JP2011099720A - Analyzer, autofocus device, and autofocusing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To detect a focus deviation direction in a contrast detection type autofocusing. <P>SOLUTION: A plurality of structures having different height or a digging is disposed near an observation target, and their respective focus evaluation values are calculated each. A focus deviation direction is identified by a difference in the focus evaluation values. By distinguishing the focus deviation direction, it is possible to continue to observe the observation target without losing images. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an autofocus apparatus for a reaction device and an analysis apparatus including the same for observing a reaction by an optical sensor element in a nucleic acid analyzer such as a genetic diagnosis / analysis apparatus.

  In recent nucleic acid analyzers, a method has been proposed in which a large number of DNA probes or polymerases are immobilized on a reaction device made of a glass substrate or the like, and a base extension reaction is performed here to determine the sequence. The region where this fixation and reaction is performed is hereinafter referred to as a reaction spot. A reaction spot may be a single molecule immobilized (single molecule system) or a plurality of molecules of the same species (multiple molecule system). A massively parallel nucleic acid analyzer has been developed in which a large number of reaction spots are arranged and base extension and sequencing are performed in parallel at each reaction spot.

  In Non-Patent Document 1, which is a specific example in which a single molecule is fixed to a reaction spot, DNA sequence decoding at a single molecule level using a total reflection evanescent irradiation detection method is performed. Lasers with wavelengths of 532 nm and 635 nm are used as excitation light, and are used for fluorescence excitation of the phosphors Cy3 and Cy5, respectively. A single target DNA molecule is immobilized on the side of the solution layer on the refractive index boundary plane using biotin-avidin protein binding to form a reaction spot. When a Cy3-labeled primer is introduced into the solution by solution exchange, a single fluorescently labeled primer molecule hybridizes to the target DNA molecule. After this hybridization reaction for a certain period of time, unreacted excess primer is washed away. After that, when total reflection evanescent irradiation using excitation light of 532 nm is performed, Cy3 is present in the evanescent field, so that the binding position of the target DNA molecule can be confirmed by fluorescence detection. After confirmation, Cy3 is irradiated with high-output excitation light to cause fluorescence fading, and subsequent fluorescence emission is suppressed. Next, when a polymerase and dNTP of one kind of base labeled with one molecule of Cy5 (N is any one of A, C, G, and T) are introduced by solution exchange, the target DNA molecule is introduced. Only when they are complementary, the fluorescently labeled dNTP molecule is incorporated into the extended strand of the primer molecule. After this extension reaction is performed for a certain period of time, excess unreacted dNTP is washed away. After that, when total reflection evanescent irradiation using excitation light of 633 nm is performed, Cy5 exists in the evanescent field, so that the complementary relationship can be confirmed by fluorescence detection at the binding position of the target DNA molecule. After the confirmation, Cy5 is irradiated with high-power excitation light to cause fluorescence fading, and subsequent fluorescence emission is suppressed. Bases complementary to the target DNA molecule are obtained by repeating the above-described dNTP incorporation reaction process in a step-by-step manner such as A → C → G → T → A → (step extension reaction). It is possible to determine the sequence. A plurality of reaction spots are formed in a region (hereinafter referred to as a measurement visual field) that can be observed at one time by a detector for measuring fluorescence, and each of the reaction spots contains a different target DNA molecule. By processing the processes in parallel, simultaneous DNA sequencing of a plurality of target DNA molecules becomes possible. It is expected that the number of simultaneous parallel processes at this time can be dramatically increased as compared with the conventional DNA sequencing based on electrophoresis.

  Further, some advanced researches include a combination of semiconductor chips having a fine structure for generating plasmon resonance or the like in decoding of a DNA base sequence in a single molecule unit. As an example, Patent Document 1 uses a fluorescence enhancement effect that is several to several tens of times that of localized surface plasmons. The range to which the effect of fluorescence enhancement is about 10 nm to 20 nm. If localized surface plasmons are generated on the surface of the metal microstructure on which the target DNA is immobilized, only the fluorescently labeled dNTP incorporated into the target DNA will benefit from fluorescence enhancement. The fluorescence intensity difference is doubled to several tens of times or more. This method makes it possible to measure the base extension reaction without removing unreacted fluorescently labeled dNTP.

  On the other hand, various methods for aligning target molecules in an arbitrary shape and arrangement have been proposed. In Non-Patent Document 2, an electrode having a desired pattern is first provided on a substrate, and PLL-g-PEG (Poly-L-Lysin-g-polyethylene glycol) is applied to the entire surface of the substrate. Thereafter, a voltage is applied to the electrode to retract the electrode part PLL-g-PEG. This presents a method for specifically adsorbing fluorescent molecules only on the electrode part. In Non-Patent Document 5, after applying photodissociable molecules to a substrate, a nanoscale target molecule fixed region pattern is produced using a lithography technique using near-field scanning light. In these techniques, a method of producing a DNA or protein pattern of 100 nm or less on a substrate is shown.

