Method for controlling imaging apparatus and imaging apparatus

Abstract

To provide a method and a device that, with a simple configuration, can reduce a focus position deviation between a plurality of wavelengths and can acquire fluorescent images in a plurality of wavelengths focused on the entire surface of an array plate.SOLUTION: A method for controlling an imaging apparatus includes: a first scan condition acquisition step of acquiring, by using a plurality of pieces of first distance information, a first scan condition corresponding to primary light in a first wavelength according to a main scanning position in a longitudinal direction; a second scan condition acquisition step of acquiring, by using at least one of the first scan condition and the pieces of first distance information, a second scan condition corresponding to primary light in a second wavelength different in the wavelength of light from the primary light in the first wavelength according to the main scanning position; a first scanning step of scanning an objective optical system along a short direction according to the first scan condition; a second scanning step of scanning the objective optical system along the short direction according to the second scan condition; and a sub-scanning step of changing the main scanning position along the longitudinal direction.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to a control method for an imaging device for measuring a sample, and to the imaging device. [Background technology]

[0002] Protein array plates or peptide array plates are known, in which a large number of biological substances with peptide bonds, such as proteins or peptides, are immobilized on a substrate. Using these plates, interactions with a large number of biological substances immobilized on the substrate can be simultaneously investigated. This array plate is effective for comprehensively analyzing the interactions between a large number of proteins or peptides and liquid samples derived from living organisms, such as blood, cell extracts, saliva, and interstitial fluid. Such analyses allow the measurement of the characteristics of the sample. Hereinafter, the protein or peptide immobilization site on the substrate will be referred to as a spot.

[0003] As a method for observing spots that have interacted with a sample, for example, a method is known in which the spots are labeled with a fluorescent probe to identify which spots have interacted. A microarray scanner is known as a device for observing an array plate labeled with a fluorescent probe (Patent Document 1).

[0004] Patent Document 1 discloses a system that includes an illumination optical system, a fluorescence objective optical system, and a two-dimensional scanning system. The illumination optical system has a function of concentrating and irradiating laser light of multiple wavelengths onto an array plate. The fluorescence objective optical system has a function of detecting the amount of fluorescent light from spots labeled with fluorescent probes.

[0005] The two-dimensional scanning system has the function of capturing an image that reflects the amount of fluorescent light from spots on the array plate by scanning the array plate or optical system in two dimensions. A confocal optical system is used as the fluorescence objective optical system. Because array plates vary in glass thickness and inclination, it is difficult to obtain a fluorescent image across the entire surface of the array plate with a confocal optical system, which has a shallow focal depth.

[0006] In contrast to this, in Patent Document 1, automatic focus adjustment is performed using a focus sensor simultaneously with two-dimensional scanning in order to obtain a focused fluorescent image over the entire surface of the array plate.

[0007] Meanwhile, Patent Document 2 discloses a technique for reducing image deviations due to differences in scanning direction by switching the wavelength of laser light between the forward and backward paths when two-dimensionally scanning an optical system. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 7,911,670 [Patent Document 2] Japanese Patent Application Laid-Open No. 2007-183313 Summary of the Invention [Problem to be solved by the invention]

[0009] In microarray scanners that detect fluorescence at multiple wavelengths, it is difficult to perfectly align the focus position of each wavelength's laser light across the entire surface of the array plate, which can lead to misalignment of the focus position between wavelengths. As a result, the brightness of the fluorescence image may decrease depending on the wavelength, or the brightness may vary significantly within the array plate. To correct for differences in protein amounts between individual array plates, corrections are made based on the fluorescence brightness values ​​or their ratios, but this can lead to a decrease in the reliability of the measurement data.

[0010] In the technology of Patent Document 1, in order to perform automatic focusing with high accuracy while scanning the array plate or optical system two-dimensionally at high speed, high-speed feedback control consisting of a high-performance focus sensor and a low-vibration actuator is required, which poses the problem of making the device configuration complex and expensive.

[0011] The technology of Patent Document 2 describes switching between a plurality of laser wavelengths in the forward and backward paths, but does not describe how to deal with focus position deviations between the plurality of wavelengths.

[0012] Therefore, an object of the present invention is to provide a method and apparatus that can reduce focus position shifts between multiple wavelengths and acquire focused fluorescence images of multiple wavelengths across the entire surface of an array plate using a simple configuration. [Means for solving the problem]

[0013] The present invention provides A control method for an imaging device in which an array plate having a longitudinal direction and a lateral direction and a spot array on one surface is scanned with an objective optical system that irradiates primary light and detects secondary light, thereby acquiring a fluorescent image, the method comprising: a first distance measuring step of measuring the distance to the one surface using the objective optical system that emits primary light of a first wavelength, and acquiring a plurality of pieces of first distance information for the primary light of the first wavelength corresponding to a plurality of positions in the longitudinal direction; a first scanning condition acquisition step of acquiring a first scanning condition corresponding to the primary light of the first wavelength according to a main scanning position in the longitudinal direction by using the plurality of first distance information; a second scanning condition acquisition step of acquiring second scanning conditions corresponding to primary light of a second wavelength different in wavelength from the primary light of the first wavelength according to the main scanning position, using at least one of the first scanning conditions and the first distance information; a first scanning step of scanning the objective optical system along the short-side direction under the first scanning condition; a second scanning step of scanning the objective optical system along the short-side direction under the second scanning condition; a sub-scanning step of changing the main scanning position along the longitudinal direction; The present invention relates to a control method for an imaging device, including: The present invention also provides An imaging device for optically imaging an array plate having a longitudinal direction and a lateral direction and a spot array on one surface thereof in two dimensions, a mounting portion on which the array plate is mounted; an objective optical system including a light emitting unit optically coupled to a light source that selectively emits primary light of different wavelengths, and that emits the primary light to the array plate, and a light collecting unit that detects secondary light from the array plate; a distance defining unit that defines a working distance between the mounting unit and the objective optical system in the optical axis direction of the primary light; a scanning unit that changes a relative position between the mounting unit and the objective optical system in a plane that intersects with the optical axis direction; a scanning condition acquisition unit that acquires, as scanning conditions, the working distances at a plurality of positions in the plane for each wavelength of the primary light; an imaging unit that acquires a fluorescent image corresponding to the primary light based on information about the wavelength of the primary light, information about the intensity of the received light from the light collecting unit, and information about the relative position; The imaging device has the following features. The present invention also provides An imaging device for optically imaging an array plate having a longitudinal direction and a lateral direction and a spot array on one surface thereof in two dimensions, a mounting portion on which the array plate is mounted; an objective optical system including a light emitting unit optically coupled to a light source that selectively emits primary light of different wavelengths, and that emits the primary light to the array plate, and a light collecting unit that detects secondary light from the array plate; a scanning unit including a main scanning unit that performs main scanning and a sub-scanning unit that performs sub-scanning along the short-side direction and the long-side direction to change a relative position between the mounting unit and the objective optical system in a plane intersecting the optical axis direction; an emission wavelength control unit that controls the light source and the scanning unit to change the wavelength of the light emitted from the light emitting unit in accordance with a sub-scanning position in the short side direction, a main scanning direction in the long side direction, and a number of main scanning passes; an imaging unit that acquires a fluorescent image corresponding to the primary light based on information about the wavelength of the primary light, information about the intensity of light received from the light collecting unit, and information about the relative position; The imaging device has the following features. [Effects of the Invention]

[0014] The specimen measurement device according to the present invention can correct focus shifts between multiple wavelengths across the entire surface of an array plate without relying on high-speed feedback control. This makes it possible to reduce focus shifts between multiple wavelengths and acquire in-focus fluorescent images at multiple wavelengths across the entire surface of the array plate with a simple configuration. As a result, it is possible to improve reliability when correcting differences in protein amounts between individuals on an array plate using the ratio of fluorescent brightness. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a diagram showing an imaging device according to the present invention. [Figure 2] 2(a) to 2(c) are diagrams showing a specimen according to the present invention. [Figure 3] FIG. 3 is a diagram illustrating an internal configuration of the controller according to the first embodiment. [Figure 4] FIG. 4 is a diagram illustrating an operation flow of the imaging process according to the first embodiment. [Figure 5] 5A and 5B are diagrams illustrating the positional relationship of the sub-scanning in the imaging process according to the first embodiment. [Figure 6] 6A and 6B are diagrams illustrating positional relationships of height scanning in the imaging process according to the first embodiment. [Figure 7] FIG. 7 is a diagram illustrating the internal configuration of the measurement system according to the first embodiment. [Figure 8] FIG. 8 is a diagram illustrating operation timing of the imaging process according to the first embodiment. [Figure 9] 9(a) to 9(c) are diagrams illustrating positional relationships of height scanning in the height information acquisition process according to the first embodiment. [Figure 10] FIG. 10 is a diagram illustrating an operation flow of the height information acquisition process according to the first embodiment. [Figure 11]FIG. 11 is a diagram illustrating a positional relationship of height scanning in the imaging process according to the second embodiment. [Figure 12] FIG. 12 is a diagram illustrating a positional relationship of height scanning in the imaging process according to the third embodiment. [Figure 13] FIG. 13 is a diagram illustrating a positional relationship of height scanning in the imaging process according to the modified example of the third embodiment. [Figure 14] FIG. 14 is a diagram illustrating a positional relationship of height scanning in the imaging process according to the fourth embodiment. [Figure 15] FIG. 15 is a diagram illustrating operation timing of the imaging process according to the modified example of the fourth embodiment. [Figure 16] FIG. 16 is a diagram illustrating the operation of the piston crank mechanism according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0016] First Embodiment The first embodiment relates to a control method for an imaging device. The method for controlling an imaging device of the present invention includes: A control method for an imaging device in which an array plate having a longitudinal direction and a lateral direction and a spot array on one surface is scanned with an objective optical system that irradiates primary light and detects secondary light, thereby acquiring a fluorescent image, the method comprising: a first distance measuring step of measuring the distance to the one surface using the objective optical system that emits primary light of a first wavelength, and acquiring a plurality of pieces of first distance information for the primary light of the first wavelength corresponding to a plurality of positions in the longitudinal direction; a first scanning condition acquisition step of acquiring a first scanning condition corresponding to the primary light of the first wavelength according to a main scanning position in the longitudinal direction by using the plurality of first distance information; a second scanning condition acquisition step of acquiring second scanning conditions corresponding to primary light of a second wavelength different in wavelength from the primary light of the first wavelength according to the main scanning position, using at least one of the first scanning conditions and the first distance information; a first scanning step of scanning the objective optical system along the short-side direction under the first scanning condition; a second scanning step of scanning the objective optical system along the short-side direction under the second scanning condition; a sub-scanning step of changing the main scanning position along the longitudinal direction; Includes.

[0017] The control method for an imaging device of the present invention is a control method for an imaging device that scans an array plate having a longitudinal direction and a lateral direction, one surface of which has a spot array, with primary light and detects secondary light, to obtain a fluorescent image. A specimen is placed on the spot array, and a primary antibody and a secondary antibody are bound to the specimen. While the objective optical system scans the array plate, it detects secondary light obtained by irradiating primary light onto fluorescent labels on the secondary antibody, for example, to obtain a fluorescent image that can be used for specimen measurement or testing. Each step is explained below. Some terms are explained in the second embodiment, so please refer to that section.

[0018] [First distance measurement process] The control method for an imaging device of the present invention includes a first distance measurement step of measuring the distance to one surface using the objective optical system that emits primary light of a first wavelength and obtaining multiple first distance information for the primary light of the first wavelength corresponding to multiple positions in the longitudinal direction.

[0019] The primary light of the first wavelength is, for example, illumination light for fluorescently labeled molecules of the spot array. To use this primary light of the first wavelength for accurate measurements such as sample measurement, it is necessary to focus the primary light of the first wavelength on the spot array on one side of the array plate. Therefore, distance measurement is performed to obtain information (first distance information) about the distance from the objective optical system to the spot array along the optical axis of the objective optical system when the primary light of the first wavelength is focused.

[0020] The array plate is fixed to a specimen stage for observation and positioned as parallel as possible to the two-dimensional scanning direction of the objective optical system. However, the positional relationship between the arrangement of the spot array and the two-dimensional scanning direction of the objective optical system is not perfectly parallel, and a slight tilt may occur. Therefore, the distance between the array plate and the objective optical system along the optical axis of the objective optical system may change, particularly in the longitudinal direction. Therefore, information on the distance from the objective optical system to the spot array along the optical axis of the objective optical system when the primary light of the first wavelength is focused (first distance information) is obtained at one or more different positions in the longitudinal direction of the array plate.

[0021] [First scanning condition acquisition step] The control method for an imaging device of the present invention includes a first scanning condition acquisition step of using the plurality of first distance information to acquire a first scanning condition corresponding to primary light of the first wavelength according to a main scanning position in the longitudinal direction.

[0022] The first distance information obtained in the first distance measurement step is information about the distance from the objective optical system to the spot array along the optical axis of the objective optical system when the primary light of the first wavelength is focused at at least one or more different positions in the longitudinal direction of the array plate. Since actual scanning is performed at multiple points in the longitudinal direction, data interpolation or extrapolation is performed from the first distance information for at least one or more positions to obtain information about the position of the objective optical system relative to each main scanning position (information at multiple points). Here, the main scanning position is, for example, the starting point when scanning begins in the short direction, and is somewhere among the multiple points in the longitudinal direction.

[0023] An example of a method for data interpolation or extrapolation is to assume a linear slope between two positions. Taking into account the warping of the array plate itself, data interpolation or extrapolation may be performed by applying a fitting function to positional information for three or more points. In this case, the fitting function is preferably a higher-order function, such as a quadratic or higher function. This allows the imaging device control method of the present invention to perform accurate sample measurement in a simpler manner.

[0024] [Second scanning condition acquisition process] The control method for an imaging device of the present invention includes a second scanning condition acquisition step of acquiring, using at least one of the first scanning condition and the first distance information, a second scanning condition corresponding to primary light of a second wavelength that is different in wavelength from the primary light of the first wavelength according to the main scanning position.

[0025] The control method for an imaging device of the present invention may further include a distance correction step of outputting second distance information for primary light of the second wavelength based on the first distance information, and the second scanning condition acquisition step may acquire the second scanning condition using the second distance information acquired from the distance correction unit using the plurality of first distance information and the first scanning condition.

[0026] In this case, for example, the second distance information can be obtained by adding or subtracting a constant to the first distance information. This allows a fluorescent image with reliable measurement results to be obtained using a simpler method. This can be achieved under the assumption that the difference between the focal length of the primary light of the first wavelength and the focal length of the primary light of the second wavelength is constant. The second scanning conditions can be obtained based on the obtained second distance information and the first scanning conditions, but the method for obtaining the second distance information is not limited to adding or subtracting a constant.

[0027] The control method for an imaging device of the present invention may further include a scanning condition correcting step of outputting a second scanning condition for primary light of the second wavelength corresponding to the first scanning condition, and the second scanning condition obtaining step may obtain the second scanning condition from the scanning condition correcting unit based on the first scanning condition.

[0028] In this case, for example, it is conceivable to set the second scanning conditions to be the same as the first scanning conditions. By doing so, a fluorescent image with reliable measurement results can be obtained using a simpler method. The method for determining the second scanning conditions is not limited to the method for determining the first scanning conditions.

[0029] [First scanning step] The method for controlling an imaging device of the present invention includes a first scanning step of scanning the objective optical system along the short-side direction under the first scanning condition.

