. A columnar structure such as a cube / cuboid / cylinder is desirable. The size of the column structure is preferably about 1 mm to 20 mm because it is not necessary to touch the laser incident / reflecting position 611 of the prism 603. When the column structure has a size of about 20 mm, it cannot be touched by hand unless the aspect ratio is large. Therefore, the size and depth of the recess 612 are not limited to this size. Naturally, the size of the indentation 612 is larger than that of the laser 604, but dust or the like accumulates over time at the laser incident / reflection position 611 of the prism 603, causing irregular reflection of the laser 604. Therefore, it is desirable that the recess 612 has a size that can clean the laser incident / reflection position 611 periodically. In other words, if it can fit a cotton swab for an optical lens, it cannot be touched by hand, but can be cleaned periodically. Therefore, as an example of the recess 612, a cube of about 5 mm can be considered. When it is difficult to process the hollow structure, a holed structure (for example, a nut) can be attached to the prism 603. This structure can be applied to all the prisms of Examples 1-12.

  FIG. 7 shows an example of the flow path when dispensing into the reagent port in FIG. However, the figure is only an example, and the present invention is not limited to this. The measurement substrate can be provided with a mechanism for holding or feeding the reagent on the surface opposite to the prism that is a matching object. This is applicable to all embodiments. FIG. 7A shows a case where the prism is arranged on the turntable. On the measurement substrate holder 701, the matching oil 702 is present between the prisms 703. In order to detect the reaction on the measurement substrate 704 with the objective lens 705, the measurement substrate holder cover 707 with a flow path is joined to the measurement substrate holder 701. At this time, the distance between the measurement substrate 704 and the objective lens 705 needs to satisfy the condition of the working distance (working distance) unique to the objective lens. By injecting the reagent into the reagent injection / discharge port 708 using the dispensing chip 206 shown in FIG. 2, the reagent is fed to the measurement substrate 704 and the reaction is detected. There are a method of feeding a reagent by making the flow path hydrophilic and utilizing a capillary phenomenon, a method of sucking one of the reagent injection / discharge ports 708 by applying a negative pressure, a method of feeding by a centrifugal force in the outer peripheral direction, etc. . FIG. 7B is a diagram when the prism is arranged on the turntable. Since the prism 703 is disposed under the measurement substrate 704, the matching oil reservoir 706 is installed.

  1 to 3 disclose a matching oil automatic filling method using a turntable method. It goes without saying that the automatic filling of the matching oil can be realized by using a method other than the turntable as shown in FIGS. That is, the turntable type is disclosed in FIGS. 1 to 3 as an example of the method of moving the measurement substrate on which the matching oil is placed to the prism. Therefore, the same function can also be realized by moving the table on which the measurement board is installed instead of the turntable. FIG. 8 discloses a substrate installation table of a prism type evanescent microscope when a prism is installed on a measurement substrate. FIG. 8A is a top view of the substrate mounting table, and FIG. 8B is a side view of the substrate mounting table, showing a state before the measurement substrate is detected. The prism is installed on the measurement substrate. The dispensing arm / substrate autoloader arm rotating shaft 801 rotates and the substrate autoloader arm accesses the substrate autoloader 802. Also, the dispensing arm / substrate autoloader arm rotating shaft 801 rotates, the dispensing arm accesses the dispensing tip mounting portion 803, the reagent is sucked in the reagent container 804, and the dispensing tip is discharged after the reagent is discharged at a desired position. The dispensing tip is discarded at the discarding unit 805. The rotating direction of the dispensing arm / substrate autoloader arm rotating shaft 801 can be either clockwise or counterclockwise. FIG. 8B is a diagram in which the matching oil 807 is dropped by the dispensing tip 806. A measurement substrate holder 809 is installed on a substrate mounting table 808 with an XYZ stage. At this time, the height of the measurement substrate 812 when the substrate mounting table 808 with an XYZ stage is moved can be made constant by pressing the substrate mounting table 808 with an XYZ stage from above and below with the table holder 810. The position and number of the table holder 810 are not limited, and the XYZ stage-equipped board mounting base 808 and the measurement board 812 can be pressed. Note that the positions of the prism 813 and the objective lens 814 are fixed except for fine adjustment of focusing in the microscopic measurement. By rotating the dispensing arm / substrate autoloader arm rotating shaft 801 and moving the substrate mounting table 808 with an XYZ stage, the reagent can be dropped at an arbitrary position. The dispensing to the reagent A port 816 and the reagent B port 817 and the matching oil 807 to the measurement substrate 812 can be dropped. In the drawing, two dispensing arm / substrate autoloader arm rotation shafts 801 are arranged, but any number can be used. Further, instead of the dispensing arm / substrate autoloader arm rotating shaft 801, a dispenser or substrate autoloader that can access any position can be used instead. The laser 818 is incident in parallel to the traveling direction of the substrate mounting table 808 with an XYZ stage. However, as shown in FIGS. 4 to 6, it is more desirable to make the laser 818 incident perpendicular to the traveling direction of the substrate mounting table 808 with an XYZ stage. This is to prevent the matching oil 807 from adhering to the laser irradiation position of the prism 813 and causing irregular reflection to prevent detection.