  Furthermore, although the extension reaction proceeds by dNTP uptake by a polymerase, recent researches have proposed various reaction systems in which dNTP uptake is continuously and automatically performed. In such a reaction system that has been newly researched, for the purpose of improving throughput and reliability, research has been conducted to extend the number of bases read in one observation to about several thousand bases. In order to increase the number of bases to be read, it is necessary not only to improve the reaction method, but also to stably and continuously observe a detection device that observes the reaction.

  In these nucleic acid analyzers, it is important to correctly capture the DNA elongation reaction using an optical detection system composed of a lens and a detector. It is essential to focus. Further, in a so-called real-time reaction system in which the base extension reaction is performed without step control, it is difficult to temporarily stop the base extension reaction. For this reason, after starting the observation, it is impossible to stop the observation until the extension reaction is completed. In the meantime, in the observation of the object on the nano order, the in-focus position may be changed every moment sensitively to the influence of a subtle change in room temperature or the vibration of the surrounding ground.

  For this reason, while observing the object, it is necessary to always detect the amount of focus deviation in parallel with it and perform autofocus to continue correcting the deviation. As this method, an autofocus method using auxiliary light such as infrared rays (hereinafter referred to as an active method autofocus) is generally used. In this method, auxiliary light such as infrared rays is irradiated onto an observation object, and the amount of focus deviation is confirmed from the characteristics or phase of the reflected light. An example of this is JP-A-61-112112. In this invention, a subject is irradiated with infrared light through an imaging lens, and a shift amount is detected based on a spot position shift amount when the reflected light is imaged. According to this method, since the amount and direction of focus deviation can be detected, the object can always be observed in a focused state by correcting the lens extension amount in the direction in which the amount of deviation is corrected.

  However, this method requires an additional optical system, a light emitting element, and a detecting element, which makes the structure complicated and increases the cost. In addition, since it is necessary to irradiate the observation target with light for autofocusing, it is necessary to give sufficient consideration to the wavelength and intensity so that the light does not have an unnecessary influence on the observation target. The nucleic acid analyzer as the object of the present invention uses a plurality of fluorescence and excitation light that excites them, and may affect the detection of a weak reaction signal.

JP 61-112112 A JP 2007-171158 A P.N.A.S. 2003, Vol. 100, pp. 3960-3964. Analytical Chemistry, Vol.78, No.3, February 1, 2006, pp. 711-717

  As an autofocus method that does not have the above-described problem in principle, there is a contrast detection type autofocus. In this method, a high frequency component is extracted from an image acquired by a camera, and the magnitude of the strength of the high frequency component is defined as a focus evaluation value. Then, the lens extension amount is adjusted so that the focus evaluation value becomes the highest while photographing an image. The high-frequency component in the image is mainly the contour of the object to be photographed, and the point at which these are detected most strongly is based on the focal point.

  When this method is applied to a nucleic acid analyzer, the optical system and detector for observation can be used as they are, so that no additional optical system or detector is required, and autofocus can be realized at low cost. . In addition, since there is no need to make auxiliary light incident unlike active type autofocus, there is no influence on the observation target.

  Because of these advantages, autofocus by contrast detection is suitable for nucleic acid analyzers. However, as a problem to be applied, when it is out of focus, it is unclear which direction the deviation is. There was a drawback of being.

  In the active type autofocus, the shift direction can be detected from the return light reflected upon the object by, for example, the knife edge method. This makes it possible to determine whether the positional relationship between the object and the focal point is too far or too close. On the other hand, in contrast detection method, although it is known that the focus is shifted, the direction of the shift is unknown, and the lens extension amount is moved in either direction, and the change in the focus evaluation value is examined on a trial basis. The only way to estimate the direction of defocus was.

  When the amount of lens extension is changed, if it is in the direction of defocusing, observation cannot be continued, and information on the base extension reaction during observation is lost. For this reason, in order to apply the contrast detection type autofocus to the nucleic acid analyzer, it is essential to have a means capable of knowing which direction the focus shift direction is.

  In order to solve the above problem, the method of the present invention arranges a plurality of convex portions or concave portions having different heights in the vicinity of the observation target, for example, on the reaction substrate, and calculates the focus evaluation values of these contrast detection portions. The focus shift direction is identified by the difference between these focus evaluation values.

  By adopting the structure as described above, the focus position can be confirmed while capturing the fluorescence emission due to the base extension reaction that is the observation object. Further, when the focus position changes due to some influence, the shift direction can be determined. For this reason, the focus position can be readjusted without stopping the reaction observation, and a stable observation over a long period of time becomes possible.

  By using this method, an additional optical system such as the active method autofocus, a light emitting element, and a detection element are not required, so that the structure of the detection optical system is simplified and adjustment points are reduced. Further, since unnecessary auxiliary light is not irradiated onto the object, the focused state on the object can be maintained without any photochemical effect.

An example of the most basic configuration of the present invention. An example of a device that performs base sequence decoding. Details around the reaction substrate 101 and the two-dimensional sensor camera 221. Procedure for manufacturing a structure that can be used in the present invention. The conceptual diagram of a focus position determination method. An example of a structure that can solve problems when shooting conditions are different. The conceptual diagram of the focus position determination method by a contrast detection system. Reaction board structure that can detect the amount of focus shift. An example of an autofocus target using beads. The conceptual diagram of the focus position discrimination method by the bead for autofocus.