[0030] In the first scanning step, the objective optical system is positioned based on the information on the position of the objective optical system relative to each main scanning position obtained in the first scanning condition acquisition step, and the objective optical system is scanned in the short direction of the array plate while irradiating primary light of a first wavelength toward the spot array.

[0031] In the control method for an imaging device of the present invention, the first scanning step preferably includes a step of acquiring a first fluorescent image by irradiating the spot array with primary light of the first wavelength based on fluorescence intensity information and scanning position information detected by the objective optical system. In this way, focus position deviations among multiple wavelengths can be corrected across the entire surface of the array plate without using high-speed feedback control.

[0032] [Second scanning process] The method for controlling an imaging device of the present invention includes a second scanning step of scanning the objective optical system along the short-side direction under the second scanning condition.

[0033] In the second scanning step, similar to the first scanning step, the objective optical system is positioned based on the information on the position of the objective optical system relative to each main scanning position obtained in the second scanning condition acquisition step, and the objective optical system is scanned in the short direction of the array plate while irradiating light of the second wavelength toward the spot array.

[0034] The second scanning step preferably includes a step of acquiring a second fluorescent image by irradiating the spot array with primary light of the second wavelength based on the fluorescent intensity information and scanning position information detected by the objective optical system. This makes it possible to correct focus position shifts among multiple wavelengths over the entire surface of the array plate without using high-speed feedback control.

[0035] [Sub-scanning process] The method for controlling an imaging device of the present invention includes a sub-scanning step of changing the main scanning position along the longitudinal direction.

[0036] Once the first scanning step and the second scanning step are completed, the longitudinal position of the array plate is changed, and the first scanning step and the second scanning step are performed again. Preferably, the first scanning condition acquisition step acquires the first scanning condition for each sub-scanning step, and the second scanning condition acquisition step acquires the second scanning condition for each sub-scanning step. This allows for accurate sample measurement using a simpler method.

[0037] In the control method for an imaging device of the present invention, the first scanning step and the second scanning step are preferably performed such that the primary light of the first wavelength and the primary light of the second wavelength are scanned along a common scanning path corresponding to a common main scanning position in the longitudinal direction, thereby making it possible to detect secondary light under the same conditions for the primary light of the first wavelength and the primary light of the second wavelength.

[0038] In the control method of an imaging device of the present invention, the first scanning step and the second scanning step are preferably performed such that the primary light of the first wavelength and the primary light of the second wavelength scan the common scanning path in opposite directions. In particular, when scanning with the primary light of the first wavelength and scanning with the primary light of the second wavelength are performed alternately for each line, positional deviation due to thermal drift can be minimized, thereby improving the reliability of the obtained fluorescence image.

[0039] In the method for controlling an imaging device of the present invention, it is preferable that the sub-scanning step is performed once each time the first scanning step and the second scanning step are performed at least once, and it is more preferable that the sub-scanning step is performed once each time the first scanning step and the second scanning step are performed once, thereby reducing positional deviation due to thermal drift.

[0040] [Second distance measurement process] It is preferable that the control method for an imaging device of the present invention further includes a second distance measurement step of using the objective optical system to measure the distance to one surface with primary light of the second wavelength and obtaining second distance information for the primary light of the second wavelength.

[0041] The primary light of the second wavelength is illumination light having a wavelength different from that of the primary light of the first wavelength. Therefore, even if the same lens is used for focusing, the primary light of the second wavelength and the primary light of the first wavelength may have different focal lengths. That is, in order to focus the primary light of the second wavelength and the primary light of the first wavelength on the array plate, the distance between the array plate and the objective optical system along the optical axis of the objective optical system may be different.

[0042] Therefore, the second distance measurement step is performed separately from the first distance measurement step and is similar to the first distance measurement step. For the same reason as the first distance measurement step, the second distance measurement step acquires information on the distance from the objective optical system to the spot array along the optical axis of the objective optical system when the primary light of the second wavelength is focused at at least one different position in the longitudinal direction of the array plate. In the control method for an imaging device of the present invention, the second distance measurement step may be performed at multiple positions in the longitudinal direction. This information may be used as second distance information.

[0043] In the second scanning condition acquisition step when the second distance measurement step is performed, for example, scanning conditions are acquired for primary light of the second wavelength, similarly to the first scanning condition acquisition step. As in the first scanning condition acquisition step, a method of data interpolation or extrapolation is, for example, assuming a linear slope between two positions. Data interpolation or extrapolation may be performed by applying a fitting function to position information of three or more points. In this case, the fitting function is preferably, for example, a quadratic or higher multi-order function. In this way, the imaging control method of the present invention can perform accurate sample measurement in a simpler manner.

[0044] In the control method for an imaging device of the present invention, the second scanning condition acquisition step may acquire the second scanning conditions using the second distance information acquired in the second distance measurement step and the first scanning conditions. In this way, it is possible to acquire fluorescent images of multiple wavelengths that are in focus across the entire surface of the array plate. The method for acquiring the second scanning conditions is not limited to one, and various methods are possible as described above.

[0045] [others] In the control method for an imaging device of the present invention, it is preferable that the primary light for capturing a fluorescent image of the spot array includes either the primary light of the first wavelength or the primary light of the second wavelength, and the primary light for capturing a fluorescent image of a secondary antibody that labels a reaction product on the spot array includes the other of the primary light of the first wavelength or the primary light of the second wavelength.

[0046] A first fluorescent image can be captured by irradiating an array plate having a plurality of arranged spots with primary light of a first wavelength, and a second fluorescent image can be captured by irradiating the array plate with primary light of a second wavelength, which is different from the first wavelength of the primary light, in order to obtain functional information about the spots. The first fluorescent image and the second fluorescent image can be captured using an image analysis PC.

[0047] On the array plate, multiple types of proteins are spotted in an array. Each spot is about 100 μm in diameter, and the spots are spaced about 40 μm apart. The spots are organized in 9x9 blocks, which are arranged in a 3x6 matrix.

[0048] The spots on the array plate of this embodiment can be fluorescently labeled with, for example, Alexa Flour 680. Some protein spots are phosphorylated, and only the phosphorylated protein spots can be fluorescently labeled with, for example, Alexa Flour 790. When each spot contains the protein BCAR1, the protein BCAR1 can be tagged with GST (glutathione S-transferase), and the GST tag can be labeled with a fluorescent probe that fluoresces when exposed to light with a wavelength of 670 nm.

[0049] To excite protein spots fluorescently labeled with Alexa Flour 680 or a GST tag, the wavelength of the primary light of the first wavelength is preferably 670 nm. To excite protein spots fluorescently labeled with Alexa Flour 790, the wavelength of the primary light of the second wavelength is preferably 780 nm. In this case, the fluorescent image acquired by exciting the array plate with light of 670 nm wavelength is the first fluorescent image, and the fluorescent image acquired by exciting the array plate with light of 780 nm wavelength is the second fluorescent image.

[0050] <Second embodiment> The second embodiment relates to an imaging device. The imaging device of the present invention comprises: An imaging device for optically imaging an array plate having a longitudinal direction and a lateral direction and a spot array on one surface thereof in two dimensions, a mounting portion on which the array plate is mounted; an objective optical system including a light emitting unit optically coupled to a light source that selectively emits primary light of different wavelengths, and that emits the primary light to the array plate, and a light collecting unit that detects secondary light from the array plate; a distance defining unit that defines a working distance between the mounting unit and the objective optical system in the optical axis direction of the primary light; a scanning unit that changes a relative position between the mounting unit and the objective optical system in a plane that intersects with the optical axis direction; a scanning condition acquisition unit that acquires, as scanning conditions, the working distances at a plurality of positions in the plane for each wavelength of the primary light; an imaging unit that acquires a fluorescent image corresponding to the primary light based on information about the wavelength of the primary light, information about the intensity of the received light from the light collecting unit, and information about the relative position; It has.

[0051] (imaging device) An imaging device 100 according to the second embodiment will be described with reference to FIGS. The imaging device 100 is an imaging device that optically captures two-dimensional images of an array plate 101 having a longitudinal direction and a lateral direction and a spot 202 on one side, and is therefore sometimes referred to as a scanner 100 or a fluorescent scanner 100.

[0052] (Placement section) The placement unit 109 includes a placement target (not shown) provided on the Y-direction linear stage 109.

[0053] (Objective optical system) The imaging device 100 includes a mounting section 109 on which the array plate 101 is mounted, an objective optical system 104 that is optically coupled to a light source that switches between emitting primary light Ip (Ip1, Ip2) of different wavelengths λ1 and λ2, and includes a light emitting section 104i that emits the primary light Ip to the array plate 101, and a light collecting section 104c that collects secondary light Is from the array plate 101. The objective optical system 104 is optically coupled to the confocal optical system 103 , and irradiates the array plate 101 , which is the object, with primary light Ip and collects secondary light Is from the array plate 101 .

[0054] (Distance regulation part) The imaging device 100 further includes a distance defining unit 316 that defines a working distance WD between the mounting unit 109 and the objective optical system 104 in the optical axis direction of the primary light Ip. The distance defining unit 316 defines the working distance WD between the mounting unit 109 and the objective optical system 104 in the optical axis direction of the primary light Ip via the height scanning motor 113, the motor driver 114, and the motor control circuit 309. In other words, the distance defining unit 316 controls the working distance WD between the mounting unit 109 and the objective optical system 104 in the optical axis direction of the primary light Ip via the height scanning motor 113, the motor driver 114, and the motor control circuit 309.

[0055] (Scanning unit) The imaging device 100 also includes a scanning unit 170 (main scanning unit 190, sub-scanning unit 180) that changes the relative positions of the mounting unit 109 and the objective optical system 104 in a plane intersecting the optical axis direction. The sub-scanning unit 180 is configured to scan the objective optical system 104 in the Y-axis direction relative to a base (not shown), and includes a Y-direction linear stage 109 and a sub-scanning motor 110. The main scanning unit 190 is configured to scan the objective optical system 104 in the X-axis direction relative to a base (not shown), and includes a motor driver 115, a main scanning motor 107, a piston-crank mechanism 120, and an encoder 108. The piston-crank mechanism 120 includes a crank 118 and a connecting rod 119.

[0056] (Scanning condition acquisition unit) The imaging device 100 further includes a scanning condition acquisition unit 315 that acquires, as scanning conditions, working distances WD at multiple positions within a plane for each of the wavelengths λ1 and λ2 of the primary light Ip (Ip1, Ip2).

[0057] (Imaging Department) The imaging device 100 further includes an imaging unit 317 that performs image reconstruction based on information Iwp about the wavelength λ1 of the primary light, information Iis about the received light intensity of the secondary light ls from the light collecting units 724 and 725, and information Irp about the relative position. The imaging unit 317 forms and acquires a fluorescent image corresponding to the primary light 1p (lp1 or lp2). The imaging unit 317 acquires information Iwp about the wavelength λ1 of the primary light from the light source control circuit 305, acquires information Iis about the received light intensity of the secondary light ls from the data acquisition control circuit 312, and acquires information Irp about the relative position from the coordinate calculation circuit 310. The scanning condition acquisition unit 315, distance determination unit 316, and imaging unit 317 can all be partially or entirely replaced by the CPU 301, memory 303, a storage unit (not shown), a communication circuit 313, a memory control circuit 304, etc.

[0058] In this case, it is preferable that the imaging device of the present invention further includes a light source, a distance correction unit, and a scanning condition correction unit. Here, the light source can be applied to the light source 102 described below, the distance correction unit can be applied to the motor driver 114 and height scanning motor 113 described below, and the scanning condition correction unit can be applied to the motor driver 115, main scanning motor 107, motor driver 111, and sub-scanning motor 110 described below.

[0059] The present invention also provides An imaging device for optically imaging an array plate having a longitudinal direction and a lateral direction and a spot array on one surface thereof in two dimensions, a mounting portion on which the array plate is mounted; an objective optical system including a light emitting unit optically coupled to a light source that selectively emits primary light of different wavelengths, and that emits the primary light to the array plate, and a light collecting unit that detects secondary light from the array plate; a scanning unit including a main scanning unit that performs main scanning and a sub-scanning unit that performs sub-scanning along the short-side direction and the long-side direction to change a relative position between the mounting unit and the objective optical system in a plane intersecting the optical axis direction; an emission wavelength control unit that controls the light source and the scanning unit to change the wavelength of the light emitted from the light emitting unit in accordance with a sub-scanning position in the short side direction, a main scanning direction in the long side direction, and a number of main scanning passes; an imaging unit that acquires a fluorescent image corresponding to the primary light based on information about the wavelength of the primary light, information about the intensity of light received from the light collecting unit, and information about the relative position; It has.

[0060] The imaging device 100 includes a light source 102 and a scanning unit 170 (main scanning unit 190, sub-scanning unit 180) to change the wavelength λ of the light emitted from the light emitting unit 104i so as to switch between primary light Ip1 of a first wavelength λ1 and primary light Ip2 of a second wavelength λ2 that is different in wavelength from the primary light of the first wavelength.

[0061] (Emission wavelength control section) The imaging device 100 includes an emission wavelength control unit 318 that controls the light source 102 and the scanning unit 170 (main scanning unit 190, sub-scanning unit 180) to change the wavelength λp (λp1 or λp2) of light emitted from the light emitting unit 104i in accordance with the sub-scanning position Ps in the longitudinal direction of the array plate 101, the main scanning direction in the lateral direction (+X direction or −X direction), and the number of main scanning passes. Like the scanning condition acquisition unit 315, the distance determination unit 316, and the imaging unit 317, the emission wavelength control unit 318 can be partially or entirely replaced by the CPU 301, the memory 303, a storage unit (not shown), a communication circuit 313, a memory control circuit 304, etc.

[0062] In this case, it is preferable that the imaging device of the present invention further includes a light source.It is preferable that the imaging device of the present invention further includes a distance defining unit that defines a working distance between the mounting unit and the objective optical system in the optical axis direction of the primary light, and the emission wavelength control unit controls the working distance defined by the distance defining unit in accordance with the wavelength of the primary light.It is preferable that the imaging device of the present invention further includes a scanning condition acquisition unit that acquires the working distances at multiple positions in the plane for each wavelength of the primary light as scanning conditions.

[0063] Here, the distance definition unit can be applied to the CPU 301 and coordinate calculation circuit 310 described below, and the scanning condition acquisition unit can be applied to the CPU 301 and coordinate calculation circuit 310 described below. These will be explained below. Note that explanations of parts that overlap with the first embodiment may be omitted.

[0064] The imaging device of the present invention optically two-dimensionally images an array plate having a longitudinal direction and a lateral direction and having a spot array on one surface. An imaging device according to the present invention will be described with reference to FIG.

[0065] The array plate 101 is the measurement target. On the array plate 101, a large number of spots of biological materials are immobilized and fluorescently labeled. The light source 102 is a semiconductor laser that emits light with wavelengths of about 670 nm and 780 nm, and the emission and stopping of each wavelength can be controlled independently.

[0066] The confocal optical system 103 guides the primary light from the light source 102 to the array plate, and also guides the fluorescence from the spots on the array plate 101 and the reflected light from the surface of the slide glass to the optical sensor. The confocal optical system 103 is composed of a pinhole, a filter, a dichroic mirror, a quarter-wave plate, a polarizing beam splitter, and a lens. Using the confocal optical system reduces the influence of autofluorescence components from the slide glass and increases the signal-to-noise ratio of the measurement of fluorescence components originating from the spots.