  FIG. 8C is a top view of the substrate mounting table, and FIG. 8D is a side view of the substrate mounting table, showing a state where the measurement substrate is detected. The prism is installed under the measurement substrate. A procedure until the measurement substrate 812a is detected will be described. First, the matching oil 807a is dropped from the dispensing tip 806a onto the measurement substrate 812a. The amount of matching oil is preferably 5 to 50 μL, but is determined by the area of the prism 813 where the matching oil 807a is filled and the distance between the prism 813 and the measurement substrate 812a. Therefore, the amount is not limited. By dispensing a liquid amount in which the liquid height of the matching oil 807a is higher than the distance between the prism 813 and the measurement substrate 812a, the prism 813 and the matching oil 807a come into contact with each other, and the prism 813 and the measurement substrate 812a are in contact with each other. The matching oil 807a can be filled in between. When the liquid height of the matching oil 807a is lower than the distance between the prism 813 and the measurement substrate 812a, the matching between the prism 813 and the measurement substrate 812a is not performed because the prism 813 and the matching oil 807a are not in contact with each other. Oil 807a cannot be filled. Therefore, microscopic observation is impossible. Next, the substrate mounting table 808 with an XYZ stage is moved in the direction of the prism 813. At this time, the matching oil 807a enters between the prism 813 and the measurement substrate 812a. When the substrate mounting table 808 with an XYZ stage is moved, the prism 813 and the matching oil 807a are in contact with each other. Therefore, the matching oil 807a has a shape extending in the moving direction of the substrate mounting table 808 with an XYZ stage. Accordingly, the position where the matching oil 807a is dropped from the dispensing tip 806a is preferably a position that passes the focal point of the objective lens 814 when the XYZ stage-equipped substrate mounting table 808 moves on the measurement substrate 812a. As a result, the matching oil 807a can be filled in the central portion of the prism 813 and the measurement substrate 812a. Further, by reducing the moving speed of the substrate mounting table 808 with an XYZ stage in the vicinity where the matching oil 807a comes into contact with the prism 813, bubbles are less likely to enter, and the central portion of the prism 813 and the measurement substrate 812a is filled with the matching oil 807a. Can do. In the case where an image position shift or a focus shift occurs due to a height shift of the measurement substrate 812a or the like, the shift is corrected by moving the substrate mounting table 808 with an XYZ stage. If focusing is still not possible, the objective lens 814 is moved up and down on the Z-axis stage for the objective lens, or the prism 813 is moved up and down. After the measurement, the measurement substrate 812a pulls the XYZ stage-equipped substrate mounting table 808 back in the direction of FIG.

  Thereafter, the dispensing arm / substrate autoloader arm rotating shaft 801 is rotated, and the substrate autoloader arm removes the measurement substrate holder 809a and installs a new measurement substrate holder 809a. The above process is performed fully automatically and continuously. However, as the number of substrates to be measured increases, matching oil 807 accumulates on the substrate mounting table 808 with an XYZ stage. This is because the matching oil 807 remaining on the prism 813 is applied onto the XYZ stage-equipped substrate installation table 808 other than the measurement substrate holder 809 as the XYZ stage-equipped substrate installation table 808 moves. As a method for preventing this, the matching oil wipe 815 can be installed on the substrate mounting table 808 with an XYZ stage. The height of the matching oil wipe 815 is a height that can reach the lower surface of the prism 813 to which the matching oil 807 is attached. As the XYZ stage-equipped substrate mounting base 808 moves, the matching oil 815 wipes the lower surface of the prism 813 to which the matching oil 807 is attached, so that the matching oil 807 can be wiped off. If the matching oil wiping 807 passes through the prism 813 and the substrate mounting table 808 with an XYZ stage is moved in the opposite direction, the matching oil 807 can be wiped a plurality of times. Can be wiped off completely. The amount of the matching oil 807 can always be kept constant by wiping off the matching oil 807 each time measurement of the measurement substrate holder 809 is completed.

  In the embodiment of FIG. 8, the measurement substrate holder 809 and the matching oil wipe 815 are spaced apart from each other, so that the XYZ between the measurement substrate holder 809 and the matching oil wipe 815 is moved by the movement of the substrate mounting table with the XYZ stage. Matching oil 807 is applied to the area of the stage-equipped substrate mounting table 808. In order to prevent this, by locating the matching oil wipe 815 near the measurement substrate holder 809 or on the measurement substrate holder 809, the area where the matching oil 819 is applied to the substrate mounting table 808 with the XYZ stage is minimized. it can. The matching oil wiping 815 is preferably a material that does not damage the prism 813 and does not adhere residue, and includes felt, optical lens paper, and the like. The matching oil wipe 815 can be installed at any place on the substrate mounting table 808 with an XYZ stage. However, when the amount of the matching oil 807 applied to the substrate mounting table 808 with an XYZ stage is small or does not affect the measurement, the matching oil wipe 815 is not necessarily provided. The matching oil wipe 815 does not need to be installed on the substrate mounting table 808 with an XYZ stage, and may be installed as the table holder 810 independently from the substrate mounting table 808 with an XYZ stage. As the objective lens 814, an immersion lens, a water immersion lens, and an oil immersion lens are known, but an immersion lens is preferably used. Of course, it is possible to use a water immersion or oil immersion lens, but a mechanism that prevents bubbles from being mixed and evaporation of liquid (particularly water) is required. FIG. 8 shows a prism type evanescent microscope. However, in the case of an objective type evanescent microscope capable of detecting one molecule, the prism 813 is a component removed from FIG. The laser is incident from the edge of the objective lens. Therefore, the present invention can be applied not only to a prism type evanescent microscope but also to a general microscope such as an objective type evanescent microscope. In the method of FIG. 8, there is a high possibility that the positional accuracy during movement is inferior compared to the turntable method of FIGS. 1 to 3, and a plurality of dispensing mechanisms may be required.