  The best mode for carrying out the present invention will be described below. Note that this embodiment is used for understanding the invention and does not limit the scope of rights to the embodiment.

  In this embodiment, three types of sights are created on the reaction substrate: (1) sight of the same height as the substrate plane, (2) sight at the position where the substrate is dug, and (3) sight at the position protruding from the substrate plane. To do. Either one of the structures (2) and (3) may be used. After producing such a structure, in the actual observation stage, the reaction substrate is observed from above, and the focus evaluation values of three or two types of aim are calculated individually. When the focus evaluation value of the contrast detection part protruding from the surface is high, it can be determined that the in-focus position is in the front direction. For this reason, in order to readjust the focus, it is only necessary to adjust the lens extension amount so as to focus farther. Further, when the focus evaluation value of the bottom surface portion of the digging structure arranged on the reaction substrate is high, it can be determined that the in-focus position is far away. For this reason, in order to readjust the focus, it is only necessary to adjust the lens extension amount so as to focus closer.

  There is no particular limitation on the shape of the sight that becomes the contrast detection region as long as the focus evaluation value can be calculated, and it may be generated in an arbitrary shape. A structure arranged for other purposes may also be used as a contrast detection part. The height of the unevenness of the contrast detection portion is preferably 10 nm or more and 100 μm or less, for example, from the depth of field derived from the optical resolution. As for the arrangement of the contrast detection parts, ideally, it is desirable to prepare a position higher and lower than the surface in addition to the substrate surface, but for simplification of the structure, only the position where the height is high or It does not matter even if it is in a low position.

  As a further developed form, the height of the contrast detection part can be changed in small increments, and a structure capable of estimating the amount of focus deviation can be obtained by arranging a plurality of aiming heights. Specifically, for example, sights having different heights are created in a range of ± 5 μm at a pitch of 1 μm with respect to the reaction substrate plane. When observing, for example, if the aim at the position of +2 μm has the highest focus evaluation value, it can be estimated that the focus has shifted by about +2 μm. For this reason, if the lens extension amount is extended by 2 μm in the minus direction, it becomes possible to focus instantaneously without unnecessary movement.

  FIG. 1 shows an example of the most basic configuration of the present invention. On the reaction substrate 101, nanostructures or pads 104 for analyzing gene sequences or gene polymorphisms are arranged on a grid, on a honeycomb, or randomly. For example, an autofocus target 103 is arranged at the center of this region. This region has a structure in which a cross target made of a different material can be confirmed at the top when viewed from the top surface, and has a structure disposed on the bottom surface of the dug portion 102 when viewed from the side surface. It has a structure with a difference in height.

  This cross target is made of a metal that emits fluorescence when irradiated with excitation light. The cross target may be a metal that does not emit fluorescence when irradiated with excitation light and has a fluorescent dye adsorbed thereto.

  FIG. 2 shows an example of an apparatus for performing base sequence decoding using this reaction device. The apparatus example of FIG. 2 is an example of a single molecule DNA sequencer, and includes an analyzer 240 and an analysis computer 222. In the analyzer 240, the reaction on the reaction substrate 214 is observed with the two-dimensional sensor camera 221. The reagent is supplied to the reaction substrate 214 by dispensing the reagent stored in each container in the reagent storage unit 216 with the dispensing unit 217 and using the liquid feeding tube 218. In addition, the temperature of the supplied reagent is appropriately adjusted by the temperature control unit 215 so that the temperature is optimal for the reaction to proceed. The waste liquid after the reaction is completed is discarded into the waste liquid container 220 via the waste liquid tube 219.

  The reaction substrate used in such an apparatus is optically coupled to the total reflection prism 229 when measured by evanescent light, for example, and illuminated by total reflection illumination by the excitation light laser units 224 and 225. . When the excitation light is irradiated, total reflection occurs on the refractive index boundary plane on the upper surface side of the substrate. At this time, the electromagnetic wave penetrates into the low medium side by the height of about one wavelength of incident light. Thereby, only an extremely limited region including the metal structure is illuminated. This area is called an evanescent field.

  When the base extension reaction proceeds on this apparatus by the above-described method, the target DNA attached on the structure of the reaction substrate 214 and the fluorescent dye incorporated by the polymerase are excited by the laser light to emit light. This is captured as a two-dimensional image by an optical detection system including a fluorescence wavelength filter 232 and an imaging lens 233 that are optical filters that transmit only the fluorescence wavelength, and a two-dimensional sensor camera 221.

  The above cross target can also be detected by the same two-dimensional sensor camera 221. This two-dimensional sensor camera 221 always observes the fluorescence of the cross target during the fluorescence observation of the base extension reaction.