[0067] The objective optical system 104 is an objective optical system for irradiating the primary light onto a spot on the array plate 101, and is composed of a mirror for directing the primary light toward the array plate 101 and a lens for focusing the primary light onto a spot on the array plate 101.

[0068] The optical sensor 105 is an optical sensor for converting light into an electrical signal, and is composed of a photomultiplier tube for acquiring fluorescence and a photodiode for acquiring reflected light. The optical sensor 105 is assumed to be capable of acquiring fluorescence from spots on the array plate and reflected light from the surface of the array plate. The optical sensor 105 is also assumed to be composed of a plurality of photomultiplier tubes, photodiodes, etc. corresponding to the plurality of wavelengths of the light source 102.

[0069] The receiving circuit 106 is a receiving circuit for amplifying the electrical signal from the optical sensor 105 and converting it into a digital value, and is composed of an amplifier circuit, a filter circuit, an AD converter, and the like.

[0070] The piston crank mechanism 120 is a piston crank mechanism for reciprocating the objective optical system 104 in the widthwise direction of the array plate 101. Due to the reciprocating motion of the objective optical system 104, the primary light from the light source 102 is scanned in the widthwise direction of the array plate. In the present invention, the widthwise direction of the array plate is referred to as the main scanning direction, and the reciprocating scanning by the piston crank mechanism 120 is referred to as main scanning. The main scanning stroke is 30 mm. The objective optical system 104 is limited in its operating direction to the main scanning direction on a guide in the main scanning direction.

[0071] The main scanning motor 107 is a pulse motor for rotating the piston crank mechanism 120 at high speed. The rotation speed of the main scanning motor 107 is set to 1200 rpm.

[0072] The encoder 108 is a linear encoder installed on the piston crank mechanism 120, and is capable of measuring the position in the main scanning direction of the objective optical system 104. The encoder 108 outputs a phase-difference pulse voltage consisting of A-phase, B-phase, and Z-phase according to the position in the main scanning direction.

[0073] The Y-direction linear stage 109 is a linear stage for moving the array plate 101 in a direction perpendicular to the main scanning direction within a horizontal plane, and is composed of a ball screw, an origin sensor, etc. Scanning in a direction perpendicular to the main scanning direction within a horizontal plane is called sub-scanning. A mounting table for placing the array plate is provided on the linear stage, and the user places the array plate to be measured on the mounting table in advance.

[0074] The sub-scanning motor 110 is a pulse motor connected to the Y-direction linear stage 109. The rotational motion of the pulse motor is converted into linear motion by the ball screw of the Y-direction linear stage 109. The motor driver 111 is a motor driver circuit for rotating the sub-scanning motor 110. When one pulse of a signal is input to the motor driver 111, the pulse motor rotates by 0.72°, and the array plate 101 moves 2 μm in the sub-scanning direction.

[0075] The Z-direction linear stage 112 is a linear stage for moving the array plate 101 in the vertical direction, and constitutes a distance determination unit 316 that is made up of a ball screw, an origin sensor, etc. Therefore, the Z-direction linear stage 112 is configured to be controllable by the distance determination unit 316 that determines the distance between the array plate 101 and the objective optical system 104. Scanning in the vertical direction is called height scanning. The height scanning motor 113 is a pulse motor connected to the Z-direction linear stage 112. The rotational motion of the height scanning motor 113 is converted into linear motion by the ball screw of the Z-direction linear stage 112.

[0076] The motor driver 114 is a motor driver circuit for rotating the height scanning motor 113. When one pulse of a signal is input to the motor driver 114, the pulse motor rotates by 0.72°, and the array plate 101 moves 1 μm vertically upward.

[0077] The motor driver 115 is a motor driver circuit for rotating the main scanning motor 107. When one pulse of a signal is input to the motor driver 115, the main scanning motor 107 rotates by 0.72°, and the objective optical system 104 moves along the main scanning direction.

[0078] The controller 116 is a controller circuit that controls the entire system and is composed of an FPGA, a CPU, a memory, embedded software, etc. It controls the light source 102, the motor driver 111, the motor driver 114, and the motor driver 115 to scan the primary light in the main scanning, sub-scanning, and height directions on the array plate 101. At the same time, it acquires position information of the objective optical system 104 measured by the encoder 108 and optical signal data from the optical sensor 105 and stores them in its internal memory.

[0079] The controller 116 includes an internal unit for acquiring height and tilt information of the array plate, a pulse train generation unit for main scanning, sub-scanning, and height scanning, an AD converter control unit, a synchronization control unit, a data acquisition control unit, and a memory. The AD converter control unit issues a conversion instruction to the AD converter of the receiving circuit 106, performs AD conversion on the electrical signal corresponding to the light intensity of the optical sensor 105, and acquires optical signal data as a digital value. The synchronization control means controls the timing of sub-scanning, height scanning, and data acquisition in synchronization with the position information of the objective optical system 104 .

[0080] This corrects the thickness and tilt of the array plate when the objective optical system 104 is outside the imaging range on the array plate, and performs height scanning so that light is focused on the surface where the spot is fixed across the entire array plate 101.

[0081] Furthermore, the synchronization control means controls the timing of light emission and wavelength switching of the light source 102. When the objective optical system 104 reaches a predetermined position close to the edge of the array plate, it also controls the start of sub-scanning and height scanning, and the start and stop of light emission of the semiconductor laser of each wavelength of the light source 102.

[0082] Specifically, on the outbound path, a laser with a wavelength of 780 nm is emitted, and the emission of the laser with a wavelength of 670 nm is stopped. On the return path, a laser with a wavelength of 670 nm is emitted, and the emission of the laser with a wavelength of 780 nm is stopped. In height scanning, the height is adjusted to match the focus position of 780 nm on the outbound path, and the height is adjusted to match the focus position of 670 nm on the return path. The focus position for each wavelength is determined by prior measurement for each array plate, and the target height across the entire array plate is calculated.

[0083] The user interface 117 is a user interface for receiving instructions from the user and displaying results, and is composed of a keyboard, a mouse, and a display.

[0084] The controller 116 can receive an instruction to capture an image of the array plate and can present image data based on the optical signal data to the user via a user interface 117. Furthermore, when capturing an image of the array plate, the user can specify the imaging range and the pixel pitch in the main scanning direction and the sub-scanning direction via the GUI on the user interface 117.

[0085] The crank 118 is a crank that constitutes a piston crank mechanism, and is connected to the rotary shaft of the main scanning motor 107 and a connecting rod 119 via joints. The length of the crank is represented by r.

[0086] The connecting rod 119 is a connecting rod that constitutes the piston-crank mechanism, and is connected to the crank 118 and the objective optical system 104 via joints. The length of the connecting rod is represented by 1. Figure 16 shows the positional relationship between the crank 118, connecting rod 119, and objective optical system 104 of the piston-crank mechanism 120.

[0087] Next, the specimen according to the present invention will be described with reference to FIG. Fig. 2(a) is a top view of the array plate 101, and Fig. 2(b) is a side view of the array plate 101. Fig. 2(c) shows the array plate in a frame attached thereto and filled with an observation liquid.

[0088] A biological substance containing a peptide bond is immobilized on each of the spots 202 on the glass slide 201. The glass slide 201 and the numerous spots 202 constitute an array plate 101. One type of biological substance is immobilized on each spot. The diameter of the spots is approximately 100 μm, and the spot spacing is 200 μm. The array plate is 25 mm long in the short direction and 75 mm long in the long direction.

[0089] The upper left point 203 of the array plate is the origin, the rightward direction in the short direction is the positive X-axis direction, and the downward direction in the long direction is the Y-axis direction. If the units of X and Y coordinates are μm, the coordinates of the four corners of the array plate are (0,0), (25000,0), (0,75000), and (25000,75000).

[0090] The stroke of the piston crank mechanism 120 is 30 mm, which is 5 mm longer than the length of the array plate 101. That is, in the main scan, a range that is 2.5 mm longer on the left and right of the array plate is scanned, and the X coordinate of the scan range is between -2500 and 27500.

[0091] Meanwhile, liquid 204 is stored in a storage area defined by the slide glass 201 and frame 203 so as to contact the spots 202 arranged in an array on one surface of the array plate 101. On the array plate 101, there are areas where spots exist and areas where spots do not exist, depending on the convenience of spot creation and the user's grip. In this embodiment, the coordinates of the four corners of the area where liquid 204 exists are (2000, 2000), (23000, 2000), (2000, 65000), and (23000, 65000). The range of the Y coordinate for the sub-scanning can be specified by the user.

[0092] Frame 203 is a frame attached to the array plate, and by surrounding all four sides, it is possible to hold a liquid inside. Liquid 204 is a liquid held inside frame 203, and serves to keep spot 202 from drying out and to reduce background noise during fluorescence measurement. Liquid 204 is called the observation liquid. It is desirable for the observation liquid to have a refractive index close to that of the array plate, protect the material on the array plate, and not emit fluorescence.

[0093] [Example 1] 3 is a block diagram showing the internal configuration of the controller 116 according to the first embodiment of the present invention. A CPU 301 is a CPU for running control software that controls the entire system, and is made up of a microprocessor, a cache memory, and the like.

[0094] The bus interface 302 is a bus interface for connecting the CPU 301 to various peripheral circuits. The memory 303 is a memory for storing imaging conditions input by the user, parameters of the specimen testing device, and optical signal data, and is configured with DDR4-SDRAM, SSD, or the like.

[0095] The memory control circuit 304 is a memory control circuit for controlling the memory 303 based on an access command to the memory via the bus interface 302 . The light source control circuit 305 is a light source control circuit that allows the CPU 301 to control the light source 102, and is composed of an interface conversion circuit, a DA converter, etc. The CPU 301 can control the on / off and light intensity of the laser irradiation of the light source 102 via the light source control circuit 305.

[0096] The AD converter control circuit (ADC control circuit) 306 is an AD converter control circuit that controls the AD converter of the receiving circuit 106 and acquires optical signal data based on instructions from the CPU 301. When the AD converter control circuit 306 receives a data acquisition trigger signal from the synchronization circuit 311, it outputs a conversion start signal to the AD converter of the receiving circuit 106. The AD converter samples the analog signal in the receiving circuit at the timing when the conversion start signal is input, converts it into optical signal data, and outputs it to the AD converter control circuit 306. The AD converter control circuit 306 transfers the optical signal data input from the AD converter to the data acquisition control circuit 312.

[0097] The motor control circuit 307 is a circuit that generates control signals to the motor driver 115 for the main scanning motor based on instructions from the CPU 301. When the CPU 301 instructs the rotation speed, acceleration, movement amount, rotation direction, and rotation start timing of the main scanning motor 107, it generates a driving pulse voltage corresponding to the instructions.

[0098] The motor control circuit 308 is a circuit that generates control signals to a motor driver 111 for the sub-scanning motor based on instructions from the CPU 301. When the CPU 301 instructs the rotation speed, acceleration, movement amount, rotation direction, and rotation start timing of the sub-scanning motor 110, the motor control circuit 308 generates a driving pulse voltage corresponding to the instructions.

[0099] The motor control circuit 309 is a circuit that generates control signals to the motor driver 114 for the height scanning motor based on instructions from the CPU 301. When the CPU 301 instructs the rotation speed, acceleration, movement amount, rotation direction, and rotation start timing of the height scanning motor 113, it generates a driving pulse voltage corresponding to the instructions.

[0100] The coordinate calculation circuit 310 is a coordinate calculation circuit that counts two phase difference pulse signals of A phase and B phase from the encoder 108 and calculates the position of the objective optical system 104. The resolution of the encoder 108 is assumed to be 1 μm. When the level of the A phase signal or B phase signal changes and the A phase signal is more phase-advanced than the B phase signal, the coordinate of the objective optical system 104 is increased by 1 μm. When the level of the A phase signal or B phase signal changes and the B phase signal is more phase-advanced than the A phase signal, the coordinate of the objective optical system 104 is decreased by 1 μm.

[0101] The synchronization circuit 311 is a synchronization circuit that generates trigger signals to the data acquisition control circuit 312, motor control circuit 308, motor control circuit 309, and light source control circuit 305 based on the coordinate information obtained from the coordinate calculation circuit 310. In the present invention, the trigger signal to the AD converter control circuit 306 is called a data acquisition trigger signal. The trigger signal to the motor control circuit 308 is called a sub-scanning trigger signal. The trigger signal to the motor control circuit 309 is called a height scanning trigger signal. The trigger signal to the light source control circuit 305 is called a wavelength switching trigger signal.

[0102] When the motor control circuit 308 receives the sub-scanning trigger signal, it outputs a drive pulse train corresponding to a movement amount equivalent to the pixel pitch in the sub-scanning direction to the motor driver 111. The pixel pitch in the sub-scanning direction is specified by the user at the start of imaging and is stored in the memory 303. When the motor control circuit 309 receives the height scanning trigger signal, it outputs a drive pulse train corresponding to the amount of movement in the height scanning direction to the motor driver 114. The amount of movement in the height scanning direction is calculated by the CPU 301. The calculation method will be described later.

[0103] The data acquisition control circuit 312 is a circuit that transfers the optical signal data output from the AD converter control circuit 306 to the memory 303 via the memory control circuit 304. It is composed of a buffer memory, a DMA controller, and the like.

[0104] The communication circuit 313 is a communication circuit for connecting the imaging device to an external network. The communication method uses a communication protocol that complies with the Ethernet standard. By connecting the imaging device to an external PC or server, it is possible to remotely control specimen imaging and store data in external mass storage.

[0105] The UI circuit 314 is a UI circuit for connecting the imaging device to the user interface 117. It is made up of input circuits for keyboards and mice and an image display circuit for controlling the display.

[0106] The peripheral circuits that make up the controller 116 are implemented on semiconductor chips such as FPGAs or ASICs, and operate in synchronization with a clock whose frequency is set to 100 MHz.

[0107] Next, the imaging operation of the array plate of this embodiment will be described with reference to FIGS. FIG. 4 shows an operational flow when imaging an array plate with the imaging device of this embodiment.

[0108] 5A and 5B are diagrams illustrating the positional relationship of the sub-scanning on the array plate during imaging operation, and are views of the array plate 101 as seen from above.

[0109] Figure 6 is a diagram explaining the positional relationship of the height scanning of the array plate during imaging operation. Figures 6(a) and 6(b) are diagrams showing an example of the array plate 101 as viewed from the side. Figures 6(a) and 6(b) show different focus positions for each wavelength.

[0110] In the imaging device of the present invention, it is preferable that the emission wavelength control unit controls the light source and the scanning unit to change the wavelength of the light emitted from the light emitting unit so as to switch between primary light of a first wavelength and primary light of a second wavelength that has a different wavelength from the primary light of the first wavelength.

[0111] In the imaging device of the present invention, it is preferable that the emission wavelength control unit controls the light source and the scanning unit so that one of the primary light of the first wavelength and the primary light of the second wavelength is main-scanned and then switched to the other of the primary light of the first wavelength and the primary light of the second wavelength, so that the primary light of the first wavelength and the primary light of the second wavelength are main-scanned on a common scanning path.