  FIG. 9 discloses a substrate installation table of the prism type evanescent microscope when the prism is installed under the measurement substrate. FIG. 9A is a top view of the substrate mounting table, and FIG. 9B is a side view of the substrate mounting table, showing a state before the measurement substrate is detected. The prism is installed on the measurement substrate. The dispensing arm / substrate autoloader arm rotating shaft 901 rotates, and the substrate autoloader arm accesses the substrate autoloader 902. In addition, the dispensing arm / substrate autoloader arm rotating shaft 901 rotates, the dispensing arm accesses the dispensing tip mounting portion 903, sucks the reagent in the reagent container 904, and dispenses the reagent at a desired position. The disposal tip 905 discards the dispensing tip. The rotating direction of the dispensing arm / substrate autoloader arm rotating shaft 901 can be either clockwise or counterclockwise. FIG. 9B is a diagram in which the matching oil 907 is dropped by the dispensing tip 906. FIG. A measurement substrate holder 909 is installed on a substrate installation table 908 with an XYZ stage. At this time, the height of the measurement substrate 912 when the XYZ stage-equipped substrate setting table 908 is moved can be made constant by pressing the substrate setting table 908 with the XYZ stage from above and below with the table holder 910. The position and number of the table holders 910 are not limited, and the XYZ stage-equipped substrate mounting table 908 and the measurement substrate 912 can be pressed. Note that the positions of the prism 913 and the objective lens 914 are fixed except for fine adjustment of focusing in the microscopic measurement. Although the objective lens 914 is a case of an objective lens for immersion, it is not limited to this, and an objective lens for water immersion and oil immersion can also be used. By rotating the dispensing arm / substrate autoloader arm rotating shaft 901 and moving the substrate mounting table 908 with an XYZ stage, the reagent can be dropped at any position such as dispensing to the reagent A port 916 and the reagent B port 917. In the drawing, two dispensing arm / substrate autoloader arm rotating shafts 901 are arranged, but any number can be used. Further, instead of the dispensing arm / substrate autoloader arm rotating shaft 901, a dispenser or substrate autoloader that can access any position can be used instead. The laser 918 is incident in parallel to the traveling direction of the substrate mounting table 908 with an XYZ stage. However, as shown in FIGS. 4 to 6, it is more desirable to make the laser 918 incident perpendicular to the traveling direction of the substrate mounting table 908 with an XYZ stage. This is to prevent the matching oil 907 from adhering to the laser irradiation position of the prism 913 and causing irregular reflection to prevent detection. The matching oil 907 is sucked by the pump 921 from the matching oil bottle 920 using the liquid feeding tube 919 and filled between the prism 913 and the measurement substrate 912. The matching oil 907 dripping from the prism 913 is collected in the matching oil sump 922. As a prism structure in which the matching oil 907 does not adhere to the laser 918 irradiated portion of the prism 913, there is a method disclosed in FIG. 6, but it is not limited thereto. FIG. 9C is a top view of the substrate mounting table, and FIG. 9D is a side view of the substrate mounting table, showing a state in which the measurement substrate is detected. The prism is installed under the measurement substrate. After the measurement, the measurement substrate 912a pulls back the substrate mounting table 908 with an XYZ stage in the direction of FIG. Thereafter, the dispensing arm / substrate autoloader arm rotating shaft 901 is rotated to remove the measurement substrate holder 909a and install a new measurement substrate holder 909a by the substrate autoloader arm.

  The above process is performed fully automatically and continuously. However, as the number of substrates to be measured increases, the matching oil 907 accumulates on the substrate mounting table 908 with an XYZ stage. This is because the matching oil 907 remaining on the prism 913 is applied onto the XYZ stage-equipped substrate installation table 908 other than the measurement substrate holder 909 in accordance with the movement of the XYZ stage-equipped substrate installation table 908. As a method for preventing this, the matching oil wipe 915 can be installed on the lower surface of the substrate mounting table 908 with an XYZ stage. The height of the matching oil wipe 915 is a height that can reach the lower surface of the prism 913 to which the matching oil 807 is attached. In accordance with the movement of the substrate mounting table 908 with the XYZ stage, the matching oil 915 can wipe off the matching oil 907 by wiping the lower surface portion of the prism 913 to which the matching oil 907 is attached. If the matching oil wiping 915 passes the prism 913 and the XYZ stage-equipped substrate mounting base 908 is moved in the opposite direction when the wiping operation cannot be performed once, the wiping operation can be performed a plurality of times. Can be wiped off completely. The amount of the matching oil 907 can always be kept constant by wiping off the matching oil 907 each time measurement of the measurement substrate holder 909 is completed. In the embodiment of FIG. 9, the measurement substrate holder 909 and the matching oil wipe 915 are spaced apart from each other, so that the XYZ between the measurement substrate holder 909 and the matching oil wipe 915 is moved by the movement of the substrate mounting table with the XYZ stage. Matching oil 907 is applied to the region of the stage-equipped substrate mounting table 908. In order to prevent this, by locating the matching oil wipe 915 near the measurement substrate holder 909 or on the measurement substrate holder 909, the area where the matching oil 907 is applied to the substrate mounting table 908 with an XYZ stage is minimized. it can. The matching oil wiping 915 is preferably a material that does not damage the prism 913 and does not adhere residue, and includes felt, optical lens paper, and the like. The matching oil wipe 915 can be installed at any place on the substrate mounting table 908 with an XYZ stage. However, when the amount of the matching oil 907 applied to the XYZ stage-equipped substrate mounting table 908 is small or does not affect the measurement, the matching oil wipe 915 is not necessarily installed. Further, the matching oil wiping 915 does not need to be installed on the XYZ stage-equipped substrate installation table 908, and may be installed as the table holder 910 independently of the XYZ stage-equipped substrate installation table 908. As the objective lens 914, an immersion, water immersion, and oil immersion lens are known, but an immersion lens is preferably used. Of course, it is possible to use a water immersion or oil immersion lens, but a mechanism that prevents bubbles from being mixed and evaporation of liquid (particularly water) is required.

  FIG. 9 shows a prism-type evanescent microscope. However, in the case of an objective-type evanescent microscope capable of detecting one molecule, the prism 913 is removed from FIG. The laser is incident from the edge of the objective lens. Therefore, the present invention can be applied not only to a prism type evanescent microscope but also to a general microscope such as an objective type evanescent microscope. In the method of FIG. 9, there is a high possibility that the positional accuracy during movement is inferior compared to the turntable method of FIGS. 1 to 3, and a plurality of dispensing mechanisms may be required.