  Details of the reaction substrate 101 and the periphery of the two-dimensional sensor camera 221 are shown in FIG. The fluorescence signal when the base extension reaction is performed on the reaction substrate 101 on which the nanostructure or the pad 104 is arranged is collected by the objective lens unit 310 and observed by the two-dimensional sensor camera 221. The objective lens unit 310 includes the objective lens 231, the fluorescence wavelength filter 232, and the imaging lens 233 shown in FIG. For focus position driving for auto-focusing, there are methods of moving the reaction substrate 301 or moving the objective lens unit 302. The method of the present invention can be applied to any focus position driving method.

  FIG. 4 shows a procedure for manufacturing a contrast detection portion that can be used in the present invention. The substrate of the present invention can be manufactured by the procedures shown and described below.

(1) Resist application 1
A resist 402 is applied on a smooth support base 401 such as quartz or silicon. As the resist 402, a negative positive for electron beam can be used. Specifically, TEBN-1 (made by Tokuyama Co., Ltd.) is mentioned. After applying the resist with a spinner, it is pre-baked with a hot plate for about 2 to 5 minutes.

(2) Patterning 1
After drawing with an electron beam having an acceleration voltage of 50 to 100 KV, development is performed with ethyl lactate, isopropanol, or ethanol.

(3) Etching 1
The support base 401 is etched using the patterned resist as a mask. Since the digging created by this etching is only used for referring to the focus evaluation value at the time of autofocusing, the depth and dimensional accuracy are not so required. For this reason, an accuracy of about 500 nm ± 10% in depth is sufficient, and the control of the digging depth may be easily managed by the etching time calculated from the etching rate.

(4) Resist removal 1
For the resist removal, a widely and commonly used ozone ashing process can be used.

(5) Metal film formation A metal film 403 is formed. The thickness of this film controls the height of the finally formed nanostructure 421. When using the localized surface plasmon, the thickness at which the height of the metal body is effectively generated varies depending on the excitation wavelength used at the time of measurement, but the desirable thickness is 1000 nm or less. As a thin film forming method, vapor deposition, sputtering, CVD, PVD, or the like can be used.

(6) Resist application 2
A resist is applied by the same method as in the procedure (1).

(7) Patterning 2
In this patterning, the shape of the nanostructure 421 necessary for base extension reaction observation is drawn, and at the same time, a target 422 for calculating a focus evaluation value in autofocus is drawn. In this case, the size of the target is desirably at least 5 μm square in consideration of a reduction in resolution due to lens performance, optical system construction accuracy, and the like, and ease of automatic search for the target.

(8) Etching 2
The metal film 403 is etched using the patterned resist as a mask. As a result, the other metal film is removed while the nanostructure 421 necessary for observation of the base extension reaction and the target 422 for calculating the focus evaluation value in autofocus remain.

(9) Resist removal 2
Finally, unnecessary resist is removed. It can be removed by the same method as in (4).

  The reaction substrate optimal for the present invention can be generated by the above method.

  In this example, a process for forming an autofocus target is added. However, when generating a reaction substrate having a complicated structure as shown in Japanese Patent Application No. 2009-088002, each process is performed. It is necessary to dig an alignment mark on the reaction substrate for alignment between them. In such a case, it is possible to reduce the number of processes by placing these marks in an observation field when actually analyzing and also serving as a contrast detection part.

  For the method of fixing the DNA probe to the reaction substrate prepared by the present method or the arrangement of the fluorescence enhancement structure using localized surface plasmons, refer to Japanese Patent Application Laid-Open No. 2007-171158, Japanese Patent Application No. 2009-084002, etc. The method shown may be used.

  FIG. 5 shows a conceptual diagram of a focus position determination method in the contrast detection method using the autofocus target 103. When the autofocus target 103 is observed with the focus on the reaction substrate, the pad is in focus and the autofocus target is slightly defocused as shown in FIG. Observed at. The magnitude relationship between the focus evaluation values at this time is as illustrated. If the reaction substrate surface is closer to a good focus position, the focus evaluation value of the autofocus target gradually increases, while the pad focus evaluation value decreases. On the other hand, when the reaction substrate surface is far from the good focus position, the focus evaluation value of both the pad and the autofocus target decreases as shown in the vicinity of FIG.

  Based on the tendency of the change in the focus evaluation value, it is possible to detect in which direction the focus is shifted, and it is possible to drive without fail in the in-focus direction.

  In order to perform these processes, it is desirable to have a function of automatically searching for the cross target from the captured image. However, there are cases where performance relating to image processing is not sufficient in a device embedded computer or the like, and it may be difficult to realize a function of automatically searching for a cross target. In such a case, the device structure can be accurately aligned so that the cross target is captured in advance at a specific coordinate position on the image, and the autofocus position determination process can be performed with the coordinate area fixed in the image. It is.

  Again, according to this example, even if the focus is shifted due to the relationship between the autofocus evaluation value of the interface fluorescence and the value of the target autofocus evaluation value, whichever is shifted? (Whether it is near or far). For example, when there is one target and the distance to the optical sensor is farther than the interface, the following is performed. If target autofocus evaluation value> interface fluorescence autofocus evaluation value, the distance between the optical sensor and the interface is too short. Further, when the target autofocus evaluation value <the interface fluorescence autofocus evaluation value and the interface fluorescence autofocus evaluation value is equal to or smaller than a predetermined threshold, the distance between the optical sensor and the interface is too long. In other cases, it is in focus.