[0112] In the imaging device of the present invention, it is preferable that the emission wavelength control unit controls the light source and the scanning unit so that the sub-scanning step is performed once each time a first scanning step of scanning with the first light and a second scanning step of scanning with the second light on a common scanning path are performed at least once, and it is more preferable that the emission wavelength control unit controls the light source and the scanning unit so that the sub-scanning step is performed once each time a first scanning step of scanning with the first light and a second scanning step of scanning with the second light on a common scanning path are performed at least once.

[0113] By doing so, it is possible to reduce the focus position shift between multiple wavelengths with a simple configuration and acquire in-focus fluorescence images of multiple wavelengths across the entire surface of the array plate. Preferred examples of these are described below using the flowchart in Figure 4.

[0114] In step S401, when the user issues an instruction to start imaging, the CPU 301 reads the imaging conditions specified by the user via the user interface 117 and stores them in the memory 303. The CPU 301 also sets information based on the imaging conditions in various circuits within the controller 116. As the imaging conditions, points 501 (X1, Y1) and 502 (X2, Y2) indicating the imaging range on the array plate 101, pixel pitch Xp in the main scanning direction, pixel pitch Yp in the sub-scanning direction, and rotation speed Xs in the main scanning direction are input.

[0115] In this embodiment, X1=500, X2=22500, Y1=500, Y2=64500, Xp=10 μm, Yp=10 μm, and Xs=1200 rpm. In the present invention, a rectangular area on the array plate 101 with points 501 and 502 as diagonals is called an imaging area 503.

[0116] The framed area 508 indicated by the dashed line indicates the inner wall of the frame 203. In the present invention, the area inside the framed area 508 is the area filled with the observation liquid during measurement. A glycerol solution is used as the observation liquid. In the framed area 508, there is little light reflected at the interface between the array plate and the observation liquid, which reduces background noise in fluorescence measurement.

[0117] Also, the number of pixels in the main scanning direction Nx=(X2-X1) / Xp and the number of pixels in the sub-scanning direction Ny=(Y2-Y1) / Yp are calculated in advance. In this embodiment, Nx=2200 and Ny=6400.

[0118] In step S402, CPU 301 acquires height information and tilt information of array plate 101 for each wavelength. In the present invention, the amount of movement of Z-direction linear stage 112 required to focus the primary light on the upper surface of slide glass 201, which constitutes array plate 101, i.e., the surface on the side where spot 202 is located, is referred to as height information. The tilt of the array plate in the sub-scanning direction is referred to as tilt information.

[0119] In step S402, height information Z3_670, Z3_780, Z4_670, and Z4_780 for each wavelength relative to two Y coordinates Y3 and Y4 is obtained by measurement. Z3_670 is height information for focusing 680 nm light at the Y3 position, and Z3_780 is height information for focusing 780 nm light at the Y3 position. Similarly, Z4_670 is height information for focusing 680 nm light at the Y4 position, and Z4_780 is height information for focusing 780 nm light at the Y4 position.

[0120] In this embodiment, Y3=750, Y4=65000. If the inclination is as shown in FIG. 6(a), Z4_670>Z3_670, Z4_780>Z3_780, and if the inclination is as shown in FIG. 6(b), Z3_670 <Z4_670、Z3_780<Z4_780となる。

[0121] 5(a), Y3 and Y4 are outside the framed area 508, and air, not the observation liquid, exists on the array plate. Therefore, at Y3 and Y4, the height of the array plate can be measured from the reflected light at the interface between the array plate and air. The method for obtaining the height information will be described later.

[0122] The slope information K_670 and K_780 for each wavelength are calculated using the following formulas: K_670 is the slope information for the primary light of 670 nm, and K_780 is the slope information for the primary light of 780 nm. K_670=(Z4_670-Z3_670) / (Y4-Y3) (Equation 1) K_780=(Z4_780-Z3_780) / (Y4-Y3) (Equation 2)

[0123] In step S403, the CPU 301 calculates the target height for each row for each wavelength based on the imaging conditions and the height information and tilt information K_670 and K_780 for each wavelength. The target height Z_670(Y) for a wavelength of 670 nm at an arbitrary sub-scanning position coordinate Y is given by the following equation. The target height 601 for a wavelength of 670 nm corresponds to the Z coordinate for focusing the primary light with a wavelength of 670 nm on the surface of the array plate 101 in Figures 6(a) and 6(b). Z_670(Y)=K_670×(Y-Y3)+Z3_670 (Formula 3)

[0124] Similarly, the target height Z_780(Y) for a wavelength of 780 nm at an arbitrary sub-scanning position coordinate Y is given by the following equation: The target height 602 for a wavelength of 780 nm corresponds to the Z coordinate for focusing the primary light with a wavelength of 780 nm on the surface of the array plate 101 in Figures 6(a) and 6(b). Z_780(Y)=K_780×(Y-Y3)+Z3_780 (Formula 4)

[0125] In step S404, the CPU 301 issues instructions to the motor control circuits 307, 308, and 309 to move the array plate 101 and the objective optical system 104 to the imaging start position.

[0126] The X coordinate of the imaging start position is the end of the scanning range of the piston-crank mechanism 120, and the X coordinate value is -2500. The Y coordinate of the imaging start position is Y1 specified in the imaging conditions. Furthermore, the Z coordinate Z1_670 of the imaging start position for primary light with a wavelength of 670 nm is expressed as Z_670(Y1) in Equation 3. Similarly, the Z coordinate Z1_780 of the imaging start position for primary light with a wavelength of 780 nm is expressed as Z_780(Y1) in Equation 4.

[0127] In step S405, the CPU 301 issues an instruction to the motor control circuit 307 to rotate the main scanning motor 107 at a rotation speed Xs. This causes the objective optical system 104 to start reciprocating in the X direction. The encoder 108 and the coordinate calculation circuit 310 calculate the X coordinate of the objective optical system 104 for each clock and output it to the synchronization circuit 311.

[0128] In step S406, the CPU 301 issues an instruction to the light source control circuit 305 to cause the light source 102 to start emitting light. This starts irradiating the array plate 101 with light via the objective optical system 104. At this time, the synchronization circuit 311 is used to irradiate the array plate 101 with light of two wavelengths, 670 nm and 780 nm, while alternately switching between the forward and backward main scanning passes. This reduces crosstalk between wavelengths and improves the measurement accuracy of optical signal data.

[0129] In step S407, the synchronization circuit 311 determines whether the objective optical system 104 has reached the wavelength switching position. It is determined that the objective optical system 104 has reached the wavelength switching position when the X coordinate of the objective optical system 104 output from the coordinate calculation circuit 310 moves to predetermined positions X3 and X4 outside the imaging area.

[0130] The coordinates of X3 and X4 are set in advance and stored in the synchronization circuit 311. The wavelength switching positions for both X3 and X4 are set to positions closer to the stroke end of the objective optical system 104 than the line feed positions X1 and X2. This is because it takes a certain amount of time to switch the wavelength, and the wavelength switching must be completed while the objective optical system is outside the imaging range. In this embodiment, X3=200, X4=22800.

[0131] If the wavelength switching position has been reached, the synchronization circuit 311 outputs a wavelength switching trigger signal to the light source control circuit 305 and also outputs a height scanning signal to the motor control circuit 309, causing steps S421 and S418 to operate in parallel. If the wavelength switching position has not been reached, the synchronization circuit 311 does not output a wavelength switching trigger signal and proceeds to step S408.

[0132] In step S408, the synchronization circuit 311 determines whether the objective optical system 104 has reached the line feed position. It is determined that the line feed position has been reached when the X coordinate of the objective optical system 104 output from the coordinate calculation circuit 310 moves from inside the imaging area to outside the imaging area. In this embodiment, if the current main scanning direction is the backward direction (the direction in which the X coordinate decreases), it is determined that the line feed position has been reached when the X coordinate of the objective optical system 104 becomes smaller than X1. In this embodiment, it is not determined that the line feed position has been reached when the X coordinate of the objective optical system 104 becomes larger than X2.

[0133] The forward scan is represented by trajectory 504 in Figure 5(b). The backward scan is represented by trajectory 506 in Figure 5(b), and follows the same trajectory as the forward scan, returning to the line feed position. The initial value of the main scan direction is the forward direction, and thereafter, the backward and forward directions are alternately repeated each time the stroke end is reached.

[0134] If the line feed position has been reached, the synchronization circuit 311 outputs a sub-scanning trigger signal and the process proceeds to step S417. If the line feed position has not been reached, the synchronization circuit 311 does not output a sub-scanning trigger signal and the process proceeds to step S409.

[0135] In step S409, the synchronization circuit 311 determines whether the objective optical system 104 has reached the target position. A point 507 in FIG.

[0136] The target position is a point on the array plate where optical signal data is acquired to generate a pixel, and the X coordinate P(N) of the Nth target position is expressed by the following equation: P(N)=X1+Xp×N (N=0,1,…Nx-1) Equation (5)

[0137] The target positions are a plurality of points on the trajectory, with a pitch in the X direction of Xp and a pitch in the Y direction of Yp, which coincide with pixel positions 507 in this embodiment. The initial value of the target position is P(0), which is stored inside the synchronization circuit 311. In determining the initial target position, it is determined that the target position has been reached when the X coordinate of the objective optical system 104 output from the coordinate calculation circuit 310 passes P(0) in the forward direction (the direction in which the X coordinate increases).

[0138] In the second and subsequent target position determinations, it is determined that the target position has been reached when the X coordinate of the objective optical system 104 output from the coordinate calculation circuit 310 passes through the target position updated in step S411, which will be described later. If the target position has been reached, the synchronization circuit 311 outputs a data acquisition trigger signal and proceeds to step S410. If the target position has not yet been reached, the synchronization circuit 311 proceeds to step S412 without outputting a data acquisition trigger signal.

[0139] In step S410, upon receiving the data acquisition trigger signal, the AD converter control circuit 306 outputs a conversion start signal, and the output voltage from the optical sensor 105 is amplified and filtered by the receiving circuit 106 before being AD converted. The AD converted optical signal data is transferred to the data acquisition control circuit 312. In this embodiment, an AD converter corresponding to each wavelength of the light source is provided, and optical signal data reflecting the amount of fluorescent light excited by the wavelength of each light source is stored.

[0140] The data acquisition control circuit 312 outputs the optical signal data from the AD converter control circuit 306 to the memory control circuit 304 via the internal DMA controller and bus interface 302. The memory control circuit 304 stores the optical signal data at a specified address in the memory 303. The address of the memory 303 is incremented each time data is output.

[0141] After storing Nx×Ny pieces of data corresponding to the wavelengths of each light source in memory 303, the data acquisition control circuit 312 sets an internal data acquisition completion register to 1. When the data acquisition control circuit 312 has not stored Nx×Ny pieces of optical signal data corresponding to the wavelengths of each light source in memory 303, it sets an internal data acquisition completion register to 0.

[0142] Next, in step S411, the synchronization circuit 311 updates the target position stored internally. If the current main scanning direction is the forward direction (the direction in which the X coordinate increases), the target position P(N) is updated to P(N+1). If the current main scanning direction is the backward direction (the direction in which the X coordinate decreases), the target position P(N) is updated to P(N-1).

[0143] In step S412, the CPU 301 determines whether the optical signal data acquisition has finished. The CPU 301 reads the data acquisition completion register of the data acquisition control circuit 312, and if the value is 1, it determines that the optical signal data acquisition has finished, and proceeds to step S413. If the value is 0, it determines that the optical signal data acquisition has not finished, and proceeds to step S407. In step S413, the CPU 301 issues an instruction to the light source control circuit 305 to stop the light emission of the light source 102. As a result, the irradiation of light onto the array plate 101 via the objective optical system 104 is stopped.

[0144] In step S414, the CPU 301 issues an instruction to the motor control circuit 307 to stop the rotation of the main scanning motor 107. This stops the reciprocating motion of the objective optical system 104 in the X direction. In step S415, the CPU 301 issues an instruction to the motor control circuits 307, 308, and 309 to move the array plate 101 and the objective optical system 104 to a stop position. The X, Y, and Z coordinates of the stop position are 0. Movement to the stop position is achieved by returning each axis to its origin using the Z-phase pulse signal of the encoder 108, the origin sensor signal in the Y-direction linear stage 109, and the origin sensor signal of the Z-direction linear stage 112. The Y-direction linear stage 109 constitutes a mount on which the array plate 101 is mounted, and is therefore sometimes referred to as the mount 109.

[0145] In step S416, the CPU 301 reads Nx×Ny pieces of optical signal data corresponding to the wavelengths of each light source from the memory 303, performs data compression and format conversion processing, and creates a captured image file in TIFF format. The captured image file is saved in the memory 303 and presented to the user via the UI circuit 314 and the user interface 117. In addition, in response to an instruction from the user, the captured image file is transferred to an external data server via the communication circuit 313.

[0146] In step S417, the CPU 301 issues an instruction to the motor control circuit 308 to move the array plate 101 by Yp in the sub-scanning direction. If the amount of movement of the array plate 101 when one pulse of a voltage pulse signal is sent to the motor driver 111 is My, the number of pulses output by the motor control circuit 308 to the motor driver 111 is Yp / My. In this embodiment, My=2 μm. The sub-scanning is represented by 505 in FIG. 5(b), and the movement distance in the Y direction is Yp.

[0147] The motor control circuit 308 outputs a pulse signal to the motor driver 111 at a speed that completes the sub-scan while the objective optical system 104 is outside the imaging area in the main scanning direction, based on an instruction from the CPU 301. When the sub-scan is completed, the process proceeds to step S420.

[0148] In step S418, the CPU 301 reads the target heights before and after the sub-scanning from the memory 303. At that time, the target height corresponding to the wavelength after the wavelength switching is read. In this embodiment, an example will be described in which a laser beam of 780 nm is irradiated on the forward path and 670 nm on the backward path. That is, when the wavelength is switched from 670 nm to 780 nm, Z_780(Y+Yp) and Z_670(Y) are read and the movement amount of the height scanning is calculated.

[0149] When the wavelength is switched from 780 nm to 670 nm, Z_670(Y) and Z_780(Y) are read out and the amount of movement in height scanning is calculated.

[0150] The amount of movement in the height scan varies depending on the current Y coordinate, but the number of output pulses and the direction of motor rotation are calculated so that the height in the Z direction after the movement is closest to the target height in the Y coordinate after the sub-scan in step S416 and the wavelength switching in step S421.

[0151] That is, when switching from the forward pass to the backward pass, the wavelength is switched from 780 nm to 670 nm, but since sub-scanning is not performed, the wavelength is set close to Z_670(Y). That is, when switching from the backward pass to the forward pass, the wavelength is switched from 670 nm to 780 nm and the sub-scanning is performed, so the value is set close to Z_780(Y+Yp).

[0152] The calculation direction is as follows: The amount of movement of the array plate 101 when one pulse of a voltage pulse signal is sent to the motor driver 114 is defined as Mz. In this embodiment, Mz=1 μm.

[0153] Let RoundMz(x) be the multiple of Mz that is closest to a number x, ABS(x) be the absolute value of the number, and Sign(x) be the sign of the number.

[0154] In the present invention, the multiple of Mz that is closest to the target height is referred to as the target pulse number. The target pulse number takes discrete values ​​and corresponds to rectangles 603 and 604 in Figures 6(a) and 6(b). The height of the hatched rectangle 603 represents the target pulse number for primary light with a wavelength of 670 nm, and the height of the white rectangle 604 represents the target pulse number for primary light with a wavelength of 780 nm.