  FIG. 10 is a top view of the turntable of FIG. 1, and the prism is arranged under the measurement substrate in the same manner as in FIG. However, unlike FIG. 3, the matching oil is attached to the lower surface of the turntable with surface tension, so that the matching oil is filled in the same manner as in FIG. The diagram is merely an example, and is not limited to this. The substrate autoloader arm can access the substrate autoloader 1002 by rotating the dispensing arm / substrate autoloader arm rotating shaft 1001. In addition, the dispensing arm can attach a dispensing tip with the dispensing tip attachment unit 1003, suck the reagent with the reagent container 1004, and discard the used dispensing tip into the dispensing tip disposal unit 1005. In addition, by disposing the dispensing arm / substrate autoloader arm rotating shaft 1001 separately, the substrate installation and the dispensing can be performed in parallel. Using the above mechanism, the matching oil 1007 is dispensed to the measurement substrate holder 1009 set on the turntable 1008 by the dispensing tip 1006. By bringing the dispensing tip 1006 close to the turntable 1008, the matching oil 1007 adheres to the lower surface of the turntable 1008 by surface tension. By inclining the dispensing tip 1006, it is possible to prevent the matching oil from adhering to the dispensing tip 1006 when the matching oil 1007 drips. Further, assuming that the matching oil 1007 hangs down, a matching oil reservoir can be installed under the matching oil 1007. After dispensing the matching oil 1007, the turntable rotating shaft 1010 with the XYZ stage is rotated 180 °. At this time, by holding the turntable 1008 from above and below with the turntable holder 1011, the height of the measurement substrate 1012 when the turntable rotating shaft 1010 with the XYZ stage is rotated can be always constant. There is no limitation on the position and number of the turntable holder 1011, and the turntable 1008 and the measurement substrate 1012 can be held. When the matching oil 1007 hangs while the turntable 1008 is rotating, the matching oil 1007 may be attached to the measurement substrate 1012 immediately before the measurement position.

  Note that the positions of the prism 1013 and the objective lens 1014 are fixed except for fine adjustment of focusing in the microscopic measurement. The matching oil 1007 enters between the prism 1013 and the measurement substrate 1012 when the turntable rotating shaft 1010 with the XYZ stage is rotated 180 °. When the turntable 1008 is rotated, since the prism 1013 and the matching oil 1007 are in contact with each other, the matching oil 1007 has a shape extending in the rotation direction of the turntable 1008 due to its viscosity. Therefore, the position where the matching oil 1007 is attached from the dispensing tip 1006 is preferably on the concentric circle of the focal position of the objective lens 1014 about the rotation table front axis on the measurement substrate 1012 and the turntable rotating shaft 1010 with an XYZ stage. . As a result, the matching oil 1007 can be filled in the central portion of the prism 1013 and the measurement substrate 1012 as a result. Further, by reducing the rotation speed of the turntable rotating shaft 1010 with the XYZ stage after the matching oil 1007 comes into contact with the prism 1013, bubbles are less likely to enter, and the matching oil 1007 is filled in the central part of the prism 1013 and the measurement substrate 1012. be able to. When an image position shift or a focus shift occurs due to a height shift of the measurement substrate 1012 or the like, the shift is corrected by moving the XYZ stage that fixes the turntable rotating shaft 1010 with the XYZ stage. After the measurement, the measurement substrate holder 1009b is moved to the position of the measurement substrate holder 1009a by rotating the turntable rotating shaft 1010 with XYZ stage 180 °. Thereafter, the substrate autoloader arm of the dispensing arm / substrate autoloader arm rotating shaft 1001 removes the measurement substrate holder 1009a from the turntable 1008 and installs the unmeasured measurement substrate holder 1009a. The above process is performed continuously and fully automatically. However, matching oil 1007 accumulates on the turntable 1008 as the number of substrates to be measured increases. This is because the matching oil 1007 remaining on the prism 1013 is applied on the turntable 1008 other than the measurement substrate holder 1009 as the turntable rotating shaft 1010 with the XYZ stage rotates. As a method for preventing this, a matching oil wipe 1015 can be installed on the turntable 1008. The height of the matching oil wipe 1015 is a height that can reach the prism 1013 to which the matching oil 1007 is attached. As the turntable rotating shaft 1010 with the XYZ stage rotates, the matching oil wipe 1015 wipes the prism 1013 to which the matching oil 1007 is attached, so that the matching oil 1007 can be wiped off. If the matching oil wipe 1015 passes through the prism 1013 and the turntable rotating shaft 1010 with the XYZ stage is rotated in the opposite direction after the matching oil wipe 1015 can not be wiped once, the matching oil 1007 can be wiped a plurality of times. Can be wiped off completely. The amount of the matching oil 1007 can always be kept constant by wiping off the matching oil 1007 each time measurement of the measurement substrate holder 1009 is completed. In the embodiment of FIG. 10, since the positions of the measurement substrate holder 1009 and the matching oil wipe 1015 are shifted by 90 °, the rotation of the turntable rotating shaft 1010 with the XYZ stage causes the measurement substrate holder 1009 and the matching oil wipe 1015 to be located. Matching oil 1007 is applied to the area of the turntable 1008. In order to prevent this, the region where the matching oil 1007 is applied to the turntable 1008 can be minimized by disposing the matching oil wipe 1015 near the measurement substrate holder 1009 or on the measurement substrate holder 1009. The matching oil 1007 that hangs down from the prism 1013 is collected by placing a matching oil reservoir container under the prism. As a prism structure in which the matching oil 1007 does not adhere to the laser irradiation portion of the prism 1013, there is a method disclosed in FIG. 6, but it is not limited thereto. The matching oil wipe 1015 is preferably a material that does not damage the prism 1013 and does not adhere residue, and includes felt, optical lens paper, and the like. The matching oil wipe 1015 can be installed at any place on the turntable 1008.

  However, the matching oil wipe 1015 is not necessarily installed when the amount of the matching oil 1007 applied to the turntable 1008 is small or when measurement is not affected. Further, the matching oil wipe 1015 does not need to be installed on the turntable, and may be installed independently of the turntable 1008 as the turntable presser 1011. In addition, a reagent A port 1016 and a reagent B port 1017 can be provided as a reagent port at an arbitrary location on the measurement substrate holder 1009. The number of reagent ports can be set arbitrarily. By rotating the dispensing arm / substrate autoloader arm rotating shaft 1001, the dispensing arm or the substrate autoloader arm is placed on the center of the measurement substrate holder 1009 or on the reagent A port 1016 and the reagent B port 1017 as shown in FIG. It can move and a reagent etc. can be dripped. As the objective lens 1014, an immersion, water immersion, and oil immersion lens are known, but an immersion lens is preferably used. Of course, it is possible to use a water immersion or oil immersion lens, but a mechanism that prevents bubbles from being mixed and evaporation of liquid (particularly water) is required. FIG. 10 shows a prism-type evanescent microscope, but in the case of an objective-type evanescent microscope capable of detecting one molecule, the prism 1013 is a component that is omitted from FIG. The laser is incident from the edge of the objective lens. Therefore, the present invention can be applied not only to a prism type evanescent microscope but also to a general microscope such as an objective type evanescent microscope.