  Note that the nanostructure shown by the circle in FIG. 5 is 100 nm, while the distance extended by the extension reaction is 0.5 nm per base, which is very small compared to the nanostructure, so that it is incorporated by the extension reaction. There is almost no effect of changing the position of the fluorescent dye.

  Further, in this embodiment, for the sake of simplicity, the description has been made assuming that there is one observation field on the reaction substrate. In this way, when there is one observation field, only one cross target is required, but when there are a plurality of observation fields on the reaction substrate, a cross target is required for each observation field. This is the same in other embodiments described below.

  In this example, the fluorescent dye incorporated during the extension reaction was also used for focusing. In addition to this, another cross target having a vertical distance different from the cross target other than the cross target described above. May be used for focusing.

  In Example 1, the autofocus target was placed at the digging position, and the defocus direction was identified using the height difference from the reaction substrate surface. In this method, when the focus position is deviated in the near direction, the focus evaluation value at the appropriate position is compared with the focus evaluation value at an appropriate position. However, the focus evaluation value can be directly compared only when the illumination method, the brightness of the entire screen, etc. are constant, but the comparison as an absolute value does not make sense when the shooting conditions are different. Absent. An example of an autofocus target that can solve this problem is shown in FIG.

  In the reaction substrate shown in FIG. 6, the digging portion target 603 is the same as the structure in the first embodiment, but in addition to that, a target 601 and a projection target 602 having the same height as the reaction substrate plane are added. ing.

  In order to manufacture a reaction substrate having this structure, (5) two different metal films are formed at the time of metal film formation, and resist coating, patterning, Etching and resist removal may be performed, and then resist coating, patterning, etching, and resist removal for pad and digging portion target creation may be performed.

  FIG. 7 shows a conceptual diagram of a focus position determination method in the contrast detection method using this structure. In a reaction substrate having the structure of this method, when a target is captured with an appropriate focus position, the focus evaluation value b of the cross target at the center of the reaction substrate surface is the highest, and the height of the reaction substrate surface is The focus evaluation values a and c of different cross targets are low. If the reaction substrate surface is closer to a good focus position, the focus evaluation value a of the cross target created as the protrusion is the highest as shown in FIG. 7 and is the same height as the reaction substrate plane. The focus evaluation value b of the cross target and the focus evaluation value c of the cross target of the digging portion are lower values. On the contrary, when the reaction substrate surface is far from a good focus position, the focus evaluation value a of the cross target created as the protrusion is the lowest, and the cross target of the same height as the reaction substrate plane The focus evaluation value increases in the order of the focus evaluation value b and the focus evaluation value c of the cross target in the digging portion.

  By using this method, even when photographing conditions such as illumination state and brightness frequently change, it is possible to determine the focus shift direction only by relative comparison of these three focus evaluation values.

  According to the present embodiment, as described above, the brightness is improved by providing the target member closer to the photographing mechanism than the interface and the target member farther from the photographing mechanism than the interface on both sides of the interface. Even if it has changed, it is possible to accurately grasp which one has shifted by simply looking at the magnitude relationship.

  In this example, the fluorescent dye incorporated during the extension reaction was also used for focusing. In addition to this, in addition to the two cross targets, the distances in the vertical direction are different from the two cross targets ( A third) cross target may be provided and used for focusing.

  In Examples 1 and 2, although the focus shift direction could be determined, it was unknown how much the shift amount was. FIG. 8 shows a reaction substrate structure that can solve this problem and detect the amount of defocus.

  In this method, five, seven, nine, or even other numbers of autofocus targets 801 having different heights are arranged. FIG. 8 shows an example of a structure in which nine targets including a target having the same height as the plane of the reaction substrate 802 are created. Such a structural example may be created by using the above-described semiconductor manufacturing process, or the MEMS technology or nanoimprint technology which is an advanced technology thereof.

  When each focus evaluation value is calculated for such an autofocus target 801, focus evaluation value profiles such as near, good, and far shown in FIG. 8 can be obtained according to the distance. By calculating the peak position of this profile by Gaussian fitting or least square approximation with a quadratic function, a shift distance coefficient proportional to the focus shift amount can be calculated. For example, if the difference in height of each target is 100 μm and the peak at the time of Gauss fitting in the near pattern in FIG. 8 is −1.8, only about 180 μm from −1.8 × 100 μm. It can be determined that the focus position is shifted to a close position. In this case, it is possible to move to the best focus position in a short time by shifting the lens extension amount by 180 μm in the long distance direction.