[0155] The number of pulses that the motor control circuit 309 outputs to the motor driver 114 when switching from the forward path to the backward path is expressed by the following formula. Zp=ABS(RoundMz(Z_670(Y)) ― RoundMz(Z_780(Y))) Equation (6)

[0156] Similarly, the number of pulses that the motor control circuit 309 outputs to the motor driver 114 when switching from the backward path to the forward path is expressed by the following formula. Zp=ABS(RoundMz(Z_780(Y+Yp)) ― RoundMz(Z_670(Y))) Equation (7)

[0157] RoundMz(Z_670(Y)) and RoundMz(Z_780(Y)) are discrete values ​​as shown by rectangles 603 and 604 in FIGS. 6(a) and 6(b), respectively.

[0158] Furthermore, the moving direction Dir that the motor control circuit 309 outputs to the motor driver 114 when switching from the forward path to the backward path is expressed by the following equation. Dir=Sign(RoundMz(Z_670(Y)) ― RoundMz(Z_780(Y))) Equation (8)

[0159] Similarly, the moving direction Dir that the motor control circuit 309 outputs to the motor driver 114 when switching from the backward path to the forward path is expressed by the following equation. Dir=Sign(RoundMz(Z_780(Y+Yp)) ― RoundMz(Z_670(Y))) Equation (9)

[0160] Let the positive direction of Dir be vertically upward, and the direction be away from the distance between the objective optical system 104 and the array plate 101.

[0161] When the inclination of the array plate is as shown in Fig. 6(a), the value of Dir is 1 when switching from the forward path to the return path, and -1 when switching from the return path to the forward path. When it is as shown in Fig. 6(b), the value of Dir is -1 when switching from the forward path to the return path, and 1 when switching from the return path to the forward path.

[0162] In Fig. 6(a), an example where Z_780(Y) < Z_670(Y) over the entire range of all Y coordinates is shown. In Fig. 6(b), an example where Z_670(Y) < Z_780(Y) over the entire range of all Y coordinates is shown. However, the magnitude relationship between Z_670(Y) and Z_780(Y) is not limited to this and may be interchanged.

[0163] In step S419, the CPU 301 issues an instruction to the motor control circuit 309 to move the array plate 101 by Zp in the direction that cancels the tilt direction Dir. When the value of Dir is positive, the array plate is moved downward, and when the value of Dir is negative, the array plate is moved upward.

[0164] The number of pulses output by the motor control circuit 309 to the motor driver 111 is Zp / Mz. Based on the instruction from the CPU 301, the motor control circuit 309 outputs a pulse signal to the motor driver 114 at a speed such that the height scanning is completed while the objective optical system 104 is outside the imaging area in the main scanning direction.

[0165] In step S420, the synchronization circuit 311 increments the current Y coordinate by Yp from the previous Y coordinate and proceeds to step S412.

[0166] In step S421, the light source control circuit 305 switches the emission and stop of the semiconductor lasers at 670 nm and 780 nm based on the information of the wavelength switching trigger signal from the synchronization circuit 311. After the height scanning in step S419 and the instruction to the light source control in step S421 are completed, it proceeds to step S412.

[0167] If the current main scanning direction is the forward direction (the direction in which the X coordinate increases), the 780 nm laser is emitted when the X coordinate of the objective optical system 104 exceeds X3, and the 780 nm laser is stopped when it exceeds X4. If the main scanning direction is the forward direction (the direction in which the X coordinate increases), the 670 nm laser is stopped from emitting light.

[0168] If the current main scanning direction is the backward direction (the direction in which the X coordinate decreases), the 670 nm laser is emitted when the X coordinate of the objective optical system 104 becomes smaller than X4, and the 670 nm laser is stopped when it becomes smaller than X3. If the main scanning direction is the forward direction (the direction in which the X coordinate increases), the 780 nm laser is stopped from emitting light.

[0169] FIG. 7 is a diagram showing details of the light source 102, the confocal optical system 103, the objective optical system 104, the optical sensor 105, and the receiving circuit 106 of the imaging device of this embodiment. The light source 102 will now be described.

[0170] The light source 102 is a light source capable of outputting light of wavelengths of 670 nm and 780 nm at independent timings, and is composed of a laser diode 701 and a laser diode 714 .

[0171] The laser diode 701 is a laser diode that generates light with a wavelength of 780 nm, and can be turned on, stopped, modulated, and the amount of light can be controlled by external electrical signals. The laser diode 714 is a laser diode that generates light with a wavelength of 670 nm, and can be turned on, stopped, modulated, and the amount of light can be controlled by external electrical signals.

[0172] Next, we will explain the confocal optical system 103. The confocal optical system 103 can separate and acquire emitted light, reflected light, and fluorescent light, and is composed of three parts: a confocal optical system for a wavelength of 780 nm, a confocal optical system for a wavelength of 670 nm, and a combining unit. The confocal optical system for a wavelength of 780 nm will be described below.

[0173] The collimator lens 702 converts the 780 nm light emitted from the laser diode 701 into parallel light. The bandpass filter 703 is a bandpass filter that passes light with a wavelength of around 780 nm.

[0174] The polarizing beam splitter 704 is a polarizing beam splitter for separating the emitted light from the reflected light. The quarter-wave plate 705 is a quarter-wave plate whose slow axis is tilted at 45° with respect to the polarization direction of the polarizing beam splitter 704 .

[0175] The light emitted from the laser diode 701 passes through a polarizing beam splitter 704 , is converted from linearly polarized light to circularly polarized light by a quarter-wave plate 705 , and reaches a dichroic mirror 706 .

[0176] Meanwhile, the light reflected from the array plate 101 is converted by the ¼λ wavelength plate 705 from circularly polarized light to linearly polarized light that is 90° perpendicular to the emitted light, reflected by the polarizing beam splitter 704 , and reaches the condenser lens 707 .

[0177] The dichroic mirror 706 is a dichroic mirror for separating the fluorescent light from the reflected light. The light emitted from the laser diode 701 is reflected by the dichroic mirror 706 and reaches the dichroic mirror 719 .

[0178] The light reflected from the array plate 101 is reflected by a dichroic mirror 706 and reaches a ¼λ wave plate 705 . The fluorescence excited by the light of 780 nm wavelength at the array plate 101 has a longer wavelength than the reflected light, passes through the dichroic mirror 706 and reaches the fluorescence filter 708 .

[0179] The condenser lens 707 is a condenser lens for the reflected light with a wavelength of 780 nm, and the reflected light is condensed and reaches the pinhole 710 . The fluorescence filter 708 is a bandpass filter that transmits fluorescence excited by light with a wavelength of 780 nm.

[0180] The condenser lens 709 is a condenser lens for the fluorescence excited by light with a wavelength of 780 nm, and the fluorescence is condensed and reaches the pinhole 711 . The pinhole 710 allows light reflected from near the surface of the array plate 101 to pass through. The light that passes through reaches the photodiode 712 of the optical sensor 105.

[0181] The pinhole 711 allows the fluorescence from near the surface of the array plate 101 to pass through. The light that passes through reaches the photomultiplier tube 713 of the optical sensor 105. Next, the confocal optical system 103 for a wavelength of 670 nm will be described below.

[0182] The laser diode 714 is a laser diode that generates light with a wavelength of 670 nm. The collimator lens 715 is a collimator lens that converts the 670 nm light into parallel light. The bandpass filter 716 is a bandpass filter that passes light with a wavelength of around 670 nm.

[0183] The polarizing beam splitter 717 is a polarizing beam splitter for separating the emitted light from the reflected light. The quarter-wave plate 718 is a quarter-wave plate whose slow axis is tilted at 45° with respect to the polarization direction of the polarizing beam splitter 717 .

[0184] The light emitted from the laser diode 714 passes through a polarizing beam splitter 717 , is converted from linearly polarized light to circularly polarized light by a quarter-wave plate 718 , and reaches a dichroic mirror 719 .

[0185] Meanwhile, the light reflected from the array plate 101 is converted by the ¼λ wavelength plate 718 from circularly polarized light to linearly polarized light that is 90° perpendicular to the emitted light, reflected by the polarizing beam splitter 717 , and reaches the condenser lens 720 .

[0186] The dichroic mirror 719 is a dichroic mirror for separating the fluorescent light from the reflected light. The light emitted from the laser diode 714 is reflected by the dichroic mirror 719 and reaches the dichroic mirror 727 .

[0187] The light reflected from the array plate 101 is reflected by a dichroic mirror 719 and reaches a ¼λ wave plate 718 .

[0188] The fluorescence excited by the light of 780 nm wavelength at the array plate 101 has a longer wavelength than the reflected light, passes through the dichroic mirror 719 and reaches the fluorescence filter 721 . The condenser lens 720 is a condenser lens for the reflected light with a wavelength of 780 nm, and the reflected light is condensed and reaches the pinhole 723 .

[0189] The fluorescence filter 721 is a bandpass filter that transmits fluorescence excited by light with a wavelength of 780 nm. The condenser lens 722 is a condenser lens for the fluorescence excited by light with a wavelength of 780 nm, and the fluorescence is condensed and reaches the pinhole 724 .

[0190] The pinhole 723 allows light reflected from the vicinity of the surface of the array plate 101 to pass through. The light that has passed through reaches the photodiode 725 of the optical sensor 105. The pinhole 724 allows the fluorescence from near the surface of the array plate 101 to pass through. The transmitted light reaches the photomultiplier tube 726 of the optical sensor 105 .

[0191] Next, we will explain the multiplexing unit. The multiplexing unit multiplexes the outgoing light with a wavelength of 670 nm and the outgoing light with a wavelength of 780 nm, and also separates the reflected light and fluorescent light originating from the 670 nm wavelength from the reflected light and fluorescent light originating from the 780 nm wavelength and the reflected light and fluorescent light originating from the 670 nm wavelength.

[0192] Dichroic mirror 727 is a dichroic mirror for separating light originating from a wavelength of 670 nm from light originating from a wavelength of 780 nm. The emitted light and reflected light of a wavelength of 670 nm and the fluorescence generated by the emitted light of a wavelength of 670 nm are reflected by dichroic mirror 727 because they have short wavelengths.

[0193] On the other hand, the emitted light with a wavelength of 780 nm, the reflected light, and the fluorescence generated by the emitted light with a wavelength of 780 nm are transmitted through the dichroic mirror 727 because they have long wavelengths.

[0194] The outgoing light having a wavelength of 670 nm reflected by the dichroic mirror 727 and the outgoing light having a wavelength of 780 nm transmitted by the dichroic mirror 727 travel along the same optical path to reach the mirror 728 .

[0195] The reflected light of 670 nm wavelength from the array plate 101 and the fluorescence generated by the emitted light of 670 nm wavelength are reflected by the dichroic mirror and reach the dichroic mirror 719 .

[0196] The reflected light having a wavelength of 780 nm from the array plate 101 and the fluorescence generated by the emitted light having a wavelength of 780 nm are transmitted through the dichroic mirror and reach the dichroic mirror 706 .

[0197] Next, the objective optical system 104 will be described. The mirror 728 is a mirror that reflects horizontal light in a vertical direction. The objective lens 729 focuses light of wavelengths 670 nm and 780 nm onto the array plate 101 .

[0198] The main scanning mechanism 730 is composed of a crank 118 , a connecting rod 119 , a piston-crank mechanism 120 , and a main scanning motor 107 .

[0199] The main scanning mechanism 730 causes the mirror 728 and the objective lens 729 to reciprocate in the main scanning direction, and the collected light beams with wavelengths of 670 nm and 780 nm are scanned onto the array plate 101 .

[0200] Next, the optical sensor 105 will be described in detail. The optical sensor 105 is an optical sensor capable of detecting the amount of reflected light with a wavelength of 670 nm, fluorescence generated by light with a wavelength of 670 nm, reflected light with a wavelength of 780 nm, and fluorescence generated by light with a wavelength of 780 nm, and is composed of photodiodes 712, 725 and photomultiplier tubes 713, 726.

[0201] The photodiode 712 detects the reflected light with a wavelength of 780 nm and outputs a current according to the amount of light. The photomultiplier tube 713 detects the fluorescence generated by light with a wavelength of 780 nm, and outputs a current according to the amount of light.

[0202] The photodiode 725 detects the reflected light with a wavelength of 670 nm and outputs a current according to the amount of light. The photomultiplier tube 726 detects the fluorescence generated by light with a wavelength of 670 nm, and outputs a current according to the amount of light.

[0203] Next, the receiving circuit 106 will be described in detail. The receiving circuit 106 is a circuit for obtaining a digital value corresponding to the amount of light from the electrical signal output from the optical sensor 105. The receiving circuit 106 can obtain digital values ​​corresponding to the amount of light: reflected light with a wavelength of 670 nm, fluorescence generated by light with a wavelength of 670 nm, reflected light with a wavelength of 780 nm, and fluorescence generated by light with a wavelength of 780 nm, and is composed of current-voltage conversion amplifiers 731, 732, 733, and 734 and AD converters 735, 736, 737, and 738.

[0204] The current-to-voltage conversion amplifier 731 is an electronic circuit that converts the current from the photomultiplier tube 713 into a voltage and amplifies it to an appropriate voltage. The current-to-voltage conversion amplifier 732 is an electronic circuit that converts the current from the photodiode 712 into a voltage and amplifies it to an appropriate voltage.

[0205] The current-to-voltage conversion amplifier 733 is an electronic circuit that converts the current from the photomultiplier tube 726 into a voltage and amplifies it to an appropriate voltage. The current-to-voltage conversion amplifier 734 is an electronic circuit that converts the current from the photodiode 725 into a voltage and amplifies it to an appropriate voltage.

[0206] The AD converter 735 is an electronic circuit that converts the output voltage from the current-voltage conversion amplifier 731 into a digital value. The AD converter 735 can obtain a digital value corresponding to the amount of fluorescent light generated by light with a wavelength of 780 nm.

[0207] The AD converter 736 is an electronic circuit that converts the output voltage from the current-voltage conversion amplifier 732 into a digital value. The AD converter 736 can obtain a digital value corresponding to the amount of reflected light with a wavelength of 780 nm.

[0208] The AD converter 737 is an electronic circuit that converts the output voltage from the current-voltage conversion amplifier 733 into a digital value. The AD converter 737 can obtain a digital value corresponding to the amount of fluorescent light generated by light with a wavelength of 670 nm.

[0209] The AD converter 738 is an electronic circuit that converts the output voltage from the current-voltage conversion amplifier 734 into a digital value. The AD converter 738 can obtain a digital value corresponding to the amount of reflected light with a wavelength of 670 nm.

[0210] AD converters 735, 736, 737, and 738 can sample input signals at independent timings according to external signals and convert them into digital values.

[0211] FIG. 8 is a timing chart showing the operational timing of main scanning, sub-scanning, height scanning, light irradiation, and optical signal data acquisition in the imaging device of this embodiment. The horizontal axis of these timing charts represents time. Timing chart 801 is a timing chart showing the operational timing of main scanning, and indicates that the piston-crank mechanism 120 rotates and main scanning is performed at the times indicated by hexagons. Timing chart 802 has the vertical axis representing the position of the objective optical system 104 in the X-axis direction and the horizontal axis representing time. The piston-crank mechanism 120 causes the objective optical system 104 to reciprocate, and the speed is shown to be fast near the center of the stroke in the X-axis position and slower near the stroke ends. Timing chart 803 is a timing chart showing the operational timing of sub-scanning. The sub-scanning motor 110 operates and sub-scanning is performed at the times indicated by hexagons.