  A method of mounting the measurement substrate holder on the turntable shown in FIGS. 1 to 3 and 10 is disclosed in FIG. However, the figure is only an example, and the present invention is not limited to this. FIG. 11A is an installation view of the measurement substrate, and shows a top view and a side view. A turntable 1102 is installed around a rotation axis 1101 with an XYZ stage as a rotation center. A measurement substrate 1104 is fixed to the measurement substrate holder 1103. The measurement substrate holder 1103 is inserted between the measurement substrate holder holder 1105 and the turntable 1102, and the position is fixed. The measurement substrate holder 1103 is transported and installed using the substrate autoloader arm described in FIG. An enlarged view of the measurement substrate 1104 is shown in FIG. A prism 1106 is provided on the measurement substrate 1104, and an objective lens 1107 is provided below the measurement substrate 1104. There is matching oil 1108 between the measurement substrate 1104 and the prism 1106. A method of filling the matching oil 1108 is disclosed in FIG. A method for installing the measurement substrate holder 1103 on the turntable 1102 will be described with reference to the side view of FIG. First, a cut is made in a portion of the turntable 1102 where the measurement substrate holder is inserted. This is because the measurement substrate holder 1103 is inserted into the turntable 1102 in a state where the measurement substrate holder 1103 is firmly sandwiched from above and below by the substrate autoloader arm, and the position is fixed. From the cross-sectional view of the turntable 1102 on the A-O-A ′ plane, the portion closest to A is only the measurement substrate holder 1103. Therefore, this region is sandwiched from above and below by the substrate autoloader arm.

  Next, one end of the measurement substrate 1104 is placed on the turntable 1102 closest to B on the B-O'-B 'plane. Since the position of the measurement substrate holder holder 1105 closest to B on the B-O′-B ′ plane is on the B ′ side from the position on the turntable 1102 closest to B, one end of the measurement substrate holder 1103 is The measurement substrate holder 1103 hits the turntable 1102 by lowering the measurement substrate holder 1103 to the turntable 1102 with the measurement substrate holder holder 1105 left (B side). Thereafter, the measurement substrate holder 1103 is pushed in the direction of the turntable rotating shaft 1101 with the XYZ stage, whereby the measurement substrate holder holder 1105 and the turntable 1102 are inserted. The measurement substrate holder 1103 can always be fixed at the same position by one end of the measurement substrate holder 1103 hitting the side wall in the vicinity of the rotation table 1101 with the XYZ stage. Since the measurement substrate holder 1103 is held by the measurement substrate holder retainer 1105, the position of the measurement substrate holder 1103 does not shift vertically and horizontally. After the measurement is complete, reverse the procedure. The measurement substrate holder 1103 can be detached from the turntable 1102 by sandwiching the measurement substrate holder 1103 from above and below with a substrate autoloader arm and pulling it from the turntable rotating shaft 1101 with an XYZ stage.

  Needless to say, this method can also be applied to the case where the substrate mounting table moves left and right instead of the turntable as shown in FIG. From the cross-sectional view of the turntable 1102 taken along the plane A-O-A ', it can be seen that the measurement substrate holder 1103 is inserted in A-O and the measurement substrate holder 1103 is not inserted in O-A'. From this, it can be seen that when the measurement substrate holder 1103 is not inserted, the turntable 1102 has a hollow portion. As can be seen in FIG. 11B, this area must be at least large enough to accommodate the prism 1106 and the objective lens 1107.

  Further, it is desirable that the thickness of this region is always the same as the measurement substrate holder 1103 when the turntable 1102 rotates. Otherwise, when the turntable 1102 rotates, there is a risk that a part of the turntable 1102 may come into contact with the prism 1106 or the objective lens 1107, and there is a possibility that the matching oil 1108 accumulates at the step portion. Because. When the measurement substrate holder 1103 is inserted, the height of the portion sandwiched between the prism 1106 and the objective lens 1107 is the same between the B-O'-B 'plane and the C-O-C' plane. This is the reason. The number of measurement substrate holders 1103 that can be inserted into the turntable 1102 is not limited to the two shown in FIG. Since it is determined by the size of the turntable 1102 and the measurement substrate holder 1103 and the number of measurement substrate holders 1103 to be measured, the turntable 1102 on which an arbitrary number of measurement substrate holders 1103 can be installed can be designed.

  1 to 11 have disclosed usage methods assuming a prism-type evanescent microscope, but these relate to a method of performing automatic measurement by automatically filling a matching oil and the like, and other analysis methods. It goes without saying that can also be applied. FIG. 12 discloses an apparatus configuration diagram of surface plasmon resonance (SPR). The diagram is merely an example, and is not limited to this. SPR is a technique in which laser light is incident on a surface of a metal thin film having a thickness of about 50 nm via a prism to detect specific light absorption (attenuation of reflected light) at a certain angle in the total reflection angle region. The refractive index of the surface can be obtained. As a result, measurement of the reaction / bonding amount between biomolecules and kinetic analysis can be performed in real time without labeling such as fluorescence. Currently, it is used for studies of interactions between various substances such as immune responses, signal transduction, proteins and nucleic acids.