  The amount of change in the height of each autofocus target in this method may be not only monotonically increased but also polynomial or exponential. For example, in the case of the above embodiment, the height of each target is set to −800 μm, −400 μm, −200 μm, −100 μm, 0 μm, 100 μm, 200 μm, 400 μm, and 800 μm. By arranging in this way, when the amount of change in height is large, focus deviation can be captured in a wider range. However, as the in-focus position becomes farther than 0 μm, the position resolution becomes coarse and the deviation amount calculation accuracy decreases. By using an exponential change amount in this way, it is possible to achieve both autofocus with high accuracy near the in-focus target while accommodating a wider range of defocus.

  In this embodiment, the fluorescent dye incorporated during the extension reaction is also used for focusing. However, as in the first and second embodiments, another target member may be provided and used for focusing.

  In order to create autofocus targets having different heights on the reaction substrate, it is necessary to repeat a process of creating and etching a mask by electron beam drawing or photolithography a plurality of times. For this reason, when the process for creating other structures and the process for creating the autofocus target cannot be combined, it is necessary to add a manufacturing process to create the autofocus target, which increases the manufacturing cost of the reaction substrate. To do.

  When the increase in cost becomes a problem, it is desired to realize this method by a cheaper method. The nucleic acid analyzer can be realized by, for example, separating beads on a reaction substrate and using this as an autofocus target. FIG. 9 shows this example. Compared with the reaction spot 903, an autofocus bead 901 having a difference in focus evaluation value is prepared. As the size of the beads, for example, microbeads having a diameter of about 100 nm to 10 μm, preferably about 1 μm are used. As the bead material, acrylic, ceramic, polystyrene, polypropylene or the like can be used.

  Such beads are introduced into the flow path in accordance with the introduction of the reagent, and are adsorbed specifically at any position on the reaction substrate 902 or using chemical bonds, or fixed at a specific position by using magnetic beads. To do. Alternatively, the flow path in the flow cell may be divided into two systems, and one of them may be dedicated to bead introduction, and the arrangement of reaction spots and beads may be completely separate. However, even in this case, the reaction spot plane and the bead arrangement plane must have the same height.

  FIG. 10 shows a conceptual diagram of a focus position determination method using the autofocus beads 901. When the autofocus bead 901 is observed with the focus on the reaction substrate, the reaction spot is in focus and the autofocus bead 901 is slightly defocused as shown in FIG. Observed as observed. The magnitude relationship between the focus evaluation values at this time is as illustrated.

  If the reaction substrate surface is closer than a good focus position, the focus evaluation values of both the pad and the autofocus target are lowered as shown in FIG. On the other hand, when the reaction substrate surface is far from a good focus position, the focus evaluation value of the autofocus beads 901 gradually increases, while the focus evaluation value of the pad decreases.

  Based on the tendency of the change in the focus evaluation value, it is possible to detect in which direction the focus is shifted, and it is possible to drive without fail in the in-focus direction.

  This autofocus target bead can also be formed of a metal that emits fluorescence, or a metal that does not emit fluorescence when irradiated with excitation light and has a fluorescent dye adsorbed thereto.

  A plurality of autofocus microbeads having different sizes may be used, and focusing may be performed based on these. That is, the reaction spot is not necessarily used for focusing.

  In the present specification, the target has been described as a cross. However, the target may be a shape that can detect the contrast of the edge, and may be a linear shape, a circular shape, a donut shape, or the like.

101, 214, 802, 902 reaction substrate 102 digging part 103 autofocus target 104 nanostructure or pad 215 temperature control unit 216 reagent storage unit 217 dispensing unit 218 liquid feeding tube 219 waste liquid tube 220 waste liquid container 221 two-dimensional sensor Camera 222 Analysis computer 223 Device control computer 224 Excitation light laser unit 1
225 Laser unit 2 for excitation light
226 λ / 4 wavelength plate 227 Mirror 228 Dichroic mirror 229 Total reflection prism 230 Measuring optical path 231 Objective lens 232 Fluorescence wavelength filter 233 Imaging lens 234 Camera controller 240 Analyzer 301 Move reaction substrate 302 Move objective lens unit 310 Objective lens unit 401 Support base 402 Resist 403 Metal film 421 Nanostructure 422 Target 601 Target 602 having the same height as the reaction substrate plane Projection target 603 Dimming target 801 Autofocus target 901 Autofocus bead 903 Reaction spot

Claims (44)