[0212] Timing charts 804 and 805 are timing charts showing the operation timing of height scanning. Timing chart 804 shows the timing of height scanning to a target height with a wavelength of 780 nm, and timing chart 805 shows the timing of height scanning to a target height with a wavelength of 670 nm. The height scanning motor 113 operates at the times indicated by hexagons, indicating that height scanning is being performed. Timing chart 806 is a timing chart showing the operation timing of acquiring 780 nm optical signal data. The photomultiplier tube 713 and receiving circuit 106 operate at the times indicated by hexagons, indicating that optical signal data derived from 780 nm primary light is being acquired.

[0213] Timing chart 807 is a timing chart showing the operation timing for acquiring 670 nm optical signal data. The photomultiplier tube 726 and receiving circuit 106 operate at the times indicated by hexagons, and optical signal data derived from the 670 nm primary light is acquired. Timing chart 808 is a timing chart showing the operation timing of the 780 nm laser diode 701 in the light source 102. The 780 nm primary light is emitted at the times indicated by hexagons, and is irradiated onto the array plate 101 via the objective optical system 104.

[0214] Timing chart 809 is a timing chart showing the operation timing of the 670 nm laser diode 714 in the light source 102. At the times indicated by the hexagons, primary light of 670 nm is emitted and irradiated onto the array plate 101 via the objective optical system 104.

[0215] In this manner, in this embodiment, when the X coordinate of the objective optical system 104 is moving in the forward direction, the array plate 101 is irradiated with primary light of 780 nm, and optical signal data generated by the 780 nm primary light is acquired. On the other hand, when the X coordinate of the objective optical system 104 is moving in the backward direction, the array plate 101 is irradiated with primary light of 670 nm, and optical signal data generated by the 670 nm primary light is acquired. In this manner, by acquiring optical signal data with different excitation wavelengths in the forward and backward directions, it is possible to reduce the effects of misalignment of images between the forward and backward directions, which occurs due to mechanical errors in the piston-crank mechanism 120, delay times in the receiving circuit 106, etc.

[0216] When the X coordinate of the main scanning acquired by the encoder 108 during the forward movement reaches X1, acquisition of 780 nm optical signal data begins. When the X coordinate of the main scanning acquired by the encoder 108 during the backward movement reaches X2, acquisition of 670 nm optical signal data begins. When the X coordinate of the main scanning reaches X1 during the backward movement, it is determined that acquisition of the optical signal data in the backward direction is completed, a sub-scanning trigger is output, and a sub-scan is performed to move to the next line.

[0217] When the X coordinate of the main scanning acquired by the encoder 108 during forward movement reaches X3, a wavelength switching trigger signal is generated to start irradiation of the 780 nm primary light. Furthermore, a height scanning trigger signal is generated to perform height scanning to a target height with a wavelength of 780 nm for the current Y coordinate. When the X coordinate of the main scanning reaches X4 during forward movement, it is determined that acquisition of the 780 nm optical signal data has been completed, and the wavelength switching trigger signal is generated to stop irradiation of the 780 nm primary light.

[0218] When the X coordinate of the main scanning acquired by the encoder 108 during the backward movement reaches X4, a wavelength switching trigger signal is generated to start irradiating the primary light of 670 nm. Also, a height scanning trigger signal is generated to perform height scanning to a target height with a wavelength of 670 nm for the current Y coordinate. When the X coordinate of the main scanning reaches X3 during the backward movement, it is determined that acquisition of the 670 nm optical signal data has been completed, and the wavelength switching trigger signal is generated to stop irradiating the primary light of 670 nm.

[0219] 9(a) to 9(c) are diagrams illustrating the positional relationship of the height scanning of the array plate during height information acquisition operation. FIGS. 9(a) and 9(b) are side views of the array plate 101. FIG. 9(c) plots the amount of reflected light acquired by the optical sensor 105 during height scanning for each height. The horizontal axis represents the amount of light, and the vertical axis represents the acquired height.

[0220] 10 shows the operational flow for acquiring height information of an array plate using the imaging device of this embodiment. The difference between the height information acquisition operation and the imaging operation is that, instead of performing a sub-scan, reflected light signal data is acquired and analyzed while performing height scanning at a constant pitch to calculate the height of the array plate surface. The photodiode 712 and photodiode 725 in the optical sensor 105 are used to acquire the height information.

[0221] In the imaging device of the present invention, it is preferable that the emission wavelength control unit controls the light source and the scanning unit so as to include a period in which the wavelength is switched from one of the first scanning step and the second scanning step to the other of the first scanning step and the second scanning step without passing through the sub-scanning step. This case will be described below.

[0222] In step S1001, the CPU 301 sets parameters for acquiring height information in the synchronization circuit 311. The parameters set include the Y coordinate Yh of the position where height information is acquired, the pixel pitch Xp in the main scanning direction, the pixel pitch Zp in the height scanning direction, points 901 (X5, Z5) and 902 (X6, Z6) that indicate the height scanning range on the XZ plane, and the rotation speed Xs in the main scanning direction. In the present invention, a rectangular area 903 with points 901 and 902 on the XZ plane as diagonals is called a height scanning area. The number of pixels in the main scanning direction Nx = (X6 - X5) / Xp and the number of pixels in the height scanning direction Nz = (Z2 - Z1) / Zp are calculated in advance.

[0223] In this example, X5=500, X6=22500, Z5=2000, Z6=6000, Xp=10 [μm], Zp=10 [μm], Xs=1200 [rpm]. In this case, Nx=2200, Nz=400. In the height information acquisition process, unlike the imaging process, the intervals at which light signal data is acquired can be large, which reduces the time required to acquire height information and the amount of data required.

[0224] In step S1002, the CPU 301 issues instructions to the motor control circuits 307, 308, and 309 to move the array plate 101 and the objective optical system 104 to the height information acquisition start position.

[0225] The X coordinate of the height information acquisition start position is the end of the scanning range of the piston crank mechanism 120, and the X coordinate value is -2500. The Y coordinate of the height information acquisition start position is Yh specified by the parameter. The Z coordinate of the height information acquisition start position is Z5 specified by the parameter. In step S1003, CPU 301 starts main scanning in the same manner as in step S405 of the imaging process, and then proceeds to step S1004.

[0226] In step S1004, the CPU 301 starts irradiating light of 780 nm and 670 nm, and the process proceeds to step S1005.

[0227] In step S1005, the synchronization circuit 311 determines whether the objective optical system 104 has reached the line feed position. It is determined that the line feed position has been reached when the X coordinate of the objective optical system 104 output from the coordinate calculation circuit 310 moves from inside the imaging area to outside the imaging area. If the current main scanning direction is the forward direction (the direction in which the X coordinate increases), it is determined that the line feed position has been reached when the X coordinate of the objective optical system 104 exceeds X6.

[0228] Scanning in the forward direction is represented by a locus 904 in Figure 9(b). If the current main scanning direction is the backward direction (the direction in which the X coordinate decreases), it is determined that the line feed position has been reached when the X coordinate of the objective optical system 104 becomes smaller than X5. Scanning in the backward direction is represented by a locus 906 in Figure 9(b). The initial value of the main scanning direction is the forward direction, and thereafter, the backward direction and forward direction are alternately repeated each time the stroke end is reached.

[0229] Unlike the imaging process, the height information acquisition process acquires data for both wavelengths on both the outbound and inbound journeys, which allows for line feeds on both the outbound and inbound journeys, thereby reducing the time required to acquire height information.

[0230] If the line feed position has been reached, the synchronization circuit 311 outputs a height scan trigger signal and the process proceeds to step S1014. If the line feed position has not been reached, the synchronization circuit 311 does not output a height scan trigger signal and the process proceeds to step S1006.

[0231] In step S1006, the synchronization circuit 311 determines whether the objective optical system 104 has reached the target position, similar to step S409 of the imaging process. The target positions are multiple points on a locus, such as 908 in Figure 9(b), with a pitch of Xp in the X direction and a pitch of Zp in the Z direction.

[0232] If the target position has been reached, the synchronization circuit 311 outputs a data acquisition trigger signal and the process proceeds to step S1007. If the target position has not yet been reached, the synchronization circuit 311 does not output a data acquisition trigger signal and the process proceeds to step S1009.

[0233] In step S1007, the data acquisition control circuit 312 acquires optical signal data in the same manner as in step S410 of the imaging process, and the process proceeds to step S1008.

[0234] However, after storing Nx×Nz pieces of optical signal data corresponding to the amount of reflected light of each wavelength acquired by AD converter 736 and AD converter 738 in memory 303, the data acquisition circuit sets the internal data acquisition completion register to 1. When the data acquisition circuit has not stored Nx×Nz pieces of optical signal data corresponding to the amount of reflected light of each wavelength in memory 303, the data acquisition circuit sets the internal data acquisition completion register to 0.

[0235] In step S1008, the synchronization circuit 311 updates the target position stored therein in the same manner as in step S411 of the imaging process, and the process proceeds to step S1009.

[0236] In step S1009, the CPU 301 determines whether or not the acquisition of optical signal data has been completed, similarly to step S412 of the imaging process. If it is determined that the acquisition of optical signal data has been completed, the process proceeds to step S1010. If it is determined that the acquisition of optical signal data has not been completed, the process proceeds to step S1005.

[0237] In step S1010, the CPU 301 stops the light irradiation in the same manner as in step S413 of the image capturing process, and the process proceeds to step S1011.

[0238] In step S1011, the CPU 301 stops the main scanning in the same manner as in step S414 of the imaging process, and the process proceeds to step S1012.

[0239] In step S1012, the CPU 301 moves the array plate 101 and the objective optical system 104 to the stop position in the same manner as in step S415 of the imaging process, and then proceeds to step S1013.

[0240] In step S1013, the CPU 301 reads and analyzes the Nx × Nz pieces of optical signal data corresponding to the amount of reflected light at each wavelength acquired by the AD converter 736 and the AD converter 738 and stored in the memory 303, and calculates height information for the array plate for each wavelength. Specifically, the Nx pieces of optical signal data acquired at the same height are averaged to determine the average light amount for each height. When the average light amounts are arranged by Z coordinate, two peaks corresponding to the front and rear surfaces of the array plate 101 are present. In FIG. 9(c), peak 909 is the peak due to reflected light from the front surface of the array plate 101, and peak 910 is the peak due to reflected light from the rear surface of the array plate 101. Of these, peak 909, which has the larger Z coordinate, i.e., Z coordinate 911 indicating the peak corresponding to the front surface, is used as height information for position Yh.

[0241] The height information obtained using the optical signal data corresponding to the amount of reflected light is the height at which the reflected light is in focus. If there is a difference in focus position between the fluorescent light and the reflected light, this difference may be corrected. For example, a known amount of focus position deviation may be added to the height information obtained using the optical signal data corresponding to the amount of reflected light, and the result may be used as the height information used in fluorescence detection.

[0242] In this embodiment, by providing a light source, optical system, optical sensor, and receiving circuit for wavelengths of 670 nm and 780 nm, respectively, optical signal data for each wavelength can be acquired simultaneously, and height information for each wavelength can be acquired in a short time.

[0243] In step S1014, the CPU 301 issues an instruction to the motor control circuit 309 to move the array plate 101 by Zp in the height scanning direction. If the amount of movement of the array plate 101 when one pulse of a voltage pulse signal is sent to the motor driver 114 is Mz, the number of pulses output from the motor control circuit 309 to the motor driver 114 is Zp / Mz. The height scanning is represented by height scanning 905 and height scanning 907 in Figure 9(b), and the movement distance in the Z direction is Zp.

[0244] Based on an instruction from the CPU 301, the motor control circuit 309 outputs a pulse signal to the motor driver 114 at a speed such that the height scan is completed while the objective optical system 104 is outside the imaging area in the main scanning direction.

[0245] In step S1015, the synchronization circuit 311 increments the current Z coordinate by Zp from the previous Z coordinate, and then the process proceeds to step S1009. When acquiring tilt information in step S402 of the imaging process, the height information acquisition process shown in FIG. 10 is performed twice, at positions Y3 and Y4, to obtain Z3_670, Z3_780, Z4_670, and Z4_780.

[0246] Height information for Y3 obtained from the optical signal data acquired by the AD converter 736 is Z3_780, and height information for Y3 obtained from the optical signal data acquired by the AD converter 738 is Z3_670.

[0247] Similarly, height information for Y4 obtained from the optical signal data acquired by the AD converter 736 is Z4_780, and height information for Y4 obtained from the optical signal data acquired by the AD converter 738 is Z4_670.

[0248] Next, the tilt information K_670 and K_780 are calculated from the height information using Equations 1 and 2.

[0249] As described above, the imaging device of this embodiment uses a synchronization circuit to perform sub-scanning, wavelength switching, and height scanning in synchronization with the position of the objective optical system. Two-dimensional scanning is performed while correcting the height for each wavelength based on the height and tilt information obtained for each wavelength. This makes it possible to acquire fluorescent images of multiple wavelengths that are in focus across the entire array plate, even if there is a difference in focus position between wavelengths due to misalignment of the optical system or the like. This allows the focus position to be optimized for each of the two wavelengths, which has the advantage of improving reliability when, for example, correcting differences in protein amount between individual array plates by measuring fluorescence brightness.

[0250] By performing sub-scanning and height scanning while the objective optical system is outside the imaging range, there is no need to move the array plate within the imaging range. This reduces the impact of vibrations associated with driving the array plate. Furthermore, because it does not require a high-speed servo control system consisting of a high-performance focus sensor or low-vibration actuator, it is possible to acquire in-focus fluorescent images across the entire array plate with a simple configuration.

[0251] Furthermore, in the height information acquisition method of this embodiment, by performing not only height scanning but also main scanning and averaging Nx pieces of data, it is possible to stably detect peaks even if the array plate is partially soiled or has liquid on it, which improves the stability of height information acquisition compared to performing the height method on a single point on the array plate.

[0252] Although the present embodiment has been described using an example in which height information is obtained using the peak position of reflected light from the surface of the glass slide, the method of obtaining height information is not limited to this. For example, height information may be obtained using the peak position of the brightness of fluorescent signals from some spots on the array plate. In this case, it is necessary to irradiate some spots with light to obtain height information, but the number of components in the optical system can be reduced because there is no need to obtain reflected light with an optical sensor.

[0253] In this embodiment, the widthwise direction of the array plate is the main scanning direction and the lengthwise direction is the sub-scanning direction, but the scanning directions are not limited to this. For example, the main scanning may be performed in the lengthwise direction and the sub-scanning may be performed in the widthwise direction. In this case, the stroke of the piston-crank mechanism 120 must be longer, but the number of sub-scans is reduced, which may shorten the imaging time.

[0254] [Modification of Example 1] In this embodiment, the target height is a straight line obtained by linearly interpolating the height information measured at two points, Y3 and Y4. However, the target height in the present invention is not limited to a straight line. It is also possible to set more measurement points outside the framed area 508 and express the target height as a higher-order function, for example. This allows for accurate correction of even more complex focus position deviations. Separate target heights may also be used for the right and left sides of the framed area 508, allowing for correction of tilt in the main scanning direction as well as the sub-scanning direction.

[0255] [Example 2] A second embodiment of the present invention will now be described. The second embodiment of the present invention differs from the first embodiment in that it deals with the case where the focus position deviation between wavelengths is large and exceeds the range in which the height can be adjusted within the sub-scanning period.