  In FIG. 12, the laser beam oscillated from the laser 1201 becomes circularly polarized light by the λ / 4 wavelength plate 1202, passes through the condenser lens 1203, and then enters the prism 304 perpendicularly. Since optical glass that can be manufactured with extremely high homogeneity is required for the glass for prism, synthetic quartz or BK7 or BSC7 having high transmittance and high homogeneity is generally used. When the laser beam is incident on the refractive index boundary plane of the measurement substrate 1205b, that is, the interface between the measurement substrate 1205b and the solution at an incident angle of about 68 °, the laser beam is totally reflected to form an evanescent wave. In FIG. 12, the measurement substrates 1205a and 1205b are set on the turntable 1207 while being fixed to the measurement substrate holder 1206b. By fixing the measurement substrate 1205 and the measurement substrate holder 1206 with high parallelism, the turntable 1207 can be set with high parallelism. Each parallelism is preferably 1 ° or less, but is not limited thereto. If the measurement substrates 1205a and 1205b are large, they can be installed directly on the turntable 1207 without using the measurement substrate holder 1206. It is to be noted that the laser beam is prevented from being totally reflected at the boundary between the prism 1204 and the measurement substrate 1205b by filling the space between the prism 1204 and the measurement substrate 1205b with matching oil 1212b having a close refractive index. Since there is no measurement substrate holder 1206 in the optical path of the laser, only matching oil exists between the prism 1204 and the measurement substrate 1205b. The distance between the prism 1204 and the measurement substrate 1205b is not limited, but if the distance is too large, the matching oil 1212b cannot be held between the prism 1204 and the measurement substrate 1205b. If an air layer enters between the prism 1204 and the measurement substrate 1205b even if the distance is small, the laser beam is totally reflected at that portion. Therefore, it is essential that the structure does not contain bubbles. The turntable 1207 can be rotated by rotating the turntable rotating shaft 1208 with an XYZ stage. The turntable rotating shaft 1208 with the XYZ stage is fixed to the XYZ stage, and the rotation center can be moved to an arbitrary position. Further, by holding the turntable 1207 from above and below using the turntable holders 1209a and 1209b, the height of the measurement substrate 1205b when the turntable rotating shaft 1208 with an XYZ stage is rotated can be always constant.

  There is no limitation on the position and number of the turntable holders 1209a and 1209b, and the turntable 1207 and the measurement boards 1205a and 1205b can be held. Note that the position of the prism 1204 is fixed. The reflected light signal detected by the SPR detector 1210 is processed by the control PC 1214 and the result is displayed on the monitor 1215. Thus, the absorption of the specific light (attenuation of the reflected light) is detected at a certain angle in the total reflection angle region to obtain the refractive index of the gold surface. Matching oil 1212 is attached to the measurement substrate 1205 from the dispensing tip 1211. This is the same method as disclosed in FIG. Further, as disclosed in FIG. 4, the prism 1204 has a tapered structure in contact with the matching oil 1212. By dispensing the liquid amount in which the liquid height of the matching oil 1212a is higher than the distance between the prism 1204 and the measurement substrate 1205b, the prism 1204 and the matching oil 1212a come into contact with each other, and the prism 1204 and the measurement substrate 1205b are in contact with each other. Between them, the matching oil 1212a can be filled. When the liquid level of the matching oil 1212a is lower than the distance between the prism 1204 and the measurement substrate 1205b, the prism 1204 and the matching oil 1212a are not in contact with each other. Therefore, the matching between the prism 1204 and the measurement substrate 1205b is performed. Oil 1212a cannot be filled. Next, the turntable 1207 is rotated 180 ° by rotating the turntable rotating shaft 1208 with an XYZ stage 180 °. At this time, the matching oil 1212a enters between the prism 1204 and the measurement substrate 1205b. After the measurement, the measurement substrate 1205b rotates the turntable 1207 through 180 ° by rotating the turntable rotation shaft 1208 through 180 °. Thereafter, the substrate autoloader 1219 is used to remove the measurement substrate holder 1206a and install a new measurement substrate holder 1206a. The above process is performed fully automatically and continuously. If necessary, the reagent sucked from the reagent container 1216 by the dispensing unit 1217 can be fed to the measurement substrate 1205a through the liquid feeding tube 1218. Further, a thing corresponding to the matching oil wipe 215 shown in FIG. 2 can be installed on the lower surface of the turntable 1207. As disclosed in FIG. 6, the matching oil reservoir container 1213 can collect the matching oil spilled from the prism 1204. The layout diagrams of the optical elements and the like disclosed in Examples 1 to 12 are merely examples, and are not limited to these. For example, the laser is circularly polarized with a λ / 4 wavelength plate, but it may be necessary to linearly polarize depending on the measurement application. In that case, a λ / 2 wavelength plate can be inserted instead of the λ / 4 wavelength plate. Although surface plasmon resonance is disclosed in FIG. 12, it can be applied to many applications such as substance identification by phosphor separation with a spectroscopic prism installed.

  As shown in FIGS. 1 and 3, the prism and the objective lens may be arranged vertically, with the prism and the objective lens being arranged vertically, instead of arranging the prism and the objective lens on or below the turntable. Of course, it is needless to say that the turntable can be inclined. FIG. 13 shows an apparatus configuration diagram of a prism type evanescent microscope when the turntable is vertical. The turntable is omitted. Matching oil 1302 is held between the measurement substrate holder 1301 and the prism 1303. A laser 1304 is irradiated onto the measurement substrate 1305 in a plane perpendicular to the traveling direction of the measurement substrate holder 1301, and is detected through the objective lens 1306. The matching oil 1302 that hangs down from the prism 1303 is collected in the matching oil reservoir 1307. The matching oil 1302 is filled between the prism 1303 and the measurement substrate 1305 using the pump 1310 from the matching oil bottle 1309 through the liquid feeding tube 1308. However, since the turntable is arranged vertically, the matching oil 1302 is likely to hang down from the prism 1303, and thus is returned to the matching oil reservoir 1307 as 1302 d through 1302 b and 1302 c. Although it is desirable that the matching oil 1302 is held at a surface tension between the prism 1303 and the measurement substrate 1305, otherwise, the amount collected in the matching oil reservoir is passed through the liquid feeding tube 1308 and the matching oil bottle. It is necessary to fill the matching oil 1302 between the prism 1303 and the measurement substrate 1305 using the pump 1310 from 1309. It goes without saying that the matching oil 1302 is prevented from adhering to the laser incident portion of the prism 1303 by the prism structure shown in FIG.