An analyzer that performs base extension reaction on a light-transmitting substrate and observes fluorescence of the incorporated fluorescent dye using an evanescent field,
An irradiation mechanism for totally reflecting the excitation light to the light-transmitting substrate;
A photographing mechanism for photographing the fluorescent dye incorporated by the base extension reaction,
On the light transmissive substrate, a target member that emits fluorescence when irradiated with excitation light at a position where the distance in the vertical direction to the imaging mechanism is different from the reaction interface,
An analyzer characterized by detecting at least a focus shift direction based on at least a focus evaluation value obtained from a target member and changing a distance between the light transmissive substrate and the irradiation mechanism. An analyzer that performs base extension reaction on a light-transmitting substrate and observes fluorescence of the incorporated fluorescent dye using an evanescent field,
An irradiation mechanism for totally reflecting the excitation light to the light-transmitting substrate;
A photographing mechanism for photographing the fluorescent dye incorporated by the base extension reaction,
A light-transmitting substrate is provided with a target member that emits fluorescence when irradiated with excitation light at a position where the distance from the imaging mechanism in the vertical direction with respect to the light-transmitting substrate is different from the reaction interface. Characteristic analyzer. An analyzer for performing base extension reaction of a nanostructure on a light-transmitting substrate and observing fluorescence of the incorporated fluorescent dye using an evanescent field,
An irradiation mechanism for totally reflecting the excitation light to the light-transmitting substrate;
And a photographing mechanism for photographing the fluorescent dye incorporated into the nanostructure by the base extension reaction,
On the light transmissive substrate, a target member that emits fluorescence when irradiated with excitation light at a position where the distance from the imaging mechanism in the vertical direction on the light transmissive substrate is different from the nanostructure,
An analyzer characterized by detecting at least a focus shift direction based on at least a focus evaluation value obtained from a target member and changing a distance between the light transmissive substrate and the irradiation mechanism. An analyzer for performing base extension reaction of a nanostructure on a light-transmitting substrate and observing fluorescence of the incorporated fluorescent dye using an evanescent field,
An irradiation mechanism for totally reflecting the excitation light to the light-transmitting substrate;
And a photographing mechanism for photographing the fluorescent dye incorporated into the nanostructure by the base extension reaction,
A light-transmitting substrate has a target member that emits fluorescence when irradiated with excitation light at a position where the distance from the imaging mechanism in the direction perpendicular to the light-transmitting substrate is different from that of the nanostructure. An analysis device characterized by. In claim 1 or 2,
A distance between a reaction interface of a light transmissive substrate and a target member in a direction perpendicular to the light transmissive substrate is 10 nm or more and 100 μm or less. In claim 1 or 2,
The distance between the nanostructure and the target member in the direction perpendicular to the light-transmitting substrate is 10 nm or more and 100 μm or less. The analyzer according to any one of claims 1 to 4,
The analyzer is characterized in that the target member is a metal. The analyzer according to any one of claims 1 to 4,
An analysis apparatus, wherein the target member is a substance on which a fluorescent dye is adsorbed. The analyzer according to any one of claims 1 to 4,
An analyzer, wherein a convex portion is provided on a light-transmitting substrate, and a target member is provided on the top of the convex portion. The analyzer according to any one of claims 1 to 4,
An analyzer, wherein a recess is provided on a light-transmitting substrate, and the target member is provided at the bottom of the recess. The analyzer according to any one of claims 1 to 4,
An analysis apparatus, wherein the target member is provided for each reaction visual field of the imaging mechanism. The analyzer according to claim 1 or 2,
An analysis apparatus, wherein a plurality of target members are provided in one reaction field of an imaging mechanism, and the vertical distances from the imaging mechanism are different from each other. The analyzer according to claim 12, comprising:
A target member closer to the photographing mechanism than the reaction interface and a target member farther from the photographing mechanism than the reaction interface are provided, and the reaction interface is arranged between these target members. Analytical device. The analyzer according to claim 13,
A plurality of target members that are closer to the imaging mechanism than the reaction interface and a plurality of target members that are further to the imaging mechanism than the reaction interface are provided, and as the distance from the reaction interface increases, An analyzer characterized in that a target member having a short distance is close to the photographing mechanism, and a target member having a distance farther from the photographing mechanism than the reaction interface is far from the photographing mechanism. The analyzer according to claim 12, comprising:
A target member closer to the imaging mechanism than the nanostructure and a target member farther from the imaging mechanism than the nanostructure are provided, and the nanostructure is disposed between these target members An analysis device characterized by. The analyzer according to claim 15, wherein
Multiple target members that are closer to the imaging mechanism than the nanostructure, and more target members that are farther to the imaging mechanism than the nanostructure, are provided. An analysis apparatus, wherein a target member that is close to the mechanism is closer to the imaging mechanism, and a target member that is farther from the imaging mechanism than the nanostructure is farther from the imaging mechanism. The analyzer according to any one of claims 1 to 4,
An analysis apparatus comprising a metal structure formed by a semiconductor manufacturing process as a target member. The analyzer according to any one of claims 1 to 4,
An analysis apparatus characterized by performing nucleic acid sequence decoding. The analyzer according to claim 9, wherein
The convex device is also used for forming a liquid flow path. The analyzer according to claim 10, wherein
The concave portion is also used for forming a liquid flow path. The analyzer according to any one of claims 1 to 4,
The photographing mechanism includes at least an objective lens, an optical filter, and an imaging lens,