[0256] In the first embodiment of the present invention, height scanning is performed to correct the difference in height of the focus position within the sub-scanning period, but there are restrictions on the speed and acceleration of the height scanning motor 113 and the Z-direction linear stage 112 for height scanning, so there is a restriction on the distance Zd that can be scanned in height within the sub-scanning period Ty. In the second embodiment of the present invention, the target position for each wavelength is adjusted taking into account the range of height scanning.

[0257] The device configuration, array plate, internal configuration of the controller, and detailed configuration of the device in this embodiment are the same as those in the first embodiment, and therefore description thereof will be omitted.

[0258] Most of the operational flow of this embodiment is the same as that of the first embodiment, but the target height calculation process for each row performed in step S403 of Fig. 4 is different. The calculation of the target height in this embodiment will be described with reference to Fig. 11.

[0259] In Figure 11, the same symbols as in Figure 6 have the same meanings and will not be explained further. Line 1101 represents the maximum movable height during the sub-scanning period, based on target height 602 for a wavelength of 780 nm. If the sub-scanning period is 8 ms and the maximum movement speed of the stage is 10 mm / s, the movable distance Zd is 80 μm. Line 1101 is obtained by adding 80 μm to target height 602 for a wavelength of 780 nm, and is expressed as Z_780(Y)+Zd.

[0260] A straight line 1102 indicates the minimum movable height during the sub-scanning period, based on the target height 602 for a wavelength of 780 nm. A straight line 1102 is obtained by subtracting 80 μm from the target height 602 for a wavelength of 780 nm, and is expressed as Z_780(Y)−Zd. The range between the straight lines 1101 and 1102 is the range that can be moved within the sub-scanning period when switching the wavelength from 780 nm to 670 nm.

[0261] In this embodiment, when determining the target height for each wavelength in step S403 of FIG. 4, adjustment is made so that it falls within a movable range.

[0262] That is, Z_780(Y) is calculated according to equation 4, but Z_670(Y) is adjusted according to the following equation so that it falls within the range between line 1101 and line 1102. Z_670(Y)=MAX(MIN(K_670×(Y-Y3)+Z3_670, Z_780(Y)+Zd), Z_780(Y)−Zd)) (Formula 10)

[0263] MIN is a function that returns the minimum value of each argument, and MAX is a function that returns the maximum value of each argument. In the example of Figure 11, in the range where the Y coordinate is smaller than the intersection 1103 of the straight line 1101 and the target height 601 for a wavelength of 670 nm, the target height 601 for a wavelength of 670 nm is Z_670(Y), and in the range where the Y coordinate is larger than the intersection 1103, the straight line 1101 is Z_670(Y).

[0264] By adjusting the target height in this way, taking into account the movable distance, it is possible to complete the height scan within the sub-scanning period and achieve a certain degree of focus even when the focus position is significantly misaligned.Since the array plate is not moved within the imaging range, the effects of vibration can be reduced.

[0265] In this embodiment, the target height Z_780(Y) for a wavelength of 780 nm is used as the reference, and the movable range is calculated by adding or subtracting the movable distance from it, but the method of setting the reference is not limited to this. Conversely, the target height Z_670(Y) for a wavelength of 670 nm may be used as the reference. Also, an intermediate value between Z_780(Y) and Z_670(Y) may be used as the reference.

[0266] [Modification of Example 2] In the second embodiment of the present invention, the target height was adjusted when the focus position deviation between wavelengths was large and exceeded the range of height adjustment within the sub-scanning period. However, the main scanning speed may also be adjusted. For example, by slowing the main scanning speed and doubling the sub-scanning period Ty, the height scan distance Zd can be doubled. In this way, the target height 601 for a wavelength of 670 nm is set to fall between the lines 1101 and 1102.

[0267] In this method, the main scanning speed is slowed down, which increases the imaging time, but even if there is a large focus position deviation between the wavelengths, it is possible to adjust the focus to the optimum value for both wavelengths.

[0268] [Example 3] A third embodiment of the present invention will now be described. The third embodiment of the present invention differs from the first embodiment in that the calculation of the target height is made simpler.

[0269] In the first embodiment, the target height 602 for the wavelength of 780 nm and the target height 601 for the wavelength of 670 nm were determined separately by measuring the reflected light of each wavelength with a photodiode, but in this embodiment, the target height for one wavelength is calculated by adding a fixed value to the target height for the other wavelength. This allows for correction of focus deviation between wavelengths in a simpler manner than in the first embodiment. The device configuration, array plate, internal configuration of the controller, and detailed configuration of the device in this embodiment are the same as those in the first embodiment, and therefore will not be described here.

[0270] Most of the operational flow of this embodiment is the same as that of the first embodiment, but the target height calculation process for each row performed in step S403 of FIG. 4 is different.

[0271] In this embodiment, the target height of 680 nm is determined by adding a constant value Zo to the target height of 780 nm in step S403 of Fig. 4. In this embodiment, the variation in focus deviation between wavelengths is small across the entire array plate and is expressed by the constant value Zo.

[0272] That is, Z_780(Y) is calculated according to equation 4, while Z_670(Y) follows the equation below. Z_670(Y)=Z_780(Y)+Zo Equation (11)

[0273] The value of Zo is measured from the height information for each wavelength when height information is acquired. It is expressed by the following formula. Note that the following formula uses height information at position Y3, but Zo may also be calculated using the difference in height information at positions other than position Y3. The value of Zo can be positive or negative. Zo = Z3_670 - Z3_780 Equation (12)

[0274] According to this method, the difference between the target height 601 for a wavelength of 670 nm and the target height 602 for a wavelength of 780 nm is a constant value across the entire array plate, as shown in Figure 12, which simplifies the calculation of the movement amount during height scanning in step S418.

[0275] In this embodiment, the Zo value was determined by measurement when acquiring height information, but the method for determining Zo is not limited to this. For example, measurement may be performed using a jig external to the imaging device during calibration. Alternatively, a calibration array plate may be measured using multiple known Zo values, and the Zo value that maximizes the 670 nm luminance may be adopted. By using these methods to omit the measurement of reflected light at 670 nm, the number of components in the optical sensor, receiving circuit, and confocal optical system can be reduced.

[0276] In this embodiment, the target height Z_780(Y) at a wavelength of 780 nm is used as a reference and a fixed value is added to it to determine Z_670(Y), but the method of determining the reference is not limited to this. Conversely, the target height Z_670(Y) at a wavelength of 670 nm may be used as a reference and a fixed value may be added to it to determine Z_780(Y).

[0277] As described above, in Example 3 of the present invention, it is possible to correct focus position shifts between wavelengths using a simpler method. When there is little variation due to the thickness or tilt position of the array plate, it becomes possible to acquire fluorescence images of multiple wavelengths that are in focus across the entire surface of the array plate.

[0278] [Modification of Example 3] In the third embodiment of the present invention, Z_670(Y) is calculated by adding a constant value to Z_780(Y), but this can be simplified further by setting a single target height across the entire surface of the array plate for both wavelengths of 780 nm and 670 nm. For example, height information acquisition processing is performed at position Y3, and Z_780(Y) and Z_670(Y) are calculated according to the following formula: Z_780(Y)=Z3_780 Equation (13) Z_670(Y)=Z3_670 Equation (14) The height information acquisition process at position Y4 will be omitted.

[0279] According to this method, the target height 601 for a wavelength of 670 nm and the target height 602 for a wavelength of 780 nm are constant values ​​over the entire array plate, as shown in FIG. 13, which simplifies the calculation of the movement amount during height scanning in step S418.

[0280] This method provides a simpler method for correcting focus position shifts between wavelengths. When the array plate is not tilted much, it becomes possible to acquire fluorescence images of multiple wavelengths that are in focus to a certain extent across the entire surface of the array plate.

[0281] [Example 4] A fourth embodiment of the present invention will be described below. The fourth embodiment of the present invention differs from the first embodiment in that an intermediate height among target heights of a plurality of wavelengths is used as a target height common to both wavelengths.

[0282] In Example 1, the target height 602 for the wavelength of 780 nm and the target height 601 for the wavelength of 670 nm were determined, and height scanning was performed to match the target heights for each wavelength after switching the wavelength. However, in this example, the average height of the target height 602 for the wavelength of 780 nm and the target height 601 for the wavelength of 670 nm is determined, and height scanning is performed to achieve the same height for both the wavelength of 780 nm and the wavelength of 670 nm.

[0283] The device configuration, array plate, internal configuration of the controller, and detailed configuration of the device in this embodiment are the same as those in the first embodiment, and therefore description thereof will be omitted.

[0284] In this embodiment, in step S403 of FIG. 4, the target height for each wavelength is calculated using the following formula. Z_670(Y)=((K_670+K_780) / 2)×(Y-Y3)+((Z3_670+Z4_780) / 2) Equation (15) Z_780(Y)= Z_670(Y) Equation (16)

[0285] These are both the average heights of the target heights at wavelengths of 670 nm and 780 nm calculated by equations 3 and 4 in Example 1, and are represented by a straight line 1401 in FIG.

[0286] In the method of Example 4 of the present invention, since Z_670(Y) and Z_780(Y) are the same, height scanning when switching wavelengths can be omitted, and height scanning can be performed when sub-scanning is performed.

[0287] As shown in FIG. 8, if sub-scanning is performed after acquiring data at a wavelength of 780 nm on the forward pass and data at a wavelength of 670 nm on the return pass, the timing chart 805 between the forward pass and the return pass can be omitted.

[0288] As explained above, in Example 4 of the present invention, although it is not possible to adjust the height to an optimum value for each wavelength, it is possible to acquire fluorescent images that are in focus to a certain extent at both wavelengths. This method reduces the amount of movement in height scanning regardless of the amount of focus shift between wavelengths, and can be applied even when there are significant restrictions on the stage movement distance.

[0289] [Modification of Example 4] In this modified example, the same height is used for both wavelengths, and therefore it is also applicable to the case where light of two wavelengths is irradiated simultaneously without wavelength switching. In the first embodiment of the present invention, light emission at two wavelengths is performed in a time-division manner, but by causing the laser diode 701 and the laser diode 714 to emit light simultaneously, and separating the fluorescence from the array plate 101 with the dichroic mirror 727 and acquiring it with the photomultiplier tube 713 and the photomultiplier tube 726, it is possible to simultaneously acquire fluorescence data at two wavelengths.

[0290] By acquiring data using both wavelengths on the forward and backward passes and performing sub-scans on both the forward and backward passes, the imaging time can be reduced.

[0291] FIG. 15 is a timing chart showing the operational timing of main scanning, sub-scanning, height scanning, light irradiation, and optical signal data acquisition in this modified example. The horizontal axis of these timing charts represents time. Timing charts 801, 802, 806, and 807 are the same as those shown in FIG. 8 of the first embodiment of the present invention, so their explanation will be omitted.

[0292] Reference numeral 1501 denotes a timing chart showing the timing of the sub-scanning operation. At the times indicated by hexagons, the sub-scanning motor 110 operates and sub-scanning is performed. Reference numeral 1502 denotes a timing chart showing the operation timing of height scanning.

[0293] At the times indicated by the hexagons, the height scanning motor 113 operates and height scanning is performed. When the X coordinate of the main scan reaches X1 during movement in the backward direction, it is determined that acquisition of optical signal data in the forward direction is complete, a sub-scan trigger is output, and a sub-scan is performed to move to the next line.

[0294] When the X coordinate of the main scan reaches X2 during movement in the forward direction, it is determined that acquisition of optical signal data in the backward direction has been completed, a sub-scan trigger is output, and a sub-scan is performed to move to the next row.

[0295] Reference numeral 1503 is a timing chart showing the operation timing of the 780 nm laser diode 701 in the light source 102. At the times indicated by the hexagons, primary light of 780 nm is emitted and irradiated onto the array plate 101 via the objective optical system 104.

[0296] Reference numeral 1504 is a timing chart showing the operation timing of the 670 nm laser diode 714 in the light source 102. At the times indicated by the hexagons, primary light of 670 nm is emitted and irradiated onto the array plate 101 via the objective optical system 104. No wavelength switching trigger signal is used, and the 780 nm primary light and the 670 nm primary light are constantly irradiated during imaging.

[0297] In this way, in this modification, primary light of 780 nm and 780 nm is simultaneously irradiated onto the array plate 101, and optical signal data generated by both wavelengths is acquired on both the forward and backward paths. This makes it possible to acquire data more efficiently and shorten the imaging time compared to acquiring optical signal data with different excitation wavelengths in the forward and backward paths.

[0298] At this time, by performing height scanning in accordance with the average position of the target heights for each wavelength, it becomes possible to acquire fluorescent images of multiple wavelengths that are in focus to a certain extent.