  FIG. 14 is a top view of a turntable with a matching oil sump. However, unlike FIG. 2, a structure is shown in which matching oil is filled in concentric circles of the turntable. As the dispensing arm / substrate autoloader arm rotating shaft 1401 rotates, the substrate autoloader arm can access the substrate autoloader 1402. A dotted line centering on the dispensing arm / substrate autoloader arm rotating shaft 1401 indicates a trajectory on which the dispensing arm or substrate autoloader arm rotates. In addition, the dispensing arm can attach the dispensing tip with the dispensing tip attachment unit 1403, suck the reagent with the reagent container 1404, and discard the used dispensing tip into the dispensing tip discarding unit 1405. It should be noted that the dispensing arm / substrate autoloader arm rotating shaft 1401 can be arranged separately, so that substrate installation and dispensing can be performed in parallel. Using the above mechanism, the matching oil 1407 is dispensed to the measurement substrate holder 1409 set on the turntable 1408 with the dispensing tip 1406. At this time, by dispensing a large amount of matching oil 1407 and filling the concentric circles of the turntable 1408, the liquid level of the matching oil 1407 becomes higher than the lower surface of the prism 1413. Matching oil 1407 can be filled between the prism 1413 and the measurement substrate 1412. When dust is mixed into the matching oil 1407, there is a possibility that the laser is diffusely reflected. Therefore, it is preferable to install a dust cover 1421 for preventing dust from entering the matching oil (not shown in the top view). Needless to say, the dust cover 1421 can take various shapes other than those disclosed in the drawings.

  After dispensing the matching oil 1407, the turntable rotating shaft 1410 with XYZ stage is rotated by 180 °. At this time, by holding the turntable 1408 from above and below with the turntable holder 1411, the height of the measurement substrate 1412 when the turntable rotating shaft 1410 with an XYZ stage is rotated can be always constant. There are no restrictions on the position and number of turntable holders 1411, and the turntable 1408 and the measurement substrate 1412 can be held down. Note that the positions of the prism 1413 and the objective lens 1414 are fixed except for fine adjustment of focusing in the microscopic measurement. In addition, a reagent A port 1415 and a reagent B port 1416 can be provided as reagent ports at arbitrary locations on the measurement substrate holder 1409. The number of reagent ports can be set arbitrarily. By rotating the dispensing arm / substrate autoloader arm rotating shaft 1401, the dispensing arm or the substrate autoloader arm is placed on the center of the measurement substrate holder 1409, the reagent A port 1415, and the reagent B port 1416 as shown in FIG. It can move and a reagent etc. can be dripped. As the objective lens 1414, an immersion, water immersion and oil immersion lens are known, but an immersion lens is preferably used. Of course, it is possible to use a water immersion or oil immersion lens, but a mechanism that prevents bubbles from being mixed and evaporation of liquid (particularly water) is required. As an example, a method of feeding water 1418 to the objective lens 1414 through the liquid feeding tube 1417 with the pump 1419 can be considered, but the present invention is not limited to this. 1418 can be other liquids such as matching oil. FIG. 14 shows a prism-type evanescent microscope, but in the case of an objective-type evanescent microscope capable of detecting one molecule, the prism 1413 is removed from FIG. In this case, the matching oil 1407 is not necessary. The laser is incident from the edge of the objective lens. Therefore, the present invention can be applied not only to a prism type evanescent microscope but also to a general microscope such as an objective type evanescent microscope.

  Further, by partially adjusting the temperature of the turntable 1408, the measurement substrate holders 1409a and 1409b can be set to different temperatures. As temperature control methods, there are air temperature control and local temperature control. Air temperature control is a method of adjusting the air temperature of a box to be kept warm by using a heating or cooling device and a temperature control box. Although the temperature stability is high, there are problems that it takes a long time to reach the temperature and that a high temperature of 60 ° C. or higher is difficult. When an air temperature control box is used in a total reflection microscope, there is a high possibility of covering all the microscope stage, prism, and objective lens that hold the slide glass. In particular, regarding the objective lens, use at a high temperature is outside the manufacturer's specification range, so it is desirable to keep it at room temperature as much as possible. The local temperature control is a method of adjusting the temperature by bringing the heating or cooling device into direct contact with the portion where temperature control is desired. Therefore, thermal expansion of the objective lens, which is a concern for air temperature control, does not occur. Although local temperature control is inferior in temperature stability to air temperature control, it has a feature that it takes a short time to reach the temperature and can control a high temperature of 60 ° C. or higher. For example, when it is necessary to change the temperature by enzyme reaction and washing, or when a highly heat-resistant enzyme is used, the local temperature control method is more suitable than air temperature control. Therefore, as a method of adjusting the temperature, a method of bringing a Peltier or heater into contact with the turntable 1408, a method of embedding a heating wire in the turntable 1408, a method of using an infrared laser, etc. are conceivable, but the method is not limited thereto. Is not to be done.

  The turntable holder 1411 may serve as a heater. As a result, while the measurement substrate 1412 sandwiched between the prism 1413 and the objective lens 1414 is being detected, the reagent is supplied to the other measurement substrate 1412 and reacted at different temperatures for a certain period of time, and then detection is performed. Bioassays are possible. For example, in PCR, a nucleic acid can be doubled by generally passing through three temperature cycles (eg, thermal denaturation: 95 ° C., hybridization: 58 ° C., extension: 72 ° C.). Therefore, by installing three measurement substrate holders 1409 on the turntable 1408 and setting the temperature to three levels, the nucleic acid is doubled every time the turntable 1408 rotates once. Quantitative PCR that measures the initial amount of DNA can be realized. In addition, as shown in the examples, there are many applications such as performing DNA sequencing by extending a fluorescently labeled base one base at a time for two strands of DNA and primer. The application of the present invention is not limited to these.