The analyzer is characterized in that the focus is controlled by moving the objective lens, the optical filter, and the imaging lens in the vertical direction. The analyzer according to any one of claims 1 to 4,
The photographing mechanism includes at least an objective lens, an optical filter, and an imaging lens,
The analyzer is characterized in that the focus is controlled by moving the light transmissive substrate in the vertical direction. The analyzer according to any one of claims 1 to 4,
An analyzer having a function of automatically searching for a target member. In claim 1 or 3,
An analyzer characterized by detecting at least a focus shift direction based on at least a focus evaluation value obtained from a target member and changing a distance between the light transmissive substrate and the irradiation mechanism. The analyzer according to any one of claims 1 to 4,
The analyzer is characterized in that the target member is a microbead. The analyzer according to claim 25, wherein
The diameter of a microbead is 100 nm or more and 10 micrometers or less, The analyzer characterized by the above-mentioned. The analyzer according to claim 25, wherein
The analyzer is characterized in that the material of the microbead is acrylic, ceramic, polystyrene, or polypropylene. The analyzer according to claim 1 or 2,
An analysis apparatus, wherein another target member is disposed on a light-transmitting substrate at a position where the distance in the vertical direction to the imaging mechanism is the same as the reaction interface. The analyzer according to claim 3 or 4,
An analysis apparatus, wherein another target member is disposed on a light-transmitting substrate at a position where the distance in the vertical direction to the imaging mechanism is the same as that of the nanostructure. The analyzer according to claim 1 or 3, wherein
It is characterized in that at least the defocus direction is detected based on the fluorescent dye incorporated by the base extension reaction and the focus evaluation value obtained from each of the target members, and the distance between the light transmitting substrate and the irradiation mechanism is changed. ,Analysis equipment. An autofocus device used in an analyzer that undergoes base extension reaction on a light-transmitting substrate and observes fluorescence of the incorporated fluorescent dye using an evanescent field,
A photographing mechanism for photographing the fluorescent dye incorporated by the base extension reaction,
On the light transmissive substrate, a target member that emits fluorescence when irradiated with excitation light at a position where the distance in the vertical direction to the imaging mechanism is different from the reaction interface,
An autofocus device characterized by detecting at least a focus shift direction based on at least a focus evaluation value obtained from a target member and changing a distance between the light transmissive substrate and the irradiation mechanism. An autofocus device used in an analyzer that undergoes base extension reaction on a light-transmitting substrate and observes fluorescence of the incorporated fluorescent dye using an evanescent field,
A photographing mechanism for photographing the fluorescent dye incorporated by the base extension reaction,
A light-transmitting substrate is provided with a target member that emits fluorescence when irradiated with excitation light at a position where the distance from the imaging mechanism in the vertical direction with respect to the light-transmitting substrate is different from the reaction interface. Features an autofocus device. An autofocus device that causes a base structure of a nanostructure on a light-transmitting substrate and observes the fluorescence of the incorporated fluorescent dye using an evanescent field,
On the light transmissive substrate, a target member that emits fluorescence when irradiated with excitation light at a position where the distance from the imaging mechanism in the vertical direction on the light transmissive substrate is different from the nanostructure,
An autofocus device characterized by detecting at least a focus shift direction based on at least a focus evaluation value obtained from a target member and changing a distance between the light transmissive substrate and the irradiation mechanism. An autofocus device that causes a base structure of a nanostructure on a light-transmitting substrate and observes the fluorescence of the incorporated fluorescent dye using an evanescent field,
And a photographing mechanism for photographing the fluorescent dye incorporated into the nanostructure by the base extension reaction,
A light-transmitting substrate has a target member that emits fluorescence when irradiated with excitation light at a position where the distance from the imaging mechanism in the direction perpendicular to the light-transmitting substrate is different from that of the nanostructure. An autofocus device characterized by   An apparatus for observing fluorescence on a light-transmitting substrate, comprising an interface that performs a fluorescence reaction, an optical sensor that is intended to image the interface, and is held in the vicinity of the interface and has a different distance from the imaging interface. An analyzer having an autofocus target and a mechanism capable of varying a distance between the optical sensor and the interface. 36. The analyzer of claim 35, wherein
An analyzer having an autofocus mechanism having an imaging interface and one autofocus target, wherein the imaging interface is also used as a second autofocus target. 36. The analyzer of claim 35, wherein
An analysis apparatus, wherein a metal structure formed by a semiconductor manufacturing process is disposed as the autofocus target. 36. The analyzer of claim 35, wherein
An analyzer comprising metal or resin beads as the autofocus target. 36. The analyzer of claim 35, wherein
An analyzer for analyzing a nucleic acid.   An apparatus for observing fluorescence on a light-transmitting substrate, which has two distance differences between an interface that performs a fluorescence reaction, an optical sensor that images the interface, and an optical sensor that is held near the interface An autofocus method comprising the above autofocus target and a mechanism capable of varying a distance between the optical sensor and the interface, and performing autofocus using a distance difference between the autofocus targets. 41. The autofocus method of claim 40, wherein
An autofocus method characterized in that a protective material for holding liquid at the interface is also used as the autofocus target. 41. The autofocus method of claim 40, wherein
2. An autofocus method characterized by digging or forming a protrusion for forming a liquid flow path as the autofocus target. 41. The analyzer of claim 40, wherein
An autofocus method characterized by using a distance difference between an observation interface and another observation object having a different distance. The autofocus method according to any one of claims 40 to 43, wherein
An autofocus method characterized by detecting a defocus direction for maintaining a focused state.

Images