[0299] The present disclosure includes the following compositions and methods. (Method 1) A control method for an imaging device in which an array plate having a longitudinal direction and a lateral direction and a spot array on one surface is scanned with an objective optical system that irradiates primary light and detects secondary light, thereby acquiring a fluorescent image, the method comprising: a first distance measuring step of measuring the distance to the one surface using the objective optical system that emits primary light of a first wavelength, and acquiring a plurality of pieces of first distance information for the primary light of the first wavelength corresponding to a plurality of positions in the longitudinal direction; a first scanning condition acquisition step of acquiring a first scanning condition corresponding to the primary light of the first wavelength according to a main scanning position in the longitudinal direction by using the plurality of first distance information; a second scanning condition acquisition step of acquiring second scanning conditions corresponding to primary light of a second wavelength different in wavelength from the primary light of the first wavelength according to the main scanning position, using at least one of the first scanning conditions and the first distance information; a first scanning step of scanning the objective optical system along the short-side direction under the first scanning condition; a second scanning step of scanning the objective optical system along the short-side direction under the second scanning condition; a sub-scanning step of changing the main scanning position along the longitudinal direction; A control method for an imaging device, comprising: (Method 2) acquiring a first fluorescent image obtained by irradiating the spot array with primary light of the first wavelength based on fluorescent intensity information and scanning position information detected by the objective optical system in the first scanning step; in the second scanning step, acquiring a second fluorescent image obtained by irradiating the spot array with primary light of the second wavelength based on fluorescent intensity information and scanning position information detected by the objective optical system; The method for controlling the imaging device according to Method 1, comprising: (Method 3) 3. The control method for an imaging device described in Method 1 or 2, wherein the first scanning step and the second scanning step are performed such that the primary light of the first wavelength and the primary light of the second wavelength are each scanned along a common scanning path corresponding to a common main scanning position in the longitudinal direction. (Method 4) The control method for an imaging device described in Method 3, wherein the first scanning step and the second scanning step are performed such that the primary light of the first wavelength and the primary light of the second wavelength scan the common scanning path in opposite directions. (Method 5) 5. The method for controlling an imaging device according to any one of Methods 1 to 4, wherein the sub-scanning step is performed once every time the first scanning step and the second scanning step are each performed once or more times. (Method 6) 5. The method for controlling an imaging device according to any one of Methods 1 to 4, wherein the sub-scanning step is performed once each time the first scanning step and the second scanning step are performed once. (Method 7) a distance correction step of outputting second distance information for the primary light of the second wavelength based on the first distance information; A control method for an imaging device described in any one of methods 1 to 6, wherein the second scanning condition acquisition process acquires the second scanning condition using the second distance information acquired from the distance correction unit using the plurality of first distance information and the first scanning condition. (Method 8) a scanning condition correcting step of outputting a second scanning condition for the primary light of the second wavelength corresponding to the first scanning condition; 8. The method for controlling an imaging device according to any one of Methods 1 to 7, wherein the second scanning condition acquisition step acquires the second scanning condition from the scanning condition correction unit based on the first scanning condition. (Method 9) A control method for an imaging device described in any one of methods 1 to 8, further comprising a second distance measurement step of using the objective optical system to measure the distance to the one surface with primary light of the second wavelength and obtaining second distance information for the primary light of the second wavelength. (Method 10) The method for controlling an imaging device according to Method 9, wherein the second distance measurement step is performed at a plurality of positions in the longitudinal direction. (Method 11) A control method for an imaging device described in any one of methods 1 to 10, wherein the second scanning condition acquisition process acquires the second scanning condition using the second distance information acquired in the second ranging process and the first scanning condition. (Method 12) the primary light for capturing a fluorescent image of the spot array includes either the primary light of the first wavelength or the primary light of the second wavelength; A method for controlling an imaging device described in any one of Methods 1 to 11, wherein the primary light for capturing a secondary antibody fluorescent image that labels the reaction product on the spot array includes the other of the primary light of the first wavelength and the primary light of the second wavelength. (Configuration 1) An imaging device for optically imaging an array plate having a longitudinal direction and a lateral direction and a spot array on one surface thereof in two dimensions, a mounting portion on which the array plate is mounted; an objective optical system including a light emitting unit optically coupled to a light source that selectively emits primary light of different wavelengths, and that emits the primary light to the array plate, and a light collecting unit that detects secondary light from the array plate; a distance defining unit that defines a working distance between the mounting unit and the objective optical system in the optical axis direction of the primary light; a scanning unit that changes a relative position between the mounting unit and the objective optical system in a plane that intersects with the optical axis direction; a scanning condition acquisition unit that acquires, as scanning conditions, the working distances at a plurality of positions in the plane for each wavelength of the primary light; an imaging unit that acquires a fluorescent image corresponding to the primary light based on information about the wavelength of the primary light, information about the intensity of the received light from the light collecting unit, and information about the relative position; An imaging device having the above configuration. (Configuration 2) 10. The imaging device of claim 1, further comprising a light source. (Configuration 3) 3. The imaging device according to configuration 1 or 2, further comprising a distance correction unit. (Configuration 4) 4. The imaging device according to any one of configurations 1 to 3, further comprising a scanning condition correction unit. (Configuration 5) An imaging device for optically imaging an array plate having a longitudinal direction and a lateral direction and a spot array on one surface thereof in two dimensions, a mounting portion on which the array plate is mounted; an objective optical system including a light emitting unit optically coupled to a light source that selectively emits primary light of different wavelengths, and that emits the primary light to the array plate, and a light collecting unit that detects secondary light from the array plate; a scanning unit including a main scanning unit that performs main scanning and a sub-scanning unit that performs sub-scanning along the short-side direction and the long-side direction to change a relative position between the mounting unit and the objective optical system in a plane intersecting the optical axis direction; an emission wavelength control unit that controls the light source and the scanning unit to change the wavelength of the light emitted from the light emitting unit in accordance with a sub-scanning position in the short side direction, a main scanning direction in the long side direction, and a number of main scanning passes; an imaging unit that acquires a fluorescent image corresponding to the primary light based on information about the wavelength of the primary light, information about the intensity of light received from the light collecting unit, and information about the relative position; An imaging device comprising: (Configuration 6) The imaging device described in configuration 5, wherein the emission wavelength control unit controls the light source and the scanning unit to change the wavelength of the light emitted from the light emitting unit so as to switch between primary light of a first wavelength and primary light of a second wavelength that has a different wavelength from the primary light of the first wavelength. (Configuration 7) The imaging device described in configuration 5 or 6, wherein the emission wavelength control unit controls the light source and the scanning unit to perform a main scan with one of the primary light of the first wavelength and the primary light of the second wavelength, and then switch to the other of the primary light of the first wavelength and the primary light of the second wavelength, so that the primary light of the first wavelength and the primary light of the second wavelength perform a main scan on a common scanning path. (Configuration 8) The imaging device according to any one of configurations 5 to 7, wherein the emission wavelength control unit controls the light source and the scanning unit to perform the sub-scanning step once each time a first scanning step of scanning with the first light and a second scanning step of scanning with the second light on a common scanning path are performed at least once. (Configuration 9) The imaging device according to any one of configurations 5 to 7, wherein the emission wavelength control unit controls the light source and the scanning unit to perform the sub-scanning step once each time a first scanning step of scanning with the first light and a second scanning step of scanning with the second light on a common scanning path are performed once. (Configuration 10) The imaging device described in configuration 8, wherein the emission wavelength control unit controls the light source and the scanning unit to include a period in which the wavelength is switched from one of the first scanning step and the second scanning step to the other of the first scanning step and the second scanning step without going through the sub-scanning step. (Configuration 11) 11. The imaging device according to any one of configurations 5 to 10, further comprising a light source. (Configuration 12) a distance defining unit that defines a working distance between the mounting unit and the objective optical system in the optical axis direction of the primary light, 12. The imaging device according to any one of configurations 5 to 11, wherein the emission wavelength control unit controls the working distance defined by the distance defining unit in accordance with the wavelength of the primary light. (Configuration 13) 13. The imaging device according to configuration 12, further comprising a scanning condition acquisition unit that acquires, as scanning conditions, the working distances at a plurality of positions in the plane for each wavelength of the primary light. [Explanation of symbols]

[0300] 100: Imaging device 101: Array plate 102: Light source 103: Confocal optical system 104: Objective optical system 105: Optical sensor 106: Receiving circuit 107: Main scanning motor 108: Encoder 109: Linear stage (mounting part) 110: Sub-scanning motor 111: Motor driver 112: Linear stage 113: Height scanning motor 114: Motor driver 115: Motor driver 116: Controller 117: User Interface 118: Crank 119: Connecting rod 120: Piston crank mechanism 170: Scanning unit 180: Sub-scanning section 190: Main scanning section 201: Glass slide 202: Spot 301:CPU 302: Bus interface 303: Memory 304: Memory control circuit 305: Light source control circuit 306: ADC control circuit 307: Motor control circuit 308: Motor control circuit 309: Motor control circuit 310: Coordinate calculation circuit 311: Synchronous circuit 312: Data acquisition control circuit 313: Communication circuit 314:UI circuit 315: Scanning condition acquisition unit 316: Distance regulation section 317: Imaging Department 318: Emission wavelength control section 503: Imaging area 601: Target height for wavelength 670nm 602: Target height for wavelength 780nm 603: Rectangle 604: Rectangle 701: Laser diode 702: Collimating lens 703: Bandpass filter 704: Polarizing beam splitter 705:1 / 4λ wavelength plate 706:Dichroic mirror 707: Condenser lens 708: Fluorescent filter 709: Condenser lens 710: Pinhole 711: Pinhole 712: Photodiode 713: Photomultiplier tube 714: Laser diode 715: Collimating lens 716: Bandpass filter 717: Polarizing beam splitter 718:1 / 4λ wavelength plate 719:Dichroic mirror 720: Condenser lens 721: Fluorescence filter 722: Condenser lens 723: Pinhole 724: Pinhole 725: Photodiode 726: Photomultiplier tube 727:Dichroic mirror 728: Mirror 729: Objective lens 730: Main scanning mechanism 731: Current-to-voltage conversion amplifier 732: Current-to-voltage conversion amplifier 733: Current-to-voltage conversion amplifier 734: Current-to-voltage conversion amplifier 735: AD converter 736: AD converter 737: AD converter 738: AD converter

Claims

1. A control method for an imaging device in which an array plate having a longitudinal direction and a lateral direction and a spot array on one surface is irradiated with primary light and an objective optical system that detects secondary light is scanned to acquire a fluorescent image, the method comprising: a first distance measuring step of measuring the distance to the one surface using the objective optical system that emits primary light of a first wavelength, and acquiring a plurality of pieces of first distance information for the primary light of the first wavelength corresponding to a plurality of positions in the longitudinal direction; a first scanning condition acquisition step of acquiring a first scanning condition corresponding to the primary light of the first wavelength according to a main scanning position in the longitudinal direction by using the plurality of first distance information; a second scanning condition acquisition step of acquiring second scanning conditions corresponding to primary light of a second wavelength, the second wavelength being different from the primary light of the first wavelength according to the main scanning position, using at least one of the first scanning condition and the first distance information; a first scanning step of scanning the objective optical system along the short-side direction under the first scanning condition; a second scanning step of scanning the objective optical system along the short-side direction under the second scanning condition; a sub-scanning step of changing the main scanning position along the longitudinal direction; A control method for an imaging device, comprising:

2. acquiring a first fluorescent image obtained by irradiating the spot array with primary light of the first wavelength based on fluorescent intensity information and scanning position information detected by the objective optical system in the first scanning step; acquiring a second fluorescent image obtained by irradiating the spot array with primary light of the second wavelength based on fluorescent intensity information and scanning position information detected by the objective optical system in the second scanning step; The method for controlling an imaging device according to claim 1 , comprising:

3. 3. The control method for an imaging device according to claim 1, wherein the first scanning step and the second scanning step are performed such that the primary light of the first wavelength and the primary light of the second wavelength are scanned along a common scanning path corresponding to a common main scanning position in the longitudinal direction.

4. 4. The control method for an imaging device according to claim 3, wherein the first scanning step and the second scanning step are performed such that the primary light of the first wavelength and the primary light of the second wavelength scan the common scanning path in opposite directions.

5. 3. The method of controlling an imaging apparatus according to claim 1, wherein the sub-scanning step is performed once every time the first scanning step and the second scanning step are each performed once or more times.

6. 3. The method of controlling an imaging apparatus according to claim 1, wherein the sub-scanning step is performed once each time the first scanning step and the second scanning step are performed once.

7. a distance correction step of outputting second distance information for the primary light of the second wavelength based on the first distance information; 3. The control method for an imaging device according to claim 1, wherein the second scanning condition acquisition step acquires the second scanning condition by using the second distance information obtained in the distance correction step using the plurality of first distance information and the first scanning condition.

8. a scanning condition correcting step of outputting a second scanning condition for the primary light of the second wavelength corresponding to the first scanning condition, 3. The method of controlling an imaging apparatus according to claim 1, wherein the second scanning condition acquisition step acquires the second scanning condition from the scanning condition correction step based on the first scanning condition.

9. 3. The control method for an imaging device according to claim 1, further comprising a second distance measurement step of measuring the distance to the one surface using the objective optical system with primary light of the second wavelength and obtaining second distance information for the primary light of the second wavelength.

10. The method of controlling an imaging device according to claim 9 , wherein the second distance measurement step is performed at a plurality of positions in the longitudinal direction.

11. 10. The control method for an imaging device according to claim 9, wherein the second scanning condition acquisition step acquires the second scanning condition using the second distance information acquired in the second ranging step and the first scanning condition.

12. the primary light for capturing a fluorescent image of the spot array includes either the primary light of the first wavelength or the primary light of the second wavelength; 3. The method for controlling an imaging device according to claim 1, wherein primary light for capturing a secondary antibody fluorescent image that labels a reaction product on the spot array includes the other of the primary light of the first wavelength and the primary light of the second wavelength.

13. An imaging device for optically imaging an array plate having a longitudinal direction and a lateral direction and a spot array on one surface thereof in two dimensions, a mounting portion on which the array plate is mounted; an objective optical system including a light emitting unit optically coupled to a light source that selectively emits primary light of different wavelengths, and that emits the primary light to the array plate, and a light collecting unit that detects secondary light from the array plate; a distance defining unit that defines a working distance between the mounting unit and the objective optical system in the optical axis direction of the primary light; a scanning unit that changes a relative position between the mounting unit and the objective optical system in a plane that intersects with the optical axis direction; a scanning condition acquisition unit that acquires, as scanning conditions, the working distances at a plurality of positions in the plane for each wavelength of the primary light; an imaging unit that acquires a fluorescent image corresponding to the primary light based on information about the wavelength of the primary light, information about the intensity of the received light from the light collecting unit, and information about the relative position; An imaging device having the above configuration.

14. The imaging device of claim 13 further comprising a light source.

15. The imaging device according to claim 13 or 14, further comprising a distance correction unit.

16. The imaging device according to claim 13 or 14, further comprising a scanning condition correction unit.

17. An imaging device for optically imaging an array plate having a longitudinal direction and a lateral direction and a spot array on one surface thereof in two dimensions, a mounting portion on which the array plate is mounted; an objective optical system including a light emitting unit optically coupled to a light source that selectively emits primary light of different wavelengths, and that emits the primary light to the array plate, and a light collecting unit that detects secondary light from the array plate; a scanning unit including a main scanning unit that performs main scanning and a sub-scanning unit that performs sub-scanning along the short-side direction and the long-side direction to change the relative positions between the mounting unit and the objective optical system in a plane that intersects with the optical axis direction; an emission wavelength control unit that controls the light source and the scanning unit to change the wavelength of the light emitted from the light emitting unit in accordance with a sub-scanning position in the short side direction, a main scanning direction in the long side direction, and a number of main scanning passes; an imaging unit that acquires a fluorescent image corresponding to the primary light based on information about the wavelength of the primary light, information about the intensity of light received from the light collecting unit, and information about the relative position; An imaging device comprising:

18. 18. The imaging device according to claim 17, wherein the emission wavelength control unit controls the light source and the scanning unit to change the wavelength of the light emitted from the light emitting unit so as to switch between primary light of a first wavelength and primary light of a second wavelength that has a different wavelength from the primary light of the first wavelength.

19. 19. The imaging device according to claim 18, wherein the emission wavelength control unit controls the light source and the scanning unit to perform a main scan with one of the primary light of the first wavelength and the primary light of the second wavelength, and then switch to the other of the primary light of the first wavelength and the primary light of the second wavelength, so that the primary light of the first wavelength and the primary light of the second wavelength perform main scanning on a common scanning path.

20. 19. The imaging device according to claim 18, wherein the emission wavelength control unit controls the light source and the scanning unit so as to perform a sub-scanning step once each time a first scanning step of scanning a common scanning path with primary light of the first wavelength and a second scanning step of scanning with primary light of the second wavelength are performed once or more times.

21. 19. The imaging device according to claim 18, wherein the emission wavelength control unit controls the light source and the scanning unit so as to perform the sub-scanning step once each time a first scanning step of scanning a common scanning path with primary light of the first wavelength and a second scanning step of scanning with primary light of the second wavelength are performed once.

22. 21. The imaging device according to claim 20, wherein the emission wavelength control unit controls the light source and the scanning unit so as to include a period in which the wavelength is switched from one of the first scanning step and the second scanning step to the other of the first scanning step and the second scanning step without passing through the sub-scanning step.

23. The imaging device of claim 17 further comprising a light source.

24. a distance defining unit that defines a working distance between the mounting unit and the objective optical system in the optical axis direction of the primary light, The imaging device according to claim 17 , wherein the emission wavelength control unit controls the working distance defined by the distance defining unit in accordance with the wavelength of the primary light.

25. The imaging device according to claim 24 , further comprising a scanning condition acquisition unit that acquires, as scanning conditions, the working distances at a plurality of positions in the plane for each wavelength of the primary light.

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