101, 301, 404, 504, 604, 818, 918, 1201, 1304 Laser 102, 302, 1202 λ / 4 wave plates 103, 303, 1203 Condensing lenses 104, 213, 304, 503, 603, 703, 813 913, 1013, 1106, 1204, 1303, 1413 Prism 105, 212, 305, 405, 505, 605, 704, 812, 1012, 1104, 1305, 1412 Measurement board 106, 209, 306, 401, 501, 601, 701 , 809, 909, 1009, 1103, 1206, 1301, 1409 Measurement board holder 107, 208, 307, 1008, 1102, 1207, 1408 Turntable 108, 210, 308, 1010, 1208, 1410 With XYZ stage Turntable rotating shaft 109, 211, 309, 1011, 1209, 1411 Turntable holder 110, 214, 310, 406, 506, 606, 705, 814, 914, 1014, 1107, 1306, 1414 Objective lens 111, 311 Objective lens Z-axis stage 112, 312 Filter unit 113, 313 Imaging lens 114, 314 CCD
115, 315, 1214 Control PC
116, 316, 1215 Monitor 117, 204, 317, 804, 904, 1004, 1216, 1404 Reagent container 118, 318, 1217 Dispensing unit 119, 218, 319, 322, 608, 919, 1218, 1308, 1417 Tube 120, 207, 323, 402, 502, 602, 702, 807, 907, 1007, 1108, 1212, 1302, 1407 Matching oil 121, 202, 321, 802, 902, 1002, 1219, 1402 Substrate autoloader 201, 801 , 901, 1001, 1401 Dispensing arm / substrate autoloader arm rotating shaft 203, 803, 903, 1003, 1403 Dispensing tip mounting portion 205, 805, 905, 1005, 1405 Dispensing tip discarding portion 206, 80 , 906, 1006, 1211, 1406 Dispensing tips 215, 815, 915, 1015 Matching oil wipes 216, 816, 916, 1016, 1415 Reagent A port 217, 817, 917, 1017, 1416 Reagent B port 219, 320, 1418 Water 220,324,610,921,1310,1419 Pump 325,607,706,922,1213,1307 Matching oil reservoir 403 Tapered prism 407 R prism 609,920,1309 Matching oil bottle 611 Laser incident / reflection position 612 Recess 707 Measurement substrate holder cover with flow path 708 Reagent injection / discharge port 808,908 Substrate installation table with XYZ stage 810,910 Table holder 1105 Measurement substrate holder holder 1205 Measurement board 1210 SPR detector 1420 Dust cover (with gold thin film)

Claims (12)

  A matching object, a mechanism for dispensing a solution, a measurement target substrate, and a moving mechanism for moving the solution between the substrate and the matching target object, the moving mechanism including a rotation shaft, As a mechanism for mounting the substrate to a mechanism for mounting the substrate, a mechanism for holding the substrate, the substrate having a mechanism for mounting the substrate, and the substrate to be mounted on the mechanism for mounting the substrate; A rotation mechanism, a parallel movement, a vertical movement, or a combination thereof, and a mechanism for separating the substrate, and the solution is made of a matching oil, immersion oil, glycerol, water, or a similar solution. The dispensing mechanism has a structure that dispenses the solution onto an upper surface or a lower surface of the moving mechanism, and the moving mechanism moves the substrate to the matching object. And a pumping light incident prism for measuring the substrate, wherein the dispensing mechanism dispenses the solution, and stops at a position that is in front of the measurement position with respect to the direction to be measured. It is either an objective lens or a component for measuring the substrate, and the matching object has a structure in contact with the solution on a taper, a curved surface, or a similar surface, and the matching object is a cube. , A rectangular parallelepiped, a cylinder, or a similar indentation structure, the excitation light can be incident on the indentation part, and the excitation light is incident from a different angle with respect to the moving direction of the moving mechanism in the vicinity of the matching object The moving mechanism has a plurality of heating and cooling structures, and the matching object has a mechanism for collecting the solution underneath, Constant target substrate, a prism type total reflection microscope objective-type total reflection microscope, one molecule fluorescence measurement, surface plasmon resonance, Raman measurement, measuring device characterized in that it is measured by any method spectroscopy. A total reflection fluorescence measuring device that causes base extension reaction on a measurement target substrate and performs fluorescence observation using an evanescent field,
An irradiation mechanism for irradiating the measurement target substrate with excitation light;
A prism for totally reflecting the excitation light from the irradiation mechanism on the measurement target substrate;
An observation mechanism for observing the fluorescent dye incorporated by the base extension reaction;
A dispensing mechanism for dispensing matching oil to be filled between the measurement target substrate and the structure;
Provided with a moving mechanism for moving matching oil between the measurement target substrate and the prism ,
The moving mechanism includes a turntable that rotates about an axis,
A total reflection fluorescence measuring apparatus, wherein the measurement target substrate is fixed to the turntable, and the measurement target substrate moves to a position facing the prism by the rotation of the turntable . It is a total reflection fluorescence measuring apparatus of Claim 2 , Comprising:
The rotation speed of the turntable is variable,
The total reflection fluorescence measuring apparatus, wherein the speed of the turntable is decreased when the matching oil contacts the prism or immediately before the contact. It is a total reflection fluorescence measuring apparatus of Claim 2 , Comprising:
A total reflection fluorescence measuring apparatus comprising a wiping mechanism for wiping matching oil on a turntable, wherein the turntable rotates and the wiping mechanism wipes the matching oil. It is a total reflection fluorescence measuring apparatus of Claim 2 , Comprising:
A total reflection fluorescence measuring apparatus characterized in that at least a part of the end of the prism on the substrate side to be measured is formed at an acute angle with respect to the surface of the turntable. It is a total reflection fluorescence measuring apparatus of Claim 5 , Comprising:
The total reflection fluorescence measuring apparatus is characterized in that the part formed at an acute angle is a part where the prism first comes into contact with the matching oil by the rotation of the turntable. It is a total reflection fluorescence measuring apparatus of Claim 2 , Comprising:
The rotary table includes a substrate fixing holder for fixing a measurement target substrate, and a reagent solution flow path is provided in the substrate fixing holder. It is a total reflection fluorescence measuring apparatus of Claim 1 , Comprising:
The rotary table includes a plurality of measurement target substrate holding units, and each measurement target substrate holding unit can be independently temperature controlled. It is a total reflection fluorescence measuring apparatus of Claim 2 , Comprising:
A total reflection fluorescence measuring apparatus, characterized in that the planar shape of the prism is such that the width becomes wider toward the outside from the center. It is a total reflection fluorescence measuring apparatus of Claim 2 , Comprising:
A total reflection fluorescence measuring apparatus characterized in that the planar shape of the prism is that of a bow tie. The total reflection fluorescence measuring apparatus according to claim 9 or 10 ,
A total reflection fluorescence measuring apparatus characterized in that a prism first comes into contact with a matching solution at a narrow part of the prism. It is a total reflection fluorescence measuring apparatus of Claim 2 , Comprising:
A total reflection fluorescence measuring apparatus, wherein a recess is provided at a laser irradiation position of a prism.